In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

PubMed Central

Palmiotti, Christopher A.; Prasad, Shikha; Naik, Pooja; Abul, Kaisar MD; Sajja, Ravi K.; Achyuta, Anilkumar H.; Cucullo, Luca

2014-01-01

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-Brain Barrier. PMID:25098812

In vitro cerebrovascular modeling in the 21st century: current and prospective technologies.

PubMed

Palmiotti, Christopher A; Prasad, Shikha; Naik, Pooja; Abul, Kaisar M D; Sajja, Ravi K; Achyuta, Anilkumar H; Cucullo, Luca

2014-12-01

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-brain Barrier.

In silico modeling on ADME properties of natural products: Classification models for blood-brain barrier permeability, its application to traditional Chinese medicine and in vitro experimental validation.

PubMed

Zhang, Xiuqing; Liu, Ting; Fan, Xiaohui; Ai, Ni

2017-08-01

In silico modeling of blood-brain barrier (BBB) permeability plays an important role in early discovery of central nervous system (CNS) drugs due to its high-throughput and cost-effectiveness. Natural products (NP) have demonstrated considerable therapeutic efficacy against several CNS diseases. However, BBB permeation property of NP is scarcely evaluated both experimentally and computationally. It is well accepted that significant difference in chemical spaces exists between NP and synthetic drugs, which calls into doubt on suitability of available synthetic chemical based BBB permeability models for the evaluation of NP. Herein poor discriminative performance on BBB permeability of NP are first confirmed using internal constructed and previously published drug-derived computational models, which warrants the need for NP-oriented modeling. Then a quantitative structure-property relationship (QSPR) study on a NP dataset was carried out using four different machine learning methods including support vector machine, random forest, Naïve Bayes and probabilistic neural network with 67 selected features. The final consensus model was obtained with approximate 90% overall accuracy for the cross-validation study, which is further taken to predict passive BBB permeability of a large dataset consisting of over 10,000 compounds from traditional Chinese medicine (TCM). For 32 selected TCM molecules, their predicted BBB permeability were evaluated by in vitro parallel artificial membrane permeability assay and overall accuracy for in vitro experimental validation is around 81%. Interestingly, our in silico model successfully predicted different BBB permeation potentials of parent molecules and their known in vivo metabolites. Finally, we found that the lipophilicity, the number of hydrogen bonds and molecular polarity were important molecular determinants for BBB permeability of NP. Our results suggest that the consensus model proposed in current work is a reliable tool for prioritizing potential CNS active NP across the BBB, which would accelerate their development and provide more understanding on their mechanisms, especially those with pharmacologically active metabolites. Copyright © 2017 Elsevier Inc. All rights reserved.

β-cyclodextrin-poly(β-amino ester) nanoparticles for sustained drug delivery across the blood-brain barrier.

PubMed

Gil, Eun Seok; Wu, Linfeng; Xu, Lichong; Lowe, Tao Lu

2012-11-12

Novel biodegradable polymeric nanoparticles composed of β-cyclodextrin and poly(β-amino ester) segments have been developed for sustained drug delivery across the blood-brain barrier (BBB). The nanoparticles have been synthesized by cross-linking β-cyclodextrin with poly(β-amino ester) via the Michael addition method. The chemical, physical, and degradation properties of the nanoparticles have been characterized by matrix-assisted laser desoption/ionization time-of-flight, attenuated total reflectance Fourier transform infrared spectroscopy, nuclear magnetic resonance, dynamic light scattering, and atomic force microscopy techniques. Bovine and human brain microvascular endothelial cell monolayers have been constructed as in vitro BBB models. Preliminary results show that the nanoparticles do not affect the integrity of the in vitro BBB models, and the nanoparticles have much higher permeability than dextran control across the in vitro BBB models. Doxorubicin has been loaded into the nanoparticles with a loading efficiency of 86%, and can be released from the nanoparticles for at least one month. The developed β-cyclodextrin-poly(β-amino ester) nanoparticles might be useful as drug carriers for transporting drugs across the BBB to treat chronic diseases in the brain.

Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model.

PubMed

Mantle, Jennifer L; Min, Lie; Lee, Kelvin H

2016-12-05

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm 2 . By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy.

PubMed

Masago, Kayo; Kihara, Yasuyuki; Yanagida, Keisuke; Hamano, Fumie; Nakagawa, Shinsuke; Niwa, Masami; Shimizu, Takao

2018-07-02

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA 6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA-LPA 6 -G 12/13 -Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema. Copyright © 2018 Elsevier Inc. All rights reserved.

Involvement of DMT1 +IRE in the transport of lead in an in vitro BBB model.

PubMed

Wang, Qiang; Luo, Wenjing; Zhang, Wenbin; Liu, Mingchao; Song, Haifeng; Chen, Jingyuan

2011-06-01

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB). The BBB is particularly vulnerable to lead (Pb) insults. This study was designed to test the hypothesis that divalent metal transporter 1 (DMT1), which is a divalent cation membrane transporter, was involved in transcellular transport across the BBB. An in vitro BBB model, which was a co-culture system of human umbilical vascular endothelial cells (ECV304) and rat glioma cells (C6), was established. Transendothelial electrical resistance (TEER) and fluoresceinisothiocyanate (FITC)-dextran permeability results showed that Pb exposure at the tested concentrations had no significant effects on intercellular tightness. Pb transport displayed properties that were associated with iron response element (IRE) positive isoform of DMT1. Accordingly, Pb transport was significantly blocked by iron (Fe). Moreover, ECV304 cells that were depleted of Fe with the chelator deferoxamine (DFO) demonstrated increased Pb transport. By transfecting ECV-304 cells with a DMT1 expression vector, overexpression of DMT1 promoted an increase in Pb transport. Treatment of ECV304 cells with DMT1 antisense oligonucleotides (ASONs) MA1 significantly inhibited the transport of Pb. Our results suggest that Pb is transported in the in vitro BBB model by a transporter with biochemical properties similar to those of the DMT1 IRE-positive isoform. Copyright © 2009 Elsevier Ltd. All rights reserved.

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

PubMed

Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon

2014-11-01

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis. © 2014 UICC.

Experimental methods and transport models for drug delivery across the blood-brain barrier.

PubMed

Fu, Bingmei M

2012-06-01

The blood-brain barrier (BBB) is a dynamic barrier essential for maintaining the micro-environment of the brain. Although the special anatomical features of the BBB determine its protective role for the central nervous system (CNS) from blood-born neurotoxins, however, the BBB extremely limits the therapeutic efficacy of drugs into the CNS, which greatly hinders the treatment of major brain diseases. This review summarized the unique structures of the BBB, described a variety of in vivo and in vitro experimental methods for determining the transport properties of the BBB, e.g., the permeability of the BBB to water, ions, and solutes including nutrients, therapeutic agents and drug carriers, and presented newly developed mathematical models which quantitatively correlate the anatomical structures of the BBB with its barrier functions. Finally, on the basis of the experimental observations and the quantitative models, several strategies for drug delivery through the BBB were proposed.

Experimental Methods and Transport Models for Drug Delivery across the Blood-Brain Barrier

PubMed Central

Fu, Bingmei M

2017-01-01

The blood-brain barrier (BBB) is a dynamic barrier essential for maintaining the micro-environment of the brain. Although the special anatomical features of the BBB determine its protective role for the central nervous system (CNS) from blood-born neurotoxins, however, the BBB extremely limits the therapeutic efficacy of drugs into the CNS, which greatly hinders the treatment of major brain diseases. This review summarized the unique structures of the BBB, described a variety of in vivo and in vitro experimental methods for determining the transport properties of the BBB, e.g., the permeability of the BBB to water, ions, and solutes including nutrients, therapeutic agents and drug carriers, and presented newly developed mathematical models which quantitatively correlate the anatomical structures of the BBB with its barrier functions. Finally, on the basis of the experimental observations and the quantitative models, several strategies for drug delivery through the BBB were proposed. PMID:22201587

Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood-brain barrier studies.

PubMed

Kamiichi, Atsuko; Furihata, Tomomi; Kishida, Satoshi; Ohta, Yuki; Saito, Kosuke; Kawamatsu, Shinya; Chiba, Kan

2012-12-07

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMEC) working together with astrocytes and pericytes, in which tight junctions and various transporters strictly regulate the penetration of diverse compounds into the brain. Clarification of the molecular machinery that provides such regulation using in vitro BBB models has provided important insights into the roles of the BBB in central nervous system (CNS) disorders and CNS drug development. In this study, we succeeded in establishing a new cell line, hereinafter referred to as human BMEC/conditionally immortalized, clone β (HBMEC/ciβ), as part of our ongoing efforts to develop an in vitro human BBB model. Our results showed that HBMEC/ciβ proliferated well. Furthermore, we found that HBMEC/ciβ exhibited the barrier property of restricting small molecule intercellular penetration and possessed effective efflux transporter functions, both of which are essential to a functioning BBB. Because higher temperatures are known to terminate immortalization signals, we specifically examined the effects of higher temperatures on the HBMEC/ciβ differentiation status. The results showed that higher temperatures stimulated HBMEC/ciβ differentiation, marked by morphological alteration and increases in several mRNA levels. To summarize, our data indicates that the newly established HBMEC/ciβ offers a promising tool for use in the development of a practical in vitro human BBB model that could make significant contributions toward understanding the molecular biology of CNS disorders, as well as to CNS drug development. It is also believed that the development of a specific culture method for HBMEC/ciβ will add significant value to the HBMEC/ciβ-based BBB model. Copyright © 2012 Elsevier B.V. All rights reserved.

Zika virus crosses an in vitro human blood brain barrier model.

PubMed

Alimonti, Judie B; Ribecco-Lutkiewicz, Maria; Sodja, Caroline; Jezierski, Anna; Stanimirovic, Danica B; Liu, Qing; Haqqani, Arsalan S; Conlan, Wayne; Bani-Yaghoub, Mahmud

2018-05-15

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.

In vitro and in vivo studies on the transport of PEGylated silica nanoparticles across the blood-brain barrier.

PubMed

Liu, Dan; Lin, Bingqian; Shao, Wei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong

2014-02-12

Transport of PEGylated silica nanoparticles (PSiNPs) with diameters of 100, 50, and 25 nm across the blood-brain barrier (BBB) was evaluated using an in vitro BBB model based on mouse cerebral endothelial cells (bEnd.3) cultured on transwell inserts within a chamber. In vivo animal experiments were further performed by noninvasive in vivo imaging and ex vivo optical imaging after injection via carotid artery. Confocal fluorescence studies were carried out to evaluate the uptake of PSiNPs by brain endothelial cells. The results showed that PSiNPs can traverse the BBB in vitro and in vivo. The transport efficiency of PSiNPs across BBB was found to be size-dependent, with increased particle size resulting in decreased efficiency. This work points to the potential application of small sized silica nanoparticles in brain imaging or drug delivery.

Uptake and transcytosis of functionalized superparamagnetic iron oxide nanoparticles in an in vitro blood brain barrier model.

PubMed

Ivask, Angela; Pilkington, Emily H; Blin, Thomas; Käkinen, Aleksandr; Vija, Heiki; Visnapuu, Meeri; Quinn, John F; Whittaker, Michael R; Qiao, Ruirui; Davis, Thomas P; Ke, Pu Chun; Voelcker, Nicolas H

2018-01-30

Two major hurdles in nanomedicine are the limited strategies for synthesizing stealth nanoparticles and the poor efficacy of the nanoparticles in translocating across the blood brain barrier (BBB). Here we examined the uptake and transcytosis of iron oxide nanoparticles (IONPs) grafted with biomimetic phosphorylcholine (PC) brushes in an in vitro BBB model system, and compared them with bare, PEG or PC-PEG mixture grafted IONPs. Hyperspectral imaging indicated IONP co-localization with cells. Quantitative analysis with total reflection X-ray fluorescence spectrometry showed that after 24 h, 78% of PC grafted, 68-69% of PEG or PC-PEG grafted, and 30% of bare IONPs were taken up by the BBB. Transcytosis of IONPs was time-dependent and after 24 h, 16-17% of PC or PC-PEG mixture grafted IONPs had passed the BBB model, significantly more than PEG grafted or bare IONPs. These findings point out that grafting of IONPs with PC is a viable strategy for improving the uptake and transcytosis of nanoparticles.

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Differences in amyloid-β clearance across mouse and human blood-brain barrier models: kinetic analysis and mechanistic modeling.

PubMed

Qosa, Hisham; Abuasal, Bilal S; Romero, Ignacio A; Weksler, Babette; Couraud, Pierre-Oliver; Keller, Jeffrey N; Kaddoumi, Amal

2014-04-01

Alzheimer's disease (AD) has a characteristic hallmark of amyloid-β (Aβ) accumulation in the brain. This accumulation of Aβ has been related to its faulty cerebral clearance. Indeed, preclinical studies that used mice to investigate Aβ clearance showed that efflux across blood-brain barrier (BBB) and brain degradation mediate efficient Aβ clearance. However, the contribution of each process to Aβ clearance remains unclear. Moreover, it is still uncertain how species differences between mouse and human could affect Aβ clearance. Here, a modified form of the brain efflux index method was used to estimate the contribution of BBB and brain degradation to Aβ clearance from the brain of wild type mice. We estimated that 62% of intracerebrally injected (125)I-Aβ40 is cleared across BBB while 38% is cleared by brain degradation. Furthermore, in vitro and in silico studies were performed to compare Aβ clearance between mouse and human BBB models. Kinetic studies for Aβ40 disposition in bEnd3 and hCMEC/D3 cells, representative in vitro mouse and human BBB models, respectively, demonstrated 30-fold higher rate of (125)I-Aβ40 uptake and 15-fold higher rate of degradation by bEnd3 compared to hCMEC/D3 cells. Expression studies showed both cells to express different levels of P-glycoprotein and RAGE, while LRP1 levels were comparable. Finally, we established a mechanistic model, which could successfully predict cellular levels of (125)I-Aβ40 and the rate of each process. Established mechanistic model suggested significantly higher rates of Aβ uptake and degradation in bEnd3 cells as rationale for the observed differences in (125)I-Aβ40 disposition between mouse and human BBB models. In conclusion, current study demonstrates the important role of BBB in the clearance of Aβ from the brain. Moreover, it provides insight into the differences between mouse and human BBB with regards to Aβ clearance and offer, for the first time, a mathematical model that describes Aβ clearance across BBB. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differences in amyloid-β clearance across mouse and human blood-brain barrier models: Kinetic analysis and mechanistic modeling

PubMed Central

Qosa, Hisham; Abuasal, Bilal S.; Romero, Ignacio A.; Weksler, Babette; Couraud, Pierre-Oliver; Keller, Jeffrey N.; Kaddoumi, Amal

2014-01-01

Alzheimer’s disease (AD) has a characteristic hallmark of amyloid-β (Aβ) accumulation in the brain. This accumulation of Aβ has been related to its faulty cerebral clearance. Indeed, preclinical studies that used mice to investigate Aβ clearance showed that efflux across blood-brain barrier (BBB) and brain degradation mediate efficient Aβ clearance. However, the contribution of each process to Aβ clearance remains unclear. Moreover, it is still uncertain how species differences between mouse and human could affect Aβ clearance. Here, a modified form of the brain efflux index method was used to estimate the contribution of BBB and brain degradation to Aβ clearance from the brain of wild type mice. We estimated that 62% of intracerebrally injected 125I-Aβ40 is cleared across BBB while 38% is cleared by brain degradation. Furthermore, in vitro and in silico studies were performed to compare Aβ clearance between mouse and human BBB models. Kinetic studies for Aβ40 disposition in bEnd3 and hCMEC/D3 cells, representative in vitro mouse and human BBB models, respectively, demonstrated 30-fold higher rate of 125I-Aβ40 uptake and 15-fold higher rate of degradation by bEnd3 compared to hCMEC/D3 cells. Expression studies showed both cells to express different levels of P-glycoprotein and RAGE, while LRP1 levels were comparable. Finally, we established a mechanistic model, which could successfully predict cellular levels of 125I-Aβ40 and the rate of each process. Established mechanistic model suggested significantly higher rates of Aβ uptake and degradation in bEnd3 cells as rationale for the observed differences in 125I-Aβ40 disposition between mouse and human BBB models. In conclusion, current study demonstrates the important role of BBB in the clearance of Aβ from the brain. Moreover, it provides insight into the differences between mouse and human BBB with regards to Aβ clearance and offer, for the first time, a mathematical model that describes Aβ clearance across BBB. PMID:24467845

Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rempe, Ralf; Cramer, Sandra; Huewel, Sabine

2011-03-04

Research highlights: {yields} Poly(n-butylcyano-acrylate) (PBCA) nanoparticles may be promising drug carriers. {yields} Influence of PBCA nanoparticles on the integrity of the blood-brain barrier in vitro. {yields} PBCA nanoparticles lead to a reversible disruption of the BBB in vitro after 4 h. {yields} Potential application as time-dependent and specific opener of the BBB. -- Abstract: In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood-brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effectmore » on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances {sup 14}C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4 h. The observed disruption of the barrier could also be confirmed by {sup 14}C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4 h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.« less

Blood-brain barrier (BBB) toxicity and permeability assessment after L-(4-¹⁰Boronophenyl)alanine, a conventional B-containing drug for boron neutron capture therapy, using an in vitro BBB model.

PubMed

Roda, E; Nion, S; Bernocchi, G; Coccini, T

2014-10-02

Since brain tumours are the primary candidates for treatment by Boron Neutron Capture Therapy, one major challenge in the selective drug delivery to CNS is the crossing of the blood-brain barrier (BBB). The present pilot study investigated (i) the transport of a conventional B-containing product (i.e., L-(4-(10)Boronophenyl)alanine, L-(10)BPA), already used in medicine but still not fully characterized regarding its CNS interactions, as well as (ii) the effects of the L-(10)BPA on the BBB integrity using an in vitro model, consisting of brain capillary endothelial cells co-cultured with glial cells, closely mimicking the in vivo conditions. The multi-step experimental strategy (i.e. Integrity test, Filter study, Transport assay) checked L-(10)BPA toxicity at 80 µg Boron equivalent/ml, and its ability to cross the BBB, additionally by characterizing the cytoskeletal and TJ's proteins by immunocytochemistry and immunoblotting. In conclusion, a lack of toxic effects of L-(10)BPA was demonstrated, nevertheless accompanied by cellular stress phenomena (e.g. vimentin expression modification), paralleled by a low permeability coefficient (0.39 ± 0.01 × 10(-3)cm min(-1)), corroborating the scarce probability that L-(10)BPA would reach therapeutically effective cerebral concentration. These findings emphasized the need for novel strategies aimed at optimizing boron delivery to brain tumours, trying to ameliorate the compound uptake or developing new targeted products suitable to safely and effectively treat head cancer. Thus, the use of in vitro BBB model for screening studies may provide a useful early safety assessment for new effective compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro.

PubMed

Kangwantas, Korakoch; Pinteaux, Emmanuel; Penny, Jeffrey

2016-02-01

The blood-brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro. We used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR. OGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1. Our data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Theranostic quantum dots for crossing blood–brain barrier in vitro and providing therapy of HIV-associated encephalopathy

PubMed Central

Xu, Gaixia; Mahajan, Supriya; Roy, Indrajit; Yong, Ken-Tye

2013-01-01

The blood–brain barrier (BBB) is a complex physiological checkpoint that restricts the free diffusion of circulating molecules from the blood into the central nervous system. Delivering of drugs and other active agents across the BBB is one of the major technical challenges faced by scientists and medical practitioners. Therefore, development of novel methodologies to address this challenge holds the key for both the diagnosis and treatment of brain diseases, such as HIV-associated encephalopathy. Bioconjugated quantum dots (QDs) are excellent fluorescent probes and nano-vectors, being designed to transverse across the BBB and visualize drug delivery inside the brain. This paper discusses the use of functionalized QDs for crossing the blood–brain barrier and treating brain disease. We highlight the guidelines for using in vitro BBB models for brain disease studies. The theranostic QDs offers a strategy to significantly improve the effective dosages of drugs to transverse across the BBB and orientate to the targets inside the brain. PMID:24298256

Neuronal and BBB damage induced by sera from patients with secondary progressive multiple sclerosis.

PubMed

Proia, Patrizia; Schiera, Gabriella; Salemi, Giuseppe; Ragonese, Paolo; Savettieri, Giovanni; Di Liegro, Italia

2009-12-01

An important component of the pathogenic process of multiple sclerosis (MS) is the blood-brain barrier (BBB) damage. We recently set an in vitro model of BBB, based on a three-cell-type co-culture system, in which rat neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological BBB. Herein we report that the serum from patients with secondary progressive multiple sclerosis (SPMS) has a damaging effect on isolated neurons. This finding suggests that neuronal damaging in MS could be a primary event and not only secondary to myelin damage, as generally assumed. SPMS serum affects the permeability of the BBB model, as indicated by the decrease of the transendothelial electrical resistance (TEER). Moreover, as shown by both immunofluorescence and Western blot analyses, BBB breaking is accompanied by a decrease of the synthesis as well as the peripheral localization of occludin, a structural protein of the tight junctions that are responsible for BBB properties.

Novel Antitransferrin Receptor Antibodies Improve the Blood-Brain Barrier Crossing Efficacy of Immunoliposomes.

PubMed

Gregori, Maria; Orlando, Antonina; Re, Francesca; Sesana, Silvia; Nardo, Luca; Salerno, Domenico; Mantegazza, Francesco; Salvati, Elisa; Zito, Andrea; Malavasi, Fabio; Masserini, Massimo; Cazzaniga, Emanuela

2016-01-01

Surface functionalization with antitransferrin receptor (TfR) mAbs has been suggested as the strategy to enhance the transfer of nanoparticles (NPs) across the blood-brain barrier (BBB) and to carry nonpermeant drugs from the blood into the brain. However, the efficiency of BBB crossing is currently too poor to be used in vivo. In the present investigation, we compared 6 different murine mAbs specific for different epitopes of the human TfR to identify the best performing one for the functionalization of NPs. For this purpose, we compared the ability of mAbs to cross an in vitro BBB model made of human brain capillary endothelial cells (hCMEC/D3). Liposomes functionalized with the best performing mAb (MYBE/4C1) were uptaken, crossed the BBB in vitro, and facilitated the BBB in vitro passage of doxorubicin, an anticancer drug, 3.9 folds more than liposomes functionalized with a nonspecific IgG, as assessed by confocal microscopy, radiochemical techniques, and fluorescence, and did not modify the cell monolayer structural or functional properties. These results show that MYBE/4C1 antihuman TfR mAb is a powerful resource for the enhancement of BBB crossing of NPs and is therefore potentially useful in the treatment of neurologic diseases and disorders including brain carcinomas. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Electro-Magnetic Nano-Particle Bound Beclin1 siRNA Crosses the Blood-Brain Barrier to Attenuate the Inflammatory Effects of HIV-1 Infection in Vitro.

PubMed

Rodriguez, Myosotys; Kaushik, Ajeet; Lapierre, Jessica; Dever, Seth M; El-Hage, Nazira; Nair, Madhavan

2017-03-01

The purpose of this study was to evaluate a novel drug delivery system comprised of ferric-cobalt electro-magnetic nano-material (CoFe2O4@ BaTiO3; MENP) bound to siRNA targeting Beclin1 (MENP-siBeclin1) to cross the blood-brain barrier (BBB) and attenuate the neurotoxic effects of HIV-1 infection in the central nervous system following on-demand release of siRNA using an in vitro primary human BBB model. Beclin1 is a key protein in the regulation of the autophagy pathway and we have recently demonstrated the importance of Beclin1 in regulating viral replication and viral-induced inflammation in HIV-1-infected microglia. The MENP-siBeclin1 nano-formulation did not compromise the physiological function or integrity of the BBB model. Furthermore, the in vitro BBB data revealed that MENP-siBeclin1 could efficiently attenuate viral replication and viral-induced inflammation, likely due to STAT1/ NF-κB signaling pathways. MENP-siBeclin1 also silenced Beclin1 protein expression in HIV-1-infected microglial cells within the model system. In addition, the cytotoxic effects of direct treatment with siBeclin1 and MENP alone or in nano-formulation on primary human neuronal cells showed a minimal amount of cell death. Overall, the data shows that the nano-formulation can silence the BECN1 gene as an effective mechanism to attenuate HIV-1 replication and viral-induced inflammation in the context of the BBB.

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

PubMed

Cecchelli, Romeo; Aday, Sezin; Sevin, Emmanuel; Almeida, Catarina; Culot, Maxime; Dehouck, Lucie; Coisne, Caroline; Engelhardt, Britta; Dehouck, Marie-Pierre; Ferreira, Lino

2014-01-01

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Human astrocytes/astrocyte conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

PubMed Central

Siddharthan, Venkatraman; V. Kim, Yuri; Liu, Suyi; Kim, Kwang Sik

2009-01-01

The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC. PMID:17368578

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

PubMed

Adriani, G; Ma, D; Pavesi, A; Goh, E L K; Kamm, R D

2015-01-01

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

PubMed

Kokubu, Yasuhiro; Yamaguchi, Tomoko; Kawabata, Kenji

2017-04-29

Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. Copyright © 2017 Elsevier Inc. All rights reserved.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

PubMed

Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

2015-08-07

Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer. The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Enhancing the Delivery of Anti Retroviral Drug “Saquinavir” Across the Blood Brain Barrier Using Nanoparticles

PubMed Central

Mahajan, Supriya D.; Roy, Indrajit; Xu, GaiXia; Yong, Ken-Tye; Ding, Hong; Aalinkeel, Ravikumar; Reynolds, Jessica L.; Sykes, Donald E.; Nair, Bindukumar B.; Lin, Elaine Y.; Prasad, Paras N.; Schwartz, Stanley A.

2010-01-01

Antiretroviral drugs are ineffective at treating viral infection in the brain because they cannot freely diffuse across the blood-brain barrier (BBB). Therefore, HIV-1 viral replication persists in the central nervous system (CNS) and continues to augment the neuropathogenesis process. Nanotechnology can play a pivotal role in HIV-1 therapeutics as it can increase drug solubility, enhance systemic bioavailability, and at the same time offer multifunctionality. Moreover, following conjugation with transferrin (Tf), these drug-loaded nanoformulations can permeate across biological barriers such as the blood brain barrier (BBB) via a receptor mediated transport mechanism. In the current study, we have stably incorporated the antiviral drug, Saquinavir, within Tf-conjugated quantum rods (QRs), which are novel nanoparticles with unique optical properties. We have evaluated the transversing ability of the QR-Tf-Saquinavir nanoformulation across an in vitro model of BBB. In addition, we have analyzed the subsequent antiviral efficacy of this targeted nanoformulation in HIV-1 infected peripheral blood mononuclear cells (PBMCs), which are cultured on the basolateral end of the in vitro BBB model. Our results show a significant uptake of QR-Tf-Saquinavir by brain microvascular endothelial cells (BMVECs), which constitute the BBB. In addition, we observed a significant enhancement in the transversing capability of QR-Tf-Saquinavir across the BBB, along with a marked decrease in HIV-1 viral replication in the PBMCs. These observations indicate that drug-loaded nanoparticles can deliver therapeutics across the BBB. These results highlight the potential of this nanoformulation in the treatment of Neuro-AIDS and other neurological disorders. PMID:20426757

bFGF Protects Against Blood-Brain Barrier Damage Through Junction Protein Regulation via PI3K-Akt-Rac1 Pathway Following Traumatic Brain Injury.

PubMed

Wang, Zhou-Guang; Cheng, Yi; Yu, Xi-Chong; Ye, Li-Bing; Xia, Qing-Hai; Johnson, Noah R; Wei, Xiaojie; Chen, Da-Qing; Cao, Guodong; Fu, Xiao-Bing; Li, Xiao-Kun; Zhang, Hong-Yu; Xiao, Jian

2016-12-01

Many traumatic brain injury (TBI) survivors sustain neurological disability and cognitive impairments due to the lack of defined therapies to reduce TBI-induced blood-brain barrier (BBB) breakdown. Exogenous basic fibroblast growth factor (bFGF) has been shown to have neuroprotective function in brain injury. The present study therefore investigates the beneficial effects of bFGF on the BBB after TBI and the underlying mechanisms. In this study, we demonstrate that bFGF reduces neurofunctional deficits and preserves BBB integrity in a mouse model of TBI. bFGF suppresses RhoA and upregulates tight junction proteins, thereby mitigating BBB breakdown. In vitro, bFGF exerts a protective effect on BBB by upregulating tight junction proteins claudin-5, occludin, zonula occludens-1, p120-catenin, and β-catenin under oxygen glucose deprivation/reoxygenation (OGD) in human brain microvascular endothelial cells (HBMECs). Both the in vivo and in vitro effects are related to the activation of the downstream signaling pathway, PI3K/Akt/Rac-1. Inhibition of the PI3K/Akt or Rac-1 by specific inhibitors LY294002 or si-Rac-1, respectively, partially reduces the protective effect of bFGF on BBB integrity. Overall, our results indicate that the protective role of bFGF on BBB involves the regulation of tight junction proteins and RhoA in the TBI model and OGD-induced HBMECs injury, and that activation of the PI3K/Akt /Rac-1 signaling pathway underlies these effects.

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

PubMed

Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette; Stensballe, Allan; Moos, Torben

2017-03-01

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] 2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789. © 2016 International Society for Neurochemistry.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

PubMed

Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

2017-04-11

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition.

PubMed

Wilson, Hannah K; Faubion, Madeline G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2016-12-01

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm 2 , equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm 2 out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.

Blood-brain barrier structure and function and the challenges for CNS drug delivery.

PubMed

Abbott, N Joan

2013-05-01

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the 'neurovascular unit' (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.

The transport of nifurtimox, an anti-trypanosomal drug, in an in vitro model of the human blood-brain barrier: evidence for involvement of breast cancer resistance protein.

PubMed

Watson, Christopher P; Dogruel, Murat; Mihoreanu, Larisa; Begley, David J; Weksler, Babette B; Couraud, Pierre O; Romero, Ignacio A; Thomas, Sarah A

2012-02-03

Human African trypanosomiasis (HAT) is a parasitic disease affecting sub-Saharan Africa. The parasites are able to traverse the blood-brain barrier (BBB), which marks stage 2 (S2) of the disease. Delivery of anti-parasitic drugs across the BBB is key to treating S2 effectively and the difficulty in achieving this goal is likely to be a reason why some drugs require highly intensive treatment regimes to be effective. This study aimed to investigate not only the drug transport mechanisms utilised by nifurtimox at the BBB, but also the impact of nifurtimox-eflornithine combination therapy (NECT) and other anti-HAT drug combination therapies (CTs) on radiolabelled-nifurtimox delivery in an in vitro model of drug accumulation and the human BBB, the hCMEC/D3 cell line. We found that nifurtimox appeared to use several membrane transporters, in particular breast-cancer resistance protein (BCRP), to exit the BBB cells. The addition of eflornithine caused no change in the accumulation of nifurtimox, nor did the addition of clinically relevant doses of the other anti-HAT drugs suramin, nifurtimox or melarsoprol, but a significant increase was observed with the addition of pentamidine. The results provide evidence that anti-HAT drugs are interacting with membrane transporters at the human BBB and suggest that combination with known transport inhibitors could potentially improve their efficacy. Copyright © 2011 Elsevier B.V. All rights reserved.

Additional increased effects of mannitol-temozolomide combined treatment on blood-brain barrier permeability.

PubMed

Choi, Chunggab; Kim, Hye Min; Shon, Jeeheun; Park, Jiae; Kim, Hyeong-Taek; Oh, Seung-Hun; Kim, Nam Keun; Kim, Ok Joon

2018-03-04

The blood-brain barrier (BBB) is major obstacle in drug or stem cell treatment in chronic stroke. We hypothesized that adding mannitol to temozolomide (TMZ) is a practically applicable method for resolving the low efficacy of intravenous mannitol therapy. In this study, we investigated whether BBB permeability could be increased by this combined treatment. First, we established a chronic ischemic stroke rat model and examined changes in leakage of Evans blue dye within a lesion site, and in expression of tight junction proteins (TJPs), by this combined treatment. Additionally, in an in vitro BBB model using trans-wells, we analyzed changes in diffusion of a fluorescent tracer and in expression of TJPs. Mannitol-TMZ combined treatment not only increased the amount of Evans blue dye within the stroke lesion site, but also reduced occludin expression in rat brain microvessels. The in vitro study also showed that combined treatment increased the permeability for two different-sized fluorescent tracers, especially large size, and decreased expression of TJPs, such as occludin and ZO-1. Increased BBB permeability effects were more prominent with combined than with single treatments. Mannitol-TMZ combined treatment induced a decrease of TJPs with a consequent increase in BBB permeability. This combined treatment is clinically useful and might provide new therapeutic options by enabling efficient intracerebral delivery of various drugs that could not otherwise be used to treat many CNS diseases due to their inability to penetrate the BBB. Copyright © 2018 Elsevier Inc. All rights reserved.

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier.

PubMed

Xie, Yi; Kim, Kee Jun; Kim, Kwang Sik

2004-11-01

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.

A method for evaluating nanoparticle transport through the blood-brain barrier in vitro.

PubMed

Guarnieri, Daniela; Muscetti, Ornella; Netti, Paolo A

2014-01-01

Blood-brain barrier (BBB) represents a formidable barrier for many therapeutic drugs to enter the brain tissue. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous system (CNS) diseases. In this context, nanoparticles are an emerging class of drug delivery systems that can be easily tailored to deliver drugs to various compartments of the body, including the brain. To identify, characterize, and validate novel nanoparticles applicable to brain delivery, in vitro BBB model systems have been developed. In this work, we describe a method to screen nanoparticles with variable size and surface functionalization in order to define the physicochemical characteristics underlying the design of nanoparticles that are able to efficiently cross the BBB.

Reliability of in vitro and in vivo methods for predicting P-glycoprotein effect on antidepressants delivery to the brain

PubMed Central

Zheng, Yi; Chen, Xijing; Benet, Leslie Z.

2017-01-01

As P-glycoprotein (P-gp) transport on antidepressant delivery has been extensively evaluated using in vitro cellular and in vivo rodent models, an increasing number of publications addressed the effect of P-gp in limiting brain penetration of antidepressants and causing treatment-resistant depression in current clinical therapies. However, contradictory results were observed in different systems. It is of vital importance to understand the potential for drug interactions related to P-gp at the blood-brain barrier (BBB), and whether co-administration of a P-gp inhibitor together with an antidepressant is a good clinical strategy for dosing of patients with treatment-resistant depression. In this review, the complicated construction of the BBB, the transport mechanisms for compounds that cross the BBB, and the basic characteristics of antidepressants are illustrated. Further, the reliability of different systems related to antidepressant brain delivery, including in vitro bidirectional transport cell lines, in vivo Mdr1 knock-out mice, and chemical inhibition studies in rodents are analyzed, supporting a low possibility that P-gp affects currently marketed antidepressants when these results are extrapolated to human BBB. These findings can also be applied to other central nervous system drugs. PMID:26293617

Nanoparticle accumulation and transcytosis in brain endothelial cell layers

NASA Astrophysics Data System (ADS)

Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A.

2013-10-01

The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin- or transferrin-conjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo-lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials.The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin- or transferrin-conjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo-lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials. Electronic supplementary information (ESI) available: Nanoparticle characterization in relevant media by Dynamic Light Scattering and SDS-PAGE. Transport study for silica nanoparticles across the BBB layer. Additional Electron Microscopy images of cells treated with the different nanoparticles investigated and details of the filters of the transwell systems. See DOI: 10.1039/c3nr02905k

Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

PubMed

Michalec, Katarzyna; Mysiorek, Caroline; Kuntz, Mélanie; Bérézowski, Vincent; Szczepankiewicz, Andrzej A; Wilczyński, Grzegorz M; Cecchelli, Roméo; Nałęcz, Katarzyna A

2014-07-15

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of uniaxial high-speed stretch as an in vitro model of mild traumatic brain injury on the blood-brain barrier.

PubMed

Rosas-Hernandez, Hector; Cuevas, Elvis; Escudero-Lourdes, Claudia; Lantz, Susan M; Sturdivant, Nasya M; Imam, Syed Z; Sarkar, Sumit; Slikker, William; Paule, Merle G; Balachandran, Kartik; Ali, Syed F

2018-04-13

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro. Published by Elsevier B.V.

Molecular determinants of blood-brain barrier permeation.

PubMed

Geldenhuys, Werner J; Mohammad, Afroz S; Adkins, Chris E; Lockman, Paul R

2015-01-01

The blood-brain barrier (BBB) is a microvascular unit which selectively regulates the permeability of drugs to the brain. With the rise in CNS drug targets and diseases, there is a need to be able to accurately predict a priori which compounds in a company database should be pursued for favorable properties. In this review, we will explore the different computational tools available today, as well as underpin these to the experimental methods used to determine BBB permeability. These include in vitro models and the in vivo models that yield the dataset we use to generate predictive models. Understanding of how these models were experimentally derived determines our accurate and predicted use for determining a balance between activity and BBB distribution.

Molecular determinants of blood–brain barrier permeation

PubMed Central

Geldenhuys, Werner J; Mohammad, Afroz S; Adkins, Chris E; Lockman, Paul R

2015-01-01

The blood–brain barrier (BBB) is a microvascular unit which selectively regulates the permeability of drugs to the brain. With the rise in CNS drug targets and diseases, there is a need to be able to accurately predict a priori which compounds in a company database should be pursued for favorable properties. In this review, we will explore the different computational tools available today, as well as underpin these to the experimental methods used to determine BBB permeability. These include in vitro models and the in vivo models that yield the dataset we use to generate predictive models. Understanding of how these models were experimentally derived determines our accurate and predicted use for determining a balance between activity and BBB distribution. PMID:26305616

Blood-brain barrier and its function during inflammation and autoimmunity.

PubMed

Sonar, Sandip Ashok; Lal, Girdhari

2018-05-01

The blood-brain barrier (BBB) is an important physiologic barrier that separates CNS from soluble inflammatory mediators and effector immune cells from peripheral circulation. The optimum function of the BBB is necessary for the homeostasis, maintenance, and proper neuronal function. The clinical and experimental findings have shown that BBB dysfunction is an early hallmark of various neurologic disorders ranging from inflammatory autoimmune, neurodegenerative, and traumatic diseases to neuroinvasive infections. Significant progress has been made in the understanding of the regulation of BBB function under homeostatic and neuroinflammatory conditions. Several neurologic disease-modifying drugs have shown to improve the BBB function. However, they have a broad-acting immunomodulatory function and can increase the risk of life-threatening infections. The recent development of in vitro multicomponent 3-dimensional BBB models coupled with fluidics chamber as well as a cell-type specific reporter and knockout mice gave a new boost to our understanding of the dynamics of the BBB. In the review, we discuss the current understanding of BBB composition and recent findings that illustrate the critical regulatory elements of the BBB function under physiologic and inflammatory conditions, and also suggested the strategies to control BBB structure and function. ©2018 Society for Leukocyte Biology.

Andrographolide-loaded nanoparticles for brain delivery: Formulation, characterisation and in vitro permeability using hCMEC/D3 cell line.

PubMed

Guccione, Clizia; Oufir, Mouhssin; Piazzini, Vieri; Eigenmann, Daniela Elisabeth; Jähne, Evelyn Andrea; Zabela, Volha; Faleschini, Maria Teresa; Bergonzi, Maria Camilla; Smiesko, Martin; Hamburger, Matthias; Bilia, Anna Rita

2017-10-01

Andrographolide (AG) is a major diterpenoid of the Asian medicinal plant Andrographis paniculata which has shown exciting pharmacological potential for the treatment of inflammation-related pathologies including neurodegenerative disorders. Conversely, the low bioavailability of AG still represents a limiting factor for its use. To overcome these limitations, AG was loaded into human serum albumin based nanoparticles (HSA NPs) and poly ethylcyanoacrylate nanoparticles (PECA NPs). HSA NPs were prepared by thermal (HSAT AG NPs) and chemical cross-linking (HSAC AG NPs), while PECA AG NPs were produced by emulsion-polymerization. NPs were characterized in terms of size, zeta (ζ)-potential, polydispersity, and release studies of AG. In addition, the ability of free AG and AG-loaded in PECA and HSAT NPs to cross the blood-brain barrier (BBB) was assessed using an in vitro BBB model based on human cerebral microvascular endothelial cell line (hCMEC/D3). For BBB drug permeability assays, a quantitative UPLC-MS/MS method for AG in Ringer HEPES buffer was developed and validated according to international regulatory guidelines for industry. Free AG did not permeate the BBB model, as also predicted by in silico studies. HSAT NPs improved by two-fold the permeation of AG while maintaining the integrity of the cell layer, while PECA NPs temporarily disrupted BBB integrity. Copyright © 2017 Elsevier B.V. All rights reserved.

Mixed oligomers and monomeric amyloid-β disrupts endothelial cells integrity and reduces monomeric amyloid-β transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model.

PubMed

Qosa, Hisham; LeVine, Harry; Keller, Jeffrey N; Kaddoumi, Amal

2014-09-01

Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-β (Aβ) assemblies. Formation of soluble Aβ assemblies is stimulated by monomeric Aβ accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aβ species. There are relatively few studies investigating toxic effects of Aβ on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aβ pool more closely resembling the in vivo situation composed of a mixture of Aβ40 monomer and Aβ42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aβ mixture significantly decreased monomeric Aβ transport across the cell culture model. Consistent with its effect on Aβ transport, Aβ mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aβ species separately failed to alter Aβ clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aβ species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

PubMed Central

2013-01-01

Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products. PMID:24262108

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

PubMed

Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

PubMed

Roe, Kelsey; Orillo, Beverly; Verma, Saguna

2014-01-01

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

Effects of Universal Mobile Telecommunications System (UMTS) electromagnetic fields on the blood-brain barrier in vitro.

PubMed

Franke, Helmut; Streckert, Joachim; Bitz, Andreas; Goeke, Johannes; Hansen, Volkert; Ringelstein, E Bernd; Nattkämper, Heiner; Galla, Hans-Joachim; Stögbauer, Florian

2005-09-01

The extensive use of mobile phone communication has raised public concerns about adverse health effects of radiofrequency (RF) electromagnetic fields (EMFs) in recent years. A central issue in this discussion is the question whether EMFs enhance the permeability of the blood-brain barrier (BBB). Here we report an investigation on the influence of a generic UMTS (Universal Mobile Telecommunications System) signal on barrier tightness, transport processes and the morphology of porcine brain microvascular endothelial cell cultures (PBEC) serving as an in vitro model of the BBB. An exposure device with integrated online monitoring system was developed for simultaneous exposure and measuring of transendothelial electrical resistance (TEER) to determine the tightness of the BBB. PBEC were exposed continuously for up to 84 h at an average electric-field strength of 3.4-34 V/m (maximum 1.8 W/kg) ensuring athermal conditions. We did not find any evidence of RF-field-induced disturbance of the function of the BBB. After and during exposure, the tightness of the BBB quantified by 14C-sucrose and serum albumin permeation as well as by TEER remained unchanged compared to sham-exposed cultures. Permeation of transporter substrates at the BBB as well as the localization and integrity of the tight-junction proteins occludin and ZO1 were not affected either.

High-Throughput Screening for Identification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity.

PubMed

Qosa, Hisham; Mohamed, Loqman A; Al Rihani, Sweilem B; Batarseh, Yazan S; Duong, Quoc-Viet; Keller, Jeffrey N; Kaddoumi, Amal

2016-07-06

The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins, and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer's disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76-4.56 μM. Of these 7 drugs, 5 were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aβ in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron, and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD.

High-throughput screening for identification of blood-brain barrier integrity enhancers: a drug repurposing opportunity to rectify vascular amyloid toxicity

PubMed Central

Qosa, Hisham; Mohamed, Loqman A.; Al Rihani, Sweilem B.; Batarseh, Yazan S.; Duong, Quoc-Viet; Keller, Jeffrey N.; Kaddoumi, Amal

2016-01-01

The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer’s disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76–4.56 μM. Of these 7 drugs, five were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aβ in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD. PMID:27392852

Effect of magnolol on cerebral injury and blood brain barrier dysfunction induced by ischemia-reperfusion in vivo and in vitro.

PubMed

Liu, Xiaoyan; Chen, Xiaoling; Zhu, Yuanjun; Wang, Kewei; Wang, Yinye

2017-08-01

Magnolol, a neolignan compound isolated from traditional Chinese medicine Magnolia officinalis, has a potentially therapeutic influence on ischemic stroke. Previous studies have demonstrated that cerebral ischemia-reperfusion (I-R) and blood-brain barrier (BBB) are involved in the pathogeneses of stroke. Therefore, in vivo and in vitro studies were designed to investigate the effects of magnolol on I-R-induced neural injury and BBB dysfunction. In cerebral I-R model of mice, cerebral infarct volumes, brain water content, and the exudation of Evans blue were significantly reduced by intravenous injection with magnolol at the doses of 1.4, 7.0, and 35.0 μg/kg. When primary cultured microglial cells were treated with 1 μg/ml lipopolysaccharide (LPS) plus increasing concentrations of magnolol, ranging from 0.01 to 10 μmol/L, magnolol could statistically inhibit LPS-induced NO release, TNF-α secretion, and expression of p65 subunit of NF-κB in the nucleus of microglial cells. In the media of brain microvascular endothelial cells (BMECs), oxygen and glucose deprivation-reperfusion (OGD-R) could remarkably lead to the elevation of TNF-α and IL-1β levels, while magnolol evidently reversed these effects. In BBB model in vitro, magnolol dose- and time-dependently declined BBB hyperpermeability induced by oxygen and glucose deprivation (OGD), OGD-R, and ephrin-A1 treatment. More importantly, magnolol could obviously inhibit phosphorylation of EphA2 (p-EphA2) not only in ephrin-A1-treated BMECs but also in cerebral I-R model of mice. In contrast to p-EphA2, magnolol significantly increased ZO-1 and occludin levels in BMECs subjected to OGD. Taken together, magnolol can protect neural damage from cerebral ischemia- and OGD-reperfusion, which may be associated with suppressing cerebral inflammation and improving BBB function.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier.

PubMed

Salmeri, Mario; Motta, Carla; Anfuso, Carmelina D; Amodeo, Andrea; Scalia, Marina; Toscano, Maria A; Alberghina, Mario; Lupo, Gabriella

2013-08-01

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention. © 2013 John Wiley & Sons Ltd.

Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity.

PubMed

Palus, Martin; Vancova, Marie; Sirmarova, Jana; Elsterova, Jana; Perner, Jan; Ruzek, Daniel

2017-07-01

Alteration of the blood-brain barrier (BBB) is a hallmark of tick-borne encephalitis (TBE), a life-threating human viral neuroinfection. However, the mechanism of BBB breakdown during TBE, as well as TBE virus (TBEV) entry into the brain is unclear. Here, primary human microvascular endothelial cells (HBMECs) were infected with TBEV to study interactions with the BBB. Although the number of infected cells was relatively low in culture (<5%), the infection was persistent with high TBEV yields (>10 6 pfu/ml). Infection did not induce any significant changes in the expression of key tight junction proteins or upregulate the expression of cell adhesion molecules, and did not alter the highly organized intercellular junctions between HBMECs. In an in vitro BBB model, the virus crossed the BBB via a transcellular pathway without compromising the integrity of the cell monolayer. The results indicate that HBMECs may support TBEV entry into the brain without altering BBB integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats II: Potential mechanisms.

PubMed

van Vliet, Erwin A; Otte, Willem M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

2016-01-01

Blood-brain barrier (BBB) leakage may play a pro-epileptogenic role after status epilepticus. In the accompanying contrast-enhanced magnetic resonance imaging (CE-MRI) study we showed that the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduced BBB leakage and seizure activity during the chronic epileptic phase. Given rapamycin's role in growth and immune response, the potential therapeutic effects of rapamycin after status epilepticus with emphasis on brain inflammation and brain vasculature were investigated. Seven weeks after kainic acid-induced status epilepticus, rats were perfusion fixed and (immuno)histochemistry was performed using several glial and vascular markers. In addition, an in vitro model for the human BBB was used to determine the effects of rapamycin on transendothelial electrical resistance as a measure for BBB integrity. (Immuno)histochemistry showed that local blood vessel density, activated microglia, and astrogliosis were reduced in rapamycin-treated rats compared to vehicle-treated rats. In vitro studies showed that rapamycin could attenuate TNFα-induced endothelial barrier breakdown. These data suggest that rapamycin improves BBB function during the chronic epileptic phase by a reduction of local brain inflammation and blood vessel density that can contribute to a milder form of epilepsy. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

[Lactobacillus rhamnosus GG conditioned medium prevents E. coli meningitis by inhibiting nuclear factor-κB pathway].

PubMed

Zeng, Qing; He, Xiao-Long; Xiao, Han-Sheng; DU, Lei; Li, Yu-Jing; Chen, Le-Cheng; Tian, Hui-Wen; Huang, Sheng-He; Cao, Hong

2017-01-20

To investigate whether Lactobacillus rhamnosus GG conditioned medium(LGG-CM)has preventive effect against E. coli K1-induced neuropathogenicity in vitro by inhibiting nuclear factor-κB (NF-κB) signaling pathway. An in vitro blood-brain barrier (BBB) model was constructed using human brain microvascular endothelial cells (HBMECs). The effect of LGG-CM on E. coli-actived NF-κB signaling pathway was assayed using Western blotting. Invasion assay and polymorphonuclear leukocyte (PMN) transmigration assay were performed to explore whether LGG-CM could inhibit E. coli invasion and PMN transmigration across the BBB in vitro. The expressions of ZO-1 and CD44 were detected using Western blotting and immunofluorescence. The changes of trans-epithelial electric resistance (TEER) and bacterial translocation were determined to evaluate the BBB permeability. Pre-treament with LGG-CM inhibited E. coli-activated NF-κB signaling pathway in HBMECs and decreased the invasion of E. coli K1 and transmigration of PMN. Western blotting showed that LGG-CM could alleviate E. coli-induced up-regulation of CD44 and down-regulation of ZO-1 expressions in HBMECs. In addition, pre-treatment with LGG-CM alleviated E. coli K1-induced reduction of TEER and suppressed bacterial translocation across the BBB in vitro. LGG-CM can block E. coli-induced activation of NF-κB signaling pathway and thereby prevents E. coli K1-induced neuropathogenicity by decreasing E. coli K1 invasion rates and PMN transmigration.

Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model.

PubMed

Patabendige, Adjanie; Michael, Benedict D; Craig, Alister G; Solomon, Tom

2018-06-01

Japanese encephalitis virus (JEV) remains a leading cause of encephalitis, globally, which continues to grow in importance despite the availability of vaccines. Viral entry into the brain can occur via the blood-brain barrier (BBB), and inflammation at the BBB is a common final pathway in many brain infections. However, the role of the BBB during JEV infection and the contribution of the endothelial and astrocytic cell inflammation in facilitating virus entry into the brain are incompletely understood. We established a BBB model using human brain endothelial cells (HBECs) and human astrocytes. HBECs are polarised, and therefore the model was inoculated by JEV from the apical side to simulate the in vivo situation. The effects of JEV on the BBB permeability and release of inflammatory mediators from both apical and basolateral sides, representing the blood and the brain side respectively were investigated. JEV infected HBECs with limited active virus production, before crossing the BBB and infecting astrocytes. Control of JEV production by HBECs was associated with a significant increase in permeability, and with elevation of many host mediators, including cytokines, chemokines, cellular adhesion molecules, and matrix metalloproteases. When compared to the controls, significantly higher amounts of mediators were released from the apical side as opposed to the basolateral side. The increased release of mediators over time also correlated with increased BBB permeability. Treatment with dexamethasone led to a significant reduction in the release of interleukin 6 (IL6), C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10) from the apical side with a reduction in BBB disruption and no change in JEV production. The results are consistent with the hypothesis that JEV infection of the BBB triggers the production of a range of host mediators from both endothelial cells and astrocytes, which control JEV production but disrupt BBB integrity thus allowing virus entry into the brain. Dexamethasone treatment controlled the host response and limited BBB disruption in the model without increasing JEV production, supporting a re-investigation of its use therapeutically. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Alternating magnetic field-induced hyperthermia increases iron oxide nanoparticle cell association/uptake and flux in blood-brain barrier models.

PubMed

Dan, Mo; Bae, Younsoo; Pittman, Thomas A; Yokel, Robert A

2015-05-01

Superparamagnetic iron oxide nanoparticles (IONPs) are being investigated for brain cancer therapy because alternating magnetic field (AMF) activates them to produce hyperthermia. For central nervous system applications, brain entry of diagnostic and therapeutic agents is usually essential. We hypothesized that AMF-induced hyperthermia significantly increases IONP blood-brain barrier (BBB) association/uptake and flux. Cross-linked nanoassemblies loaded with IONPs (CNA-IONPs) and conventional citrate-coated IONPs (citrate-IONPs) were synthesized and characterized in house. CNA-IONP and citrate-IONP BBB cell association/uptake and flux were studied using two BBB Transwell(®) models (bEnd.3 and MDCKII cells) after conventional and AMF-induced hyperthermia exposure. AMF-induced hyperthermia for 0.5 h did not alter CNA-IONP size but accelerated citrate-IONP agglomeration. AMF-induced hyperthermia for 0.5 h enhanced CNA-IONP and citrate-IONP BBB cell association/uptake. It also enhanced the flux of CNA-IONPs across the two in vitro BBB models compared to conventional hyperthermia and normothermia, in the absence of cell death. Citrate-IONP flux was not observed under these conditions. AMF-induced hyperthermia also significantly enhanced paracellular pathway flux. The mechanism appears to involve more than the increased temperature surrounding the CNA-IONPs. Hyperthermia induced by AMF activation of CNA-IONPs has potential to increase the BBB permeability of therapeutics for the diagnosis and therapy of various brain diseases.

Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

PubMed

Al-Ahmad, Abraham J

2017-10-01

Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells. Copyright © 2017 the American Physiological Society.

Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo.

PubMed

Kafa, Houmam; Wang, Julie Tzu-Wen; Rubio, Noelia; Klippstein, Rebecca; Costa, Pedro M; Hassan, Hatem A F M; Sosabowski, Jane K; Bansal, Sukhvinder S; Preston, Jane E; Abbott, N Joan; Al-Jamal, Khuloud T

2016-03-10

Brain glioblastoma and neurodegenerative diseases are still largely untreated due to the inability of most drugs to cross the blood-brain barrier (BBB). Nanoparticles have emerged as promising tools for drug delivery applications to the brain; in particular carbon nanotubes (CNTs) that have shown an intrinsic ability to cross the BBB in vitro and in vivo. Angiopep-2 (ANG), a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), has also shown promising results as a targeting ligand for brain delivery using nanoparticles (NPs). Here, we investigate the ability of ANG-targeted chemically-functionalised multi-walled carbon nanotubes (f-MWNTs) to cross the BBB in vitro and in vivo. ANG was conjugated to wide and thin f-MWNTs creating w-MWNT-ANG and t-MWNT-ANG, respectively. All f-MWNTs were radiolabelled to facilitate quantitative analyses by γ-scintigraphy. ANG conjugation to f-MWNTs enhanced BBB transport of w- and t-MWNTs-ANG compared to their non-targeted equivalents using an in vitro co-cultured BBB model consisting of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Additionally, following intravenous administration w-MWNTs-ANG showed significantly higher whole brain uptake than the non-targeted w-MWNT in vivo reaching ~2% injected dose per g of brain (%ID/g) within the first hour post-injection. Furthermore, using a syngeneic glioma model, w-MWNT-ANG showed enhanced uptake in glioma brain compared to normal brain at 24h post-injection. t-MWNTs-ANG, on the other hand, showed higher brain accumulation than w-MWNTs. However, no significant differences were observed between t-MWNT and t-MWNT-ANG indicating the importance of f-MWNTs diameter towards their brain accumulation. The inherent brain accumulation ability of f-MWNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-MWNT-ANG to deliver active therapeutics for brain glioma therapy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation.

PubMed

Rom, Slava; Zuluaga-Ramirez, Viviana; Reichenbach, Nancy L; Erickson, Michelle A; Winfield, Malika; Gajghate, Sachin; Christofidou-Solomidou, Melpo; Jordan-Sciutto, Kelly L; Persidsky, Yuri

2018-01-27

Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet's post hoc tests. We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1β in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation.

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

PubMed Central

Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

2018-01-01

The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

PubMed

Molino, Yves; David, Marion; Varini, Karine; Jabès, Françoise; Gaudin, Nicolas; Fortoul, Aude; Bakloul, Karima; Masse, Maxime; Bernard, Anne; Drobecq, Lucile; Lécorché, Pascaline; Temsamani, Jamal; Jacquot, Guillaume; Khrestchatisky, Michel

2017-05-01

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. © FASEB.

Analytical and Biological Methods for Probing the Blood-Brain Barrier

PubMed Central

Sloan, Courtney D. Kuhnline; Nandi, Pradyot; Linz, Thomas H.; Aldrich, Jane V.; Audus, Kenneth L.; Lunte, Susan M.

2013-01-01

The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB. PMID:22708905

Pregnane X Receptor and P-glycoprotein: a connexion for Alzheimer's disease management.

PubMed

Jain, Sumit; Rathod, Vijay; Prajapati, Rameshwar; Nandekar, Prajwal P; Sangamwar, Abhay T

2014-11-01

The translational failure between preclinical animal models and clinical outcome has alarmed us to search for a new strategy in the treatment of Alzheimer's disease (AD). Interlink between Pregnane X Receptor (PXR) and P-glycoprotein (Pgp) at the blood brain barrier (BBB) has raised hope toward a new disease modifying therapy in AD. Pgp is a major efflux transporter for beta amyloid (Aβ) at human BBB. A literature survey reveals diminished expression of Pgp transporter at the BBB in AD patients. Pregnane X Receptor is a major transcriptional regulator of Pgp. Restoration of Pgp at the BBB enhances the elimination of the Aβ from brain alongside and inhibits the apical to basolateral movement of Aβ from the circulatory blood. This review concentrates on in vitro, in vivo, and in silico advancements on the study of the PXR in context to Pgp and discusses the substrate and inhibitor specificity between PXR and Pgp.

A 3D Real-Scale, Biomimetic, and Biohybrid Model of the Blood-Brain Barrier Fabricated through Two-Photon Lithography.

PubMed

Marino, Attilio; Tricinci, Omar; Battaglini, Matteo; Filippeschi, Carlo; Mattoli, Virgilio; Sinibaldi, Edoardo; Ciofani, Gianni

2018-02-01

The investigation of the crossing of exogenous substances through the blood-brain barrier (BBB) is object of intensive research in biomedicine, and one of the main obstacles for reliable in vitro evaluations is represented by the difficulties at the base of developing realistic models of the barrier, which could resemble as most accurately as possible the in vivo environment. Here, for the first time, a 1:1 scale, biomimetic, and biohybrid BBB model is proposed. Microtubes inspired to the brain capillaries were fabricated through two-photon lithography and used as scaffolds for the co-culturing of endothelial-like bEnd.3 and U87 glioblastoma cells. The constructs show the maturation of tight junctions, good performances in terms of hindering dextran diffusion through the barrier, and a satisfactory trans-endothelial electrical resistance. Moreover, a mathematical model is developed, which assists in both the design of the 3D microfluidic chip and its characterization. Overall, these results show the effective formation of a bioinspired cellular barrier based on microtubes reproducing brain microcapillaries to scale. This system will be exploited as a realistic in vitro model for the investigation of BBB crossing of nanomaterials and drugs, envisaging therapeutic and diagnostic applications for several brain pathologies, including brain cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A dual-targeting nanocarrier based on poly(amidoamine) dendrimers conjugated with transferrin and tamoxifen for treating brain gliomas.

PubMed

Li, Yan; He, Hai; Jia, Xinru; Lu, Wan-Liang; Lou, Jinning; Wei, Yen

2012-05-01

A pH-sensitive dual-targeting drug carrier (G4-DOX-PEG-Tf-TAM) was synthesized with transferrin (Tf) conjugated on the exterior and Tamoxifen (TAM) in the interior of the fourth generation PAMAM dendrimers for enhancing the blood-brain barrier (BBB) transportation and improving the drug accumulation in the glioma cells. It was found that, on average, 7 doxorubicine (DOX) molecules, over 30 PEG(1000) and PEG(2000) chains and one Tf group were bonded on the periphery of each G4 PAMAM dendrimer, while 29 TAM molecules were encapsulated into the interior of per dendrimer. The pH-triggered DOX release was 32% at pH 4.5 and 6% at pH 7.4, indicating a comparatively fast drug release at weak acidic condition and stable state of the carrier at physiological environment. The in vitro assay of the drug transport across the BBB model showed that G4-DOX-PEG-Tf-TAM exhibited higher BBB transportation ability with the transporting ratio of 6.06% in 3 h. The carrier was internalized into C6 glioma cells upon crossing the BBB model by the coactions of TfR-mediated endocytosis and the inhibition effect of TAM to the drug efflux transports. Moreover, it also displayed the in vitro accumulation of DOX in the avascular C6 glioma spheroids made the tumor volume effectively reduced. Copyright © 2012 Elsevier Ltd. All rights reserved.

NIH workshop report on the trans-agency blood-brain interface workshop 2016: exploring key challenges and opportunities associated with the blood, brain and their interface.

PubMed

Ochocinska, Margaret J; Zlokovic, Berislav V; Searson, Peter C; Crowder, A Tamara; Kraig, Richard P; Ljubimova, Julia Y; Mainprize, Todd G; Banks, William A; Warren, Ronald Q; Kindzelski, Andrei; Timmer, William; Liu, Christina H

2017-05-01

A trans-agency workshop on the blood-brain interface (BBI), sponsored by the National Heart, Lung and Blood Institute, the National Cancer Institute and the Combat Casualty Care Research Program at the Department of Defense, was conducted in Bethesda MD on June 7-8, 2016. The workshop was structured into four sessions: (1) blood sciences; (2) exosome therapeutics; (3) next generation in vitro blood-brain barrier (BBB) models; and (4) BBB delivery and targeting. The first day of the workshop focused on the physiology of the blood and neuro-vascular unit, blood or biofluid-based molecular markers, extracellular vesicles associated with brain injury, and how these entities can be employed to better evaluate injury states and/or deliver therapeutics. The second day of the workshop focused on technical advances in in vitro models, BBB manipulations and nanoparticle-based drug carrier designs, with the goal of improving drug delivery to the central nervous system. The presentations and discussions underscored the role of the BBI in brain injury, as well as the role of the BBB as both a limiting factor and a potential conduit for drug delivery to the brain. At the conclusion of the meeting, the participants discussed challenges and opportunities confronting BBI translational researchers. In particular, the participants recommended using BBI translational research to stimulate advances in diagnostics, as well as targeted delivery approaches for detection and therapy of both brain injury and disease.

Brain vascular heterogeneity: implications for disease pathogenesis and design of in vitro blood-brain barrier models.

PubMed

Noumbissi, Midrelle E; Galasso, Bianca; Stins, Monique F

2018-04-23

The vertebrate blood-brain barrier (BBB) is composed of cerebral microvascular endothelial cells (CEC). The BBB acts as a semi-permeable cellular interface that tightly regulates bidirectional molecular transport between blood and the brain parenchyma in order to maintain cerebral homeostasis. The CEC phenotype is regulated by a variety of factors, including cells in its immediate environment and within functional neurovascular units. The cellular composition of the brain parenchyma surrounding the CEC varies between different brain regions; this difference is clearly visible in grey versus white matter. In this review, we discuss evidence for the existence of brain vascular heterogeneity, focusing on differences between the vessels of the grey and white matter. The region-specific differences in the vasculature of the brain are reflective of specific functions of those particular brain areas. This BBB-endothelial heterogeneity may have implications for the course of pathogenesis of cerebrovascular diseases and neurological disorders involving vascular activation and dysfunction. This heterogeneity should be taken into account when developing BBB-neuro-disease models representative of specific brain areas.

Regulatory mechanisms for iron transport across the blood-brain barrier.

PubMed

Duck, Kari A; Simpson, Ian A; Connor, James R

2017-12-09

Many critical metabolic functions in the brain require adequate and timely delivery of iron. However, most studies when considering brain iron uptake have ignored the iron requirements of the endothelial cells that form the blood-brain barrier (BBB). Moreover, current models of BBB iron transport do not address regional regulation of brain iron uptake or how neurons, when adapting to metabolic demands, can acquire more iron. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and the iron chelator, deferoxamine, stimulate release of iron from iron-loaded endothelial cells in an in vitro BBB model. The role of the endosomal divalent metal transporter 1 (DMT1) in BBB iron acquisition and transport has been questioned. Here, we show that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells. These data support an endosome-mediated model of Tf-bound iron uptake into the brain and identifies mechanisms for local regional regulation of brain iron uptake. Moreover, our data provide an explanation for the disparity in the ratio of Tf to iron transport into the brain that has confounded the field. Copyright © 2017 Elsevier Inc. All rights reserved.

The blood-brain barrier as a target in traumatic brain injury treatment.

PubMed

Thal, Serge C; Neuhaus, Winfried

2014-11-01

Traumatic brain injury (TBI) is one of the most frequent causes of death in the young population. Several clinical trials have unsuccessfully focused on direct neuroprotective therapies. Recently immunotherapeutic strategies shifted into focus of translational research in acute CNS diseases. Cross-talk between activated microglia and blood-brain barrier (BBB) could initiate opening of the BBB and subsequent recruitment of systemic immune cells and mediators into the brain. Stabilization of the BBB after TBI could be a promising strategy to limit neuronal inflammation, secondary brain damage and acute neurodegeneration. This review provides an overview on the pathophysiology of TBI and brain edema formation including definitions and classification of TBI, current clinical treatment strategies, as well as current understanding on the underlying cellular processes. A summary of in vivo and in vitro models to study different aspects of TBI is presented. Three mechanisms proposed for stabilization of the BBB, myosin light chain kinases, glucocorticoid receptors and peroxisome proliferator-activated receptors are reviewed for their influence on barrier-integrity and outcome after TBI. In conclusion, the BBB is recommended as a promising target for the treatment of traumatic brain injury, and it is suggested that a combination of BBB stabilization and neuroprotectants may improve therapeutic success. Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.

A choline derivate-modified nanoprobe for glioma diagnosis using MRI

NASA Astrophysics Data System (ADS)

Li, Jianfeng; Huang, Shixian; Shao, Kun; Liu, Yang; An, Sai; Kuang, Yuyang; Guo, Yubo; Ma, Haojun; Wang, Xuxia; Jiang, Chen

2013-04-01

Gadolinium (Gd) chelate contrast-enhanced magnetic resonance imaging (MRI) is a preferred method of glioma detection and preoperative localisation because it offers high spatial resolution and non-invasive deep tissue penetration. Gd-based contrast agents, such as Gd-diethyltriaminepentaacetic acid (DTPA-Gd, Magnevist), are widely used clinically for tumor diagnosis. However, the Gd-based MRI approach is limited for patients with glioma who have an uncompromised blood-brain barrier (BBB). Moreover, the rapid renal clearance and non-specificity of such contrast agents further hinders their prevalence. We present a choline derivate (CD)-modified nanoprobe with BBB permeability, glioma specificity and a long blood half-life. Specific accumulation of the nanoprobe in gliomas and subsequent MRI contrast enhancement are demonstrated in vitro in U87 MG cells and in vivo in a xenograft nude model. BBB and glioma dual targeting by this nanoprobe may facilitate precise detection of gliomas with an uncompromised BBB and may offer better preoperative and intraoperative tumor localization.

Regulation of Human Brain Microvascular Endothelial Cell Adhesion and Barrier Functions by Memantine.

PubMed

Wang, Fei; Zou, Zhirong; Gong, Yi; Yuan, Dong; Chen, Xun; Sun, Tao

2017-05-01

Vascular risk factors have been linked to cognitive decline and dementia in the elderly. Microvascular inflammation, especially of the endothelium, may contribute to the progression of neurodegenerative events in Alzheimer's disease (AD). Memantine, an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is a licensed drug used for the treatment of moderate to severe AD. However, little information is available regarding its anti-inflammatory effects on the endothelium. In this study, we investigated the effects of memantine on human brain microvascular endothelial dysfunction induced by the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Our results show that memantine prevents the attachment of monocyte THP-1 cells to human brain microvascular endothelial cells (HBMVEs). An in vitro BBB model experiment displayed that memantine could rescue TNF-α-induced disruption of the in vitro BBB model. In addition, memantine also interferes with monocyte transmigration across the BBB model. Our results indicate that TNF-α significantly increased the expression of cell adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin, which was prevented by pretreatment with memantine. Mechanistically, memantine reversed activation of the transcription factor NF-κB by preventing the phosphorylation and degradation of its inhibitor IκBα. Our data is the first to describe a novel anti-inflammatory mechanism driven by the endothelial cell-mediated neuroprotective effects of memantine.

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier

PubMed Central

Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted. PMID:29059256

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.

PubMed

Yang, Shu; Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.

Effects of electromagnetic pulse exposure on gelatinase of blood-brain barrier in vitro.

PubMed

Zhou, Yan; Qiu, Lian-Bo; An, Guang-Zhou; Zhou, Jia-Xing; Du, Le; Ma, Ya-Hong; Guo, Guo-Zhen; Ding, Gui-Rong

2017-01-01

The biological effects of electromagnetic pulse (EMP) on the brain have been focused on for years. It was reported that gelatinase played an important role in maintaining brain function through regulating permeability in the blood-brain barrier (BBB). To investigate the effects of EMP on gelatinase of BBB, an in vitro BBB model was established using primary cultured rat brain microvascular endothelial cells (BMVEC), astrocytes and half-contact culture of these cells in a transwell chamber. Cultured supernatant and cells were collected at different time points after exposure to EMP (peak intensity 400 kV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps and 200 pulses). Protein levels of cellular gelatinase MMP-2 and MMP-9, and endogenous inhibitor TIMP-1 and TIMP-2 were detected by Western blot. The activity of gelatinase in culture supernatant was detected by gelatin zymography. It was found that compared with the sham-exposed group, the protein level of MMP-2 was significantly increased at 6 h (p < 0.05), and the protein level of its endogenous inhibitor TIMP-2 did not change after EMP exposure. In addition, the protein levels of MMP-9 and its endogenous inhibitor TIMP-1 did not change after EMP exposure. Gelatin zymography results showed that the activity of MMP-2 in the inner pool and the outer pool of the transwell chamber was significantly increased at 6 h after EMP exposure compared with that of the sham group. These results suggested that EMP exposure could affect the expression and activity of MMP-2 in the BBB model.

miR-98 and let-7g* protect the blood–brain barrier under neuroinflammatory conditions

PubMed Central

Rom, Slava; Dykstra, Holly; Zuluaga-Ramirez, Viviana; Reichenbach, Nancy L; Persidsky, Yuri

2015-01-01

Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood–brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier ‘leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation. PMID:26126865

miR-98 and let-7g* protect the blood-brain barrier under neuroinflammatory conditions.

PubMed

Rom, Slava; Dykstra, Holly; Zuluaga-Ramirez, Viviana; Reichenbach, Nancy L; Persidsky, Yuri

2015-12-01

Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.

Blood brain barrier permeability of (-)-epigallocatechin gallate, its proliferation-enhancing activity of human neuroblastoma SH-SY5Y cells, and its preventive effect on age-related cognitive dysfunction in mice.

PubMed

Pervin, Monira; Unno, Keiko; Nakagawa, Aimi; Takahashi, Yuu; Iguchi, Kazuaki; Yamamoto, Hiroyuki; Hoshino, Minoru; Hara, Aya; Takagaki, Akiko; Nanjo, Fumio; Minami, Akira; Imai, Shinjiro; Nakamura, Yoriyuki

2017-03-01

The consumption of green tea catechins (GTCs) suppresses age-related cognitive dysfunction in mice. GTCs are composed of several catechins, of which epigallocatechin gallate (EGCG) is the most abundant, followed by epigallocatechin (EGC). Orally ingested EGCG is hydrolyzed by intestinal biota to EGC and gallic acid (GA). To understand the mechanism of action of GTCs on the brain, their permeability of the blood brain barrier (BBB) as well as their effects on cognitive function in mice and on nerve cell proliferation in vitro were examined. The BBB permeability of EGCG, EGC and GA was examined using a BBB model kit. SAMP10, a mouse model of brain senescence, was used to test cognitive function in vivo . Human neuroblastoma SH-SY5Y cells were used to test nerve cell proliferation and differentiation. The in vitro BBB permeability (%, in 30 min) of EGCG, EGC and GA was 2.8±0.1, 3.4±0.3 and 6.5±0.6, respectively. The permeability of EGCG into the BBB indicates that EGCG reached the brain parenchyma even at a very low concentration. The learning ability of SAMP10 mice that ingested EGCG (20 mg/kg) was significantly higher than of mice that ingested EGC or GA. However, combined ingestion of EGC and GA showed a significant improvement comparable to EGCG. SH-SY5Y cell growth was significantly enhanced by 0.05 µM EGCG, but this effect was reduced at higher concentrations. The effect of EGC and GA was lower than that of EGCG at 0.05 µM. Co-administration of EGC and GA increased neurite length more than EGC or GA alone. Cognitive dysfunction in mice is suppressed after ingesting GTCs when a low concentration of EGCG is incorporated into the brain parenchyma via the BBB. Nerve cell proliferation/differentiation was enhanced by a low concentration of EGCG. Furthermore, the additive effect of EGC and GA suggests that EGCG sustains a preventive effect after the hydrolysis to EGC and GA.

Amphiphilic Copolymers Shuttle Drugs Across the Blood-Brain Barrier.

PubMed

Clemens-Hemmelmann, Mirjam; Kuffner, Christiane; Metz, Verena; Kircher, Linda; Schmitt, Ulrich; Hiemke, Christoph; Postina, Rolf; Zentel, Rudolf

2016-05-01

Medical treatment of diseases of the central nervous system requires transport of drugs across the blood-brain barrier (BBB). Here, it is extended previously in vitro experiments with a model compound to show that the non-water-soluble and brain-impermeable drug domperidone (DOM) itself can be enriched in the brain by use of an amphiphilic copolymer as a carrier. This carrier consists of poly(N-(2-hydroxypropyl)-methacrylamide), statistically copolymerized with 10 mol% hydrophobic lauryl methacrylate, into whose micellar aggregates DOM is noncovalently absorbed. As tested in a BBB model efficient transport of DOM across, the BBB is achievable over a wide range of formulations, containing 0.8 to 35.5 wt% domperidone per copolymer. In neither case, the polymer itself is translocated across the BBB model. In vivo experiments in mice show that already 10 min after intraperitoneal injection of the polymer/domperidone (PolyDOM) formulation, domperidone can be detected in blood and in the brain. Highest serum and brain levels of domperidone are detected 40 min after injection. At that time point serum domperidone is increased 48-fold. Most importantly, domperidone is exclusively detectable in high amounts in the brain of PolyDOM injected mice and not in mice injected with bare domperidone. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo

PubMed Central

Choi, James J.; Wang, Shougang; Tung, Yao-Sheng; Morrison, Barclay; Konofagou, Elisa E.

2009-01-01

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525 MHz, peak-rarefactional pressure in situ: 569 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared to 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa, into the brain parenchyma. This noninvasive and localized BBB opening technique could thus provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro, but whose in vivo translation has been hampered by their associated BBB impermeability. PMID:19900750

Effect of surface charge of immortalized mouse cerebral endothelial cell monolayer on transport of charged solutes.

PubMed

Yuan, Wei; Li, Guanglei; Gil, Eun Seok; Lowe, Tao Lu; Fu, Bingmei M

2010-04-01

Charge carried by the surface glycocalyx layer (SGL) of the cerebral endothelium has been shown to significantly modulate the permeability of the blood-brain barrier (BBB) to charged solutes in vivo. The cultured monolayer of bEnd3, an immortalized mouse cerebral endothelial cell line, is becoming a popular in vitro BBB model due to its easy growth and maintenance of many BBB characteristics over repeated passages. To test whether the SGL of bEnd3 monolayer carries similar charge as that in the intact BBB and quantify this charge, which can be characterized by the SGL thickness (L(f)) and charge density (C(mf)), we measured the solute permeability of bEnd3 monolayer to neutral solutes and to solutes with similar size but opposite charges: negatively charged alpha-lactalbumin (-11) and positively charged ribonuclease (+3). Combining the measured permeability data with a transport model across the cell monolayer, we predicted the L(f) and the C(mf) of bEnd3 monolayer, which is approximately 160 nm and approximately 25 mEq/L, respectively. We also investigated whether orosomucoid, a plasma glycoprotein modulating the charge of the intact BBB, alters the charge of bEnd3 monolayer. We found that 1 mg/mL orosomucoid would increase SGL charge density of bEnd3 monolayer to approximately 2-fold of its control value.

JAM-A and ALCAM are therapeutic targets to inhibit diapedesis across the BBB of CD14+CD16+ monocytes in HIV-infected individuals.

PubMed

Williams, Dionna W; Anastos, Kathryn; Morgello, Susan; Berman, Joan W

2015-02-01

Monocyte transmigration across the BBB is a critical step in the development of cognitive deficits termed HAND that affect 40-70% of HIV-infected individuals, even with successful antiretroviral therapy. The monocyte subsets that enter the CNS during HIV infection are not fully characterized. We examined PBMC from HIV-positive individuals from 2 distinct cohorts and enumerated monocyte populations, characterized their transmigration properties across an in vitro human BBB model, and identified surface proteins critical for the entry of these cells into the CNS. We demonstrated that the frequency of peripheral blood CD14(+)CD16(+) and CD14(low)CD16(+) monocytes was increased in HIV-seropositive compared with -seronegative individuals, despite virologic control. We showed that CD14(+)CD16(+) monocytes selectively transmigrated across our BBB model as a result of their increased JAM-A and ALCAM expression. Antibody blocking of these proteins inhibited diapedesis of CD14(+)CD16(+) monocytes but not of T cells from the same HIV-infected people across the BBB. Our data indicate that JAM-A and ALCAM are therapeutic targets to decrease the entry of CD14(+)CD16(+) monocytes into the CNS of HIV-seropositive individuals, contributing to the eradication of neuroinflammation, HAND, and CNS viral reservoirs. © Society for Leukocyte Biology.

Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models.

PubMed

Jähne, Evelyn A; Eigenmann, Daniela E; Culot, Maxime; Cecchelli, Roméo; Walter, Fruzsina R; Deli, Mária A; Tremmel, Robin; Fricker, Gert; Smiesko, Martin; Hamburger, Matthias; Oufir, Mouhssin

2014-09-01

The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent. Copyright © 2014 Elsevier B.V. All rights reserved.

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.

PubMed

Banks, William A; Gray, Alicia M; Erickson, Michelle A; Salameh, Therese S; Damodarasamy, Mamatha; Sheibani, Nader; Meabon, James S; Wing, Emily E; Morofuji, Yoichi; Cook, David G; Reed, May J

2015-11-25

Disruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain. We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using (14)C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to (14)C-sucrose and radioactive albumin. In comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with (14)C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and (14)C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes. The BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

Transport of a GABAA receptor modulator and its derivatives from Valeriana officinalis L. s. l. across an in vitro cell culture model of the blood-brain barrier.

PubMed

Neuhaus, Winfried; Trauner, Gabriele; Gruber, Daniela; Oelzant, Silvester; Klepal, Waltraud; Kopp, Brigitte; Noe, Christian R

2008-09-01

The roots and rhizome of Valeriana officinalis L . s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA (A) receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA (A) modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 +/- 0.29 microm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 +/- 0.63 microm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 +/- 0.58 microm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. S. L. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.

Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage.

PubMed

Liu, Jie; Jin, Xinchun; Liu, Ke J; Liu, Wenlan

2012-02-29

Blood-brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here, we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran and promoted the secretion of metalloproteinase-2 and -9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT (2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane) or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity, and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2 h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.

Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage

PubMed Central

Liu, Jie; Jin, Xinchun; Liu, Ke J.; Liu, Wenlan

2012-01-01

Blood brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran, and promoted the secretion of metalloproteinase-2 and 9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2-h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis. PMID:22378877

Blood-brain barrier transport and neuroprotective potential of blackberry-digested polyphenols: an in vitro study.

PubMed

Figueira, Inês; Tavares, Lucélia; Jardim, Carolina; Costa, Inês; Terrasso, Ana P; Almeida, Andreia F; Govers, Coen; Mes, Jurriaan J; Gardner, Rui; Becker, Jörg D; McDougall, Gordon J; Stewart, Derek; Filipe, Augusto; Kim, Kwang S; Brites, Dora; Brito, Catarina; Brito, M Alexandra; Santos, Cláudia N

2017-11-18

Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood-brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC-MS n . BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.

Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality.

PubMed

Patel, Ronak; Page, Shyanne; Al-Ahmad, Abraham Jacob

2017-07-01

The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes, and neurons. Recently, isogenic in vitro models of the BBB based on human pluripotent stem cells have been documented, yet the impact of inter-individual variability on the yield and phenotype of such models remains to be documented. In this study, we investigated the impact of inter-individual variability on the yield and phenotype of isogenic models of the BBB, using patient-derived induced pluripotent stem cells (iPSCs). Astrocytes, BMECs, and neurons were differentiated from four asymptomatic patient-derived iPSCs (two males, two females). We differentiated such cells using existing differentiation protocols and quantified expression of cell lineage markers, as well as BBB phenotype, barrier induction, and formation of neurite processes. iPSC-derived BMECs showed barrier properties better than hCMEC/D3 monolayers; however, we noted differences in the expression and activity among iPSC lines. In addition, we noted differences in the differentiation efficiency of these cells into neural stem cells and progenitor cells (as noted by differences in expression of cell lineage markers). Such differences were reflected later in the terminal differentiation, as seen as ability to induce barrier function and to form neurite processes. Although we demonstrated our ability to obtain an isogenic model of the BBB with different patients' iPSCs, we also noted subtle differences in the expression of cell lineage markers and cell maturation processes, suggesting the presence of inter-individual polymorphisms. © 2017 International Society for Neurochemistry.

Evaluation of blood-brain barrier-stealth nanocomposites for in situ glioblastoma theranostics applications

NASA Astrophysics Data System (ADS)

Su, Chia-Hao; Tsai, Ching-Yi; Tomanek, Boguslaw; Chen, Wei-Yu; Cheng, Fong-Yu

2016-04-01

The blood-brain barrier (BBB) is a physiological structure of the blood vessels in the brain. The BBB efficiently traps most therapeutic drugs in the blood vessels and stops them from entering the brain tissue, resulting in a decreased therapeutic efficiency. In this study, we developed BBB-stealth nanocomposites composed of iron oxide (Fe3O4) nanoparticles (NPs) as a safe nanocarrier for glioblastoma therapy. We showed the antitumor activity of Dox/alg-Fe3O4 NPs using in vitro and in vivo tests. We demonstrated that G23-alg-Fe3O4 NPs crossed the BBB and entered the brain. In situ glioblastoma tumor-bearing mice were used to successfully evaluate the antitumor activity of G23-Dox/alg-Fe3O4 NPs. Magnetic resonance imaging (MRI) and bioluminescence imaging (BLI) confirmed the BBB crossing. The BBB-stealth nanocomposites show great potential for a proof-of-concept clinical trial as a theranostics platform for human brain tumor therapy.The blood-brain barrier (BBB) is a physiological structure of the blood vessels in the brain. The BBB efficiently traps most therapeutic drugs in the blood vessels and stops them from entering the brain tissue, resulting in a decreased therapeutic efficiency. In this study, we developed BBB-stealth nanocomposites composed of iron oxide (Fe3O4) nanoparticles (NPs) as a safe nanocarrier for glioblastoma therapy. We showed the antitumor activity of Dox/alg-Fe3O4 NPs using in vitro and in vivo tests. We demonstrated that G23-alg-Fe3O4 NPs crossed the BBB and entered the brain. In situ glioblastoma tumor-bearing mice were used to successfully evaluate the antitumor activity of G23-Dox/alg-Fe3O4 NPs. Magnetic resonance imaging (MRI) and bioluminescence imaging (BLI) confirmed the BBB crossing. The BBB-stealth nanocomposites show great potential for a proof-of-concept clinical trial as a theranostics platform for human brain tumor therapy. Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c6nr00280c

Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.

PubMed

Hawkins, Brian T; Grego, Sonia; Sellgren, Katelyn L

2015-05-22

Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit. Copyright © 2015 Elsevier B.V. All rights reserved.

Phospholipid Transfer Protein Is Expressed in Cerebrovascular Endothelial Cells and Involved in High Density Lipoprotein Biogenesis and Remodeling at the Blood-Brain Barrier*

PubMed Central

Chirackal Manavalan, Anil Paul; Kober, Alexandra; Metso, Jari; Lang, Ingrid; Becker, Tatjana; Hasslitzer, Karin; Zandl, Martina; Fanaee-Danesh, Elham; Pippal, Jyotsna Brijesh; Sachdev, Vinay; Kratky, Dagmar; Stefulj, Jasminka; Jauhiainen, Matti; Panzenboeck, Ute

2014-01-01

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral (“brain parenchymal”) compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-β-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB. PMID:24369175

Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.

PubMed

Ohtsuki, Sumio; Hirayama, Mio; Ito, Shingo; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2014-06-01

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.

Passage of Magnetic Tat-Conjugated Fe3O4@SiO2 Nanoparticles Across In Vitro Blood-Brain Barrier

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Shang, Ting; Zhang, Xiaodan; Ye, Ting; Wang, Dajin; Rei, Lei

2016-10-01

Delivery of diagnostic or therapeutic agents across the blood-brain barrier (BBB) remains a major challenge of brain disease treatment. Magnetic nanoparticles are actively being developed as drug carriers due to magnetic targeting and subsequently reduced off-target effects. In this paper, we developed a magnetic SiO2@Fe3O4 nanoparticle-based carrier bound to cell-penetrating peptide Tat (SiO2@Fe3O4 -Tat) and studied its fates in accessing BBB. SiO2@Fe3O4-Tat nanoparticles (NPs) exhibited suitable magnetism and good biocompatibility. NPs adding to the apical chamber of in vitro BBB model were found in the U251 glioma cells co-cultured at the bottom of the Transwell, indicating that particles passed through the barrier and taken up by glioma cells. Moreover, the synergistic effects of Tat and magnetic field could promote the efficient cellular internalization and the permeability across the barrier. Besides, functionalization with Tat peptide allowed particles to locate into the nucleus of U251 cells than the non-conjugated NPs. These results suggest that SiO2@Fe3O4-Tat NPs could penetrate the BBB through the transcytosis of brain endothelial cells and magnetically mediated dragging. Therefore, SiO2@Fe3O4-Tat NPs could be exploited as a potential drug delivery system for chemotherapy and gene therapy of brain disease.

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

PubMed Central

Jiang, Xiaoyan; Zhang, Lili; Pu, Hongjian; Hu, Xiaoming; Zhang, Wenting; Cai, Wei; Gao, Yanqin; Leak, Rehana K.; Keep, Richard F.; Bennett, Michael V. L.; Chen, Jun

2017-01-01

The damage borne by the endothelial cells (ECs) forming the blood–brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. PMID:28137866

Amphiphilic HPMA-LMA copolymers increase the transport of Rhodamine 123 across a BBB model without harming its barrier integrity.

PubMed

Hemmelmann, Mirjam; Metz, Verena V; Koynov, Kaloian; Blank, Kerstin; Postina, Rolf; Zentel, Rudolf

2012-10-28

The successful non-invasive treatment of diseases associated with the central nervous system (CNS) is generally limited by poor brain permeability of various developed drugs. The blood-brain barrier (BBB) prevents the passage of therapeutics to their site of action. Polymeric drug delivery systems are promising solutions to effectively transport drugs into the brain. We recently showed that amphiphilic random copolymers based on the hydrophilic p(N-(2-hydroxypropyl)-methacrylamide), pHPMA, possessing randomly distributed hydrophobic p(laurylmethacrylate), pLMA, are able to mediate delivery of domperidone into the brain of mice in vivo. To gain further insight into structure-property relations, a library of carefully designed polymers based on p(HPMA) and p(LMA) was synthesized and tested applying an in vitro BBB model which consisted of human brain microvascular endothelial cells (HBMEC). Our model drug Rhodamine 123 (Rh123) exhibits, like domperidone, a low brain permeability since both substances are recognized by efflux transporters at the BBB. Transport studies investigating the impact of the polymer architecture in relation to the content of hydrophobic LMA revealed that random p(HPMA)-co-p(LMA) having 10mol% LMA is the most auspicious system. The copolymer significantly increased the permeability of Rh123 across the HBMEC monolayer whereas transcytosis of the polymer was very low. Further investigations on the mechanism of transport showed that integrity and barrier function of the BBB model were not harmed by the polymer. According to our results, p(HPMA)-co-p(LMA) copolymers are a promising delivery system for neurological therapeutics and their application might open alternative treatment strategies. Copyright © 2012 Elsevier B.V. All rights reserved.

Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood-brain barrier penetration capabilities.

PubMed

Lu, Shousi; Guo, Shanshan; Xu, Pingxiang; Li, Xiaorong; Zhao, Yuming; Gu, Wei; Xue, Ming

Nitrogen-doped carbon dots (N-CDs) were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood-brain barrier (BBB) in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain.

Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood–brain barrier penetration capabilities

PubMed Central

Lu, Shousi; Guo, Shanshan; Xu, Pingxiang; Li, Xiaorong; Zhao, Yuming; Gu, Wei; Xue, Ming

2016-01-01

Nitrogen-doped carbon dots (N-CDs) were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood–brain barrier (BBB) in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain. PMID:27932880

Glioma targeting and blood-brain barrier penetration by dual-targeting doxorubincin liposomes.

PubMed

Gao, Jian-Qing; Lv, Qing; Li, Li-Ming; Tang, Xin-Jiang; Li, Fan-Zhu; Hu, Yu-Lan; Han, Min

2013-07-01

Effective chemotherapy for glioblastoma requires a carrier that can penetrate the blood-brain barrier (BBB) and subsequently target the glioma cells. Dual-targeting doxorubincin (Dox) liposomes were produced by conjugating liposomes with both folate (F) and transferrin (Tf), which were proven effective in penetrating the BBB and targeting tumors, respectively. The liposome was characterized by particle size, Dox entrapment efficiency, and in vitro release profile. Drug accumulation in cells, P-glycoprotein (P-gp) expression, and drug transport across the BBB in the dual-targeting liposome group were examined by using bEnd3 BBB models. In vivo studies demonstrated that the dual-targeting Dox liposomes could transport across the BBB and mainly distribute in the brain glioma. The anti-tumor effect of the dual-targeting liposome was also demonstrated by the increased survival time, decreased tumor volume, and results of both hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis. The dual-targeting Dox liposome could improve the therapeutic efficacy of brain glioma and were less toxic than the Dox solution, showing a dual-targeting effect. These results indicate that this dual-targeting liposome can be used as a potential carrier for glioma chemotherapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen

PubMed Central

Santiago-Tirado, Felipe H.; Onken, Michael D.; Cooper, John A.; Klein, Robyn S.

2017-01-01

ABSTRACT The blood-brain barrier (BBB) protects the central nervous system (CNS) by restricting the passage of molecules and microorganisms. Despite this barrier, however, the fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that is estimated to kill over 600,000 people annually. Cryptococcal infection begins in the lung, and experimental evidence suggests that host phagocytes play a role in subsequent dissemination, although this role remains ill defined. Additionally, the disparate experimental approaches that have been used to probe various potential routes of BBB transit make it impossible to assess their relative contributions, confounding any integrated understanding of cryptococcal brain entry. Here we used an in vitro model BBB to show that a “Trojan horse” mechanism contributes significantly to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. We also, for the first time, directly imaged C. neoformans-containing phagocytes crossing the BBB, showing that they do so via transendothelial pores. Finally, we found that Trojan horse crossing enables CNS entry of fungal mutants that cannot otherwise traverse the BBB, and we demonstrate additional intercellular interactions that may contribute to brain entry. Our work elucidates the mechanism of cryptococcal brain invasion and offers approaches to study other neuropathogens. PMID:28143979

Brain Inositol Is a Novel Stimulator for Promoting Cryptococcus Penetration of the Blood-Brain Barrier

PubMed Central

Wang, Yina; Toffaletti, Dena L.; Eugenin, Eliseo; Perfect, John R.; Kim, Kee Jun; Xue, Chaoyang

2013-01-01

Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection. PMID:23592982

Nanotoxicity of poly(n-butylcyano-acrylate) nanoparticles at the blood-brain barrier, in human whole blood and in vivo.

PubMed

Kolter, Marise; Ott, Melanie; Hauer, Christian; Reimold, Isolde; Fricker, Gert

2015-01-10

Therapy of diseases of the central nervous system is a major challenge since drugs have to overcome the blood-brain barrier (BBB). A powerful strategy to enhance cerebral drug concentration is administration of drug-loaded poly(n-butylcyano-acrylate) (PBCA) nanoparticles coated with polysorbate 80 (PS80). This study evaluates the toxicity of PBCA-nanoparticles at the BBB, representing the target organ, the inflammatory response in human whole blood, as the site of administration and in a rat model in vivo. PBCA-nanoparticles were prepared by a mini-emulsion method and characterized concerning size, surface charge, shape and PS80-adsorption. The influence on metabolic activity, cell viability and integrity of the BBB was analyzed in an in vitro model of the BBB. In ex vivo experiments in human whole blood the release of 12 inflammatory cytokines was investigated. In addition, the inflammatory response was studied in vivo in rats and complemented with the analysis of different organ toxicity parameters. PBCA-nanoparticles showed time- and concentration-dependent effects on metabolic activity, cell viability and BBB integrity. No cell death or loss of metabolic activity was observed for nanoparticle-concentrations ≤500μg/ml up to 3h of treatment. Within 12 tested inflammatory cytokines, only interleukin-8 displayed a significant release after nanoparticle exposure in human blood. No severe inflammatory processes or organ damages were identified in rats in vivo. Thus, PBCA-nanoparticles are a promising drug delivery system to overcome the BBB since they showed hardly any cytotoxic or inflammatory effect at therapeutic concentrations and incubation times. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro developmental neurotoxicity (DNT) testing: relevant models and endpoints.

PubMed

Bal-Price, Anna K; Hogberg, Helena T; Buzanska, Leonora; Lenas, Petros; van Vliet, Erwin; Hartung, Thomas

2010-09-01

Environmental chemicals have a potential impact on children's health as the developing brain is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that very complex processes of cell development and maturation take place within a tightly controlled time frame. So different stages of brain development are susceptible to toxic effects at different time points. Additionally the adult brain is well protected against chemicals by the blood brain barrier (BBB) whereas the placenta only partially protects against harmful chemical exposure. Many metals easily cross the placenta and BBB barrier since even after the birth BBB is not entirely differentiated (until about 6 months after birth). Additionally, the susceptibility of infants and children is due to increased exposure, augmented absorption rates, and less efficient ability of defense mechanism in comparison to adults. The In Vitro Session during the 12th International Neurotoxicology Association meeting (Jerusalem, June, 2009) provided the opportunity to discuss the new challenges that have to be faced to create new type of safety assessments for regulatory requirements. The integration of various tests into testing strategies as well as combination of information-rich approaches with bioinformatics was discussed. Furthermore relevant models and endpoints for developmental neurotoxicity (DNT) evaluation using in vitro approach were presented. The primary neuronal cultures of cerebellar granule cells (CGCs) as well as 3D aggregate model and the possible application of human embryonic and adult stem cells was discussed pointing out the potential of these models to be used for DNT testing. The presented systems are relevant for DNT evaluation as the key processes of brain development such cell proliferation, migration and neuronal/glial differentiation are present. Furthermore, emerging technologies such as gene expression, electrical activity measurements and metabonomics have been identified as promising tools. In a combination with other assays the in vitro approach could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by initial prioritization of chemicals with DNT potential for further testing. Copyright © 2009 Elsevier Inc. All rights reserved.

Pharmacokinetics, brain distribution, release and blood-brain barrier transport of Shunaoxin pills.

PubMed

Wu, Kai; Wang, Zhan-Zhang; Liu, Dan; Qi, Xian-Rong

2014-02-12

Shunaoxin pills, a traditional Chinese medicine (TCM) product, have been used to treat cerebrovascular diseases in China since 2005. The main active components of Shunaoxin pills are ferulic acid and ligustilide from Chuanxiong (Ligusticum chuanxiong Hort, Umbelliferae) and Danggui (Angelica sinensis radix, Umbelliferae). As Shunaoxin shows excellent activity in the central nervous system (CNS), the extent to which the major constituents of Shunaoxin reach the CNS should be investigated. Moreover, the in vivo-in vitro correlations (IVIVC) of the formulation should be studied to elucidate the mechanisms of action of TCM in the CNS. However, these data have not previously been available. Thus we intended to investigate what the extent when these constituents of Shunaoxin pills reach the CNS, and evaluate the IVIVC of release and pharmacokinetics. In this study, we evaluated the release of ferulic acid and ligustilide from Shunaoxin pills, and their transport across an in vitro model of the BBB. We also evaluated their pharmacokinetics and brain distribution in vivo. High-performance liquid chromatography (HPLC) was used to quantify both compounds simultaneously. Based on the release in vitro and absorption of ferulic acid and ligustilide in vivo, IVIVC permitted prediction of the pharmacokinetics of these compounds. The release of ferulic acid and ligustilide reached a platform phase within 1h. Ferulic acid and ligustilide rapidly crossed the BBB in different patterns; the transport ratio increased over time. After intragastric (i.g.) administration of Shunaoxin pills, ferulic acid and ligustilide were rapidly absorbed and distributed into brain, which may result in a rapid onset of action. Ferulic acid and ligustilide were transported across a model BBB. After i.g. administration of Shunaoxin pills, ferulic acid and ligustilide were rapidly absorbed and distributed in brain; this may lead to rapid pharmacological onset. The IVIVC can be used to predict in vivo pharmacokinetics from in vitro experimental results. These results provide support for the clinical use of Shunaoxin pills. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Wnt/β-catenin signaling controls development of the blood–brain barrier

PubMed Central

Liebner, Stefan; Corada, Monica; Bangsow, Thorsten; Babbage, Jane; Taddei, Andrea; Czupalla, Cathrin J.; Reis, Marco; Felici, Angelina; Wolburg, Hartwig; Fruttiger, Marcus; Taketo, Makoto M.; von Melchner, Harald; Plate, Karl Heinz; Gerhardt, Holger; Dejana, Elisabetta

2008-01-01

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown. PMID:18955553

Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core.

PubMed

Clark, Andrew J; Davis, Mark E

2015-10-06

Most therapeutic agents are excluded from entering the central nervous system by the blood-brain barrier (BBB). Receptor mediated transcytosis (RMT) is a common mechanism used by proteins, including transferrin (Tf), to traverse the BBB. Here, we prepared Tf-containing, 80-nm gold nanoparticles with an acid-cleavable linkage between the Tf and the nanoparticle core to facilitate nanoparticle RMT across the BBB. These nanoparticles are designed to bind to Tf receptors (TfRs) with high avidity on the blood side of the BBB, but separate from their multidentate Tf-TfR interactions upon acidification during the transcytosis process to allow release of the nanoparticle into the brain. These targeted nanoparticles show increased ability to cross an in vitro model of the BBB and, most important, enter the brain parenchyma of mice in greater amounts in vivo after systemic administration compared with similar high-avidity nanoparticles containing noncleavable Tf. In addition, we investigated this design with nanoparticles containing high-affinity antibodies (Abs) to TfR. With the Abs, the addition of the acid-cleavable linkage provided no improvement to in vivo brain uptake for Ab-containing nanoparticles, and overall brain uptake was decreased for all Ab-containing nanoparticles compared with Tf-containing ones. These results are consistent with recent reports of high-affinity anti-TfR Abs trafficking to the lysosome within BBB endothelium. In contrast, high-avidity, Tf-containing nanoparticles with the acid-cleavable linkage avoid major endothelium retention by shedding surface Tf during their transcytosis.

Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers.

PubMed

Coyne, Carolyn B; Bozym, Rebecca; Morosky, Stefanie A; Hanna, Sheri L; Mukherjee, Amitava; Tudor, Matthew; Kim, Kwang Sik; Cherry, Sara

2011-01-20

Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an in vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly antienteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses. Copyright © 2011 Elsevier Inc. All rights reserved.

Nootropic nanocomplex with enhanced blood-brain barrier permeability for treatment of traumatic brain injury-associated neurodegeneration.

PubMed

Park, Jeongmin; Choi, Eunshil; Shin, Seulgi; Lim, Sungsu; Kim, Dohee; Baek, Suji; Lee, Kang Pa; Lee, Jae Jun; Lee, Byeong Han; Kim, Bokyung; Jeong, Keunsoo; Baik, Ja-Hyun; Kim, Yun Kyung; Kim, Sehoon

2018-06-15

Traumatic brain injury (TBI) is an intracranial injury which can induce immediate neuroinflammation and long-term neurological deficits. Methylene blue (MB) as a nootropic has a great potential to treat neurodegeneration after TBI because of its anti-inflmmatory and neuroprotective functions. However, its limited accumulation to the brain across the blood-brain barrier (BBB) remains a major hurdle to be overcome. In this paper, we present a polymer surfactant-encapsulated nanocomplex of MB as a delivery system with high BBB permeability for efficacious treatment of TBI-induced neurodegeneration. MB was formulated via electrostatically/hydrophobically directed assembly with fatty acid and Pluronic surfactant (F-127 or F-68) to construct nanocomplexes of two different colloidal sizes (<10 nm and ~108 nm in hydrodynamic diameter for NanoMB-127 and NanoMB-68, respectively). Compared to uncomplexed free MB, formulation into the ultrasmall nanocomplex (NanoMB-127) significantly enhanced the uptake of MB by blood-brain vascular endothelial bEnd3 cells in vitro, and indeed improved its BBB penetration upon systemic administration to normal mice in vivo. However, large-size NanoMB-68 showed negligible BBB crossing despite the efficient bEnd3 cell internalization in vitro, probably due to the unfavorable pharmacokinetic profile associated with its large particle size. By virtue of the efficient BBB penetration and cellular uptake, ultrasmall NanoMB-127 was shown to distinctively reduce the expression level of an inflammatory cytokine with no notable toxicity in vitro and also considerably prevent the neurodegeneration after TBI in mice at much lower doses than free MB. Overall, the Pluronic-supported nanocomplexation method allows efficient brain delivery of MB, offering a novel way of enhancing the efficacy of neurotherapeutics to treat brain diseases. Copyright © 2018. Published by Elsevier B.V.

Intestinal and Blood-Brain Barrier Permeability of Ginkgolides and Bilobalide: In Vitro and In Vivo Approaches

USDA-ARS?s Scientific Manuscript database

In this study intestinal and blood brain barrier (BBB) transport of ginkgolides A, B, C, J and bilobalide, isolated from Ginkgo biloba (Family-Ginkgoaceae), was evaluated in Caco-2 and MDR1-MDCK cell monolayer models. Transepithelial transport was examined for 2 hours in both absorptive and secretor...

Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier

PubMed Central

Wang, Xiangru; Maruvada, Ravi; Morris, Andrew J.; Liu, Jun O.; Baek, Dong Jae; Kim, Kwang Sik

2016-01-01

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood–brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics. PMID:27711202

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.

PubMed

Nielsen, Simone S E; Siupka, Piotr; Georgian, Ana; Preston, Jane E; Tóth, Andrea E; Yusof, Siti R; Abbott, N Joan; Nielsen, Morten S

2017-09-24

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm 2 , and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10 -6 ± 0.13 10 -6 cm sec -1 (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Influence of borneol and muscone on geniposide transport through MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model.

PubMed

Chen, Zhen-Zhen; Lu, Yang; Du, Shou-Ying; Shang, Ke-Xin; Cai, Cheng-Bo

2013-11-01

The objective of this study was (1) to characterize geniposide transport through MDCK and MDCK-MDR1 cell lines to confirm its transport mechanism and (2) to evaluate the effect of borneol and muscone as enhancers of geniposide transport in the BBB models so as to explore the enhancement mechanism. Transport studies of geniposide were performed in both directions, from apical to basolateral and from basolateral to apical sides. Drug concentrations were analyzed by HPLC. Geniposide showed relatively poor absorption in MDCK and MDCK-MDR1 cells, apparent permeability coefficients ranging from 0.323×10(-6) to 0.422×10(-6) cm/s. The in vitro experiments showed that geniposide transport in both directions was not concentration dependent and saturable, indicating purely passive diffusion. The efflux ratio of geniposide was less than 2 in the two cell models, which suggested that geniposide was not P-gp substrates. Geniposide transport in both directions significantly increased when co-administrated with increasing concentrations of borneol and muscone. Actin staining results indicated that borneol and muscone increased geniposide transport in the BBB models may attribute to disassembly effect on tight junction integrity. Copyright © 2013 Elsevier B.V. All rights reserved.

Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells.

PubMed

Saint-Pol, Julien; Candela, Pietra; Boucau, Marie-Christine; Fenart, Laurence; Gosselet, Fabien

2013-06-23

It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry. Copyright © 2013 Elsevier B.V. All rights reserved.

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

PubMed

Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

2015-01-01

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

A radical scavenger edaravone inhibits matrix metalloproteinase-9 upregulation and blood-brain barrier breakdown in a mouse model of prolonged cerebral hypoperfusion

PubMed Central

Maki, Takakuni; Liang, Anna C.; Arai, Ken

2014-01-01

Matrix metalloproteinase-9 (MMP-9) plays key roles in the brain pathophysiology, especially in blood-brain barrier (BBB) breakdown. Therefore, inhibiting MMP-9 activity may be a promising therapy for protecting brains in cerebrovascular diseases. Here we show that in a mouse prolonged cerebral hypoperfusion model, a clinically proven radical scavenger edaravone suppressed MMP-9 and reduced BBB damage in cerebral white matter. Prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in male adult C57BL/6J mice (10 weeks old). After 7 days of cerebral hypoperfusion, white matter region (e.g. corpus callosum) exhibited significant BBB leakage, assessed by IgG staining. Correspondingly, immunostaining and western blotting showed that MMP-9 was upregulated in the white matter. Edaravone treatment (3 mg/kg, i.p. at day 0 and 3) inhibited both BBB leakage and MMP-9 increase. Under the early phase of cerebral hypoperfusion conditions, oligodendrocyte precursor cells (OPCs) mainly contribute to the MMP-9 increase, but our immunostaining data showed that very little OPCs expressed MMP-9 in the edaravone-treated animals at day 7. Therefore, in vitro studies with primary rat OPCs were conducted to examine whether edaravone would directly suppressed MMP-9 expressions in OPCs. OPC cultures were exposed to sub-lethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Prolonged chemical hypoxic stress increased MMP-9 expression in OPCs, and radical scavenging with edaravone (10 μM for 7 days) ameliorated the increase. Taken together, our proof-of-concept study demonstrates that radical scavengers may provide a potential therapeutic approach for white matter injury by suppressing BBB damage. PMID:24820542

A radical scavenger edaravone inhibits matrix metalloproteinase-9 upregulation and blood-brain barrier breakdown in a mouse model of prolonged cerebral hypoperfusion.

PubMed

Miyamoto, Nobukazu; Pham, Loc-Duyen D; Maki, Takakuni; Liang, Anna C; Arai, Ken

2014-06-24

Matrix metalloproteinase-9 (MMP-9) plays key roles in the brain pathophysiology, especially in blood-brain barrier (BBB) breakdown. Therefore, inhibiting MMP-9 activity may be a promising therapy for protecting brains in cerebrovascular diseases. Here we show that in a mouse prolonged cerebral hypoperfusion model, a clinically proven radical scavenger edaravone suppressed MMP-9 and reduced BBB damage in cerebral white matter. Prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in male adult C57BL/6J mice (10 weeks old). After 7 days of cerebral hypoperfusion, white matter region (e.g. corpus callosum) exhibited significant BBB leakage, assessed by IgG staining. Correspondingly, immunostaining and western blotting showed that MMP-9 was upregulated in the white matter. Edaravone treatment (3mg/kg, i.p. at days 0 and 3) inhibited both BBB leakage and MMP-9 increase. Under the early phase of cerebral hypoperfusion conditions, oligodendrocyte precursor cells (OPCs) mainly contribute to the MMP-9 increase, but our immunostaining data showed that very little OPCs expressed MMP-9 in the edaravone-treated animals at day 7. Therefore, in vitro studies with primary rat OPCs were conducted to examine whether edaravone would directly suppressed MMP-9 expressions in OPCs. OPC cultures were exposed to sub-lethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Prolonged chemical hypoxic stress increased MMP-9 expression in OPCs, and radical scavenging with edaravone (10μM for 7 days) ameliorated the increase. Taken together, our proof-of-concept study demonstrates that radical scavengers may provide a potential therapeutic approach for white matter injury by suppressing BBB damage. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Memantine transport by a proton-coupled organic cation antiporter in hCMEC/D3 cells, an in vitro human blood-brain barrier model.

PubMed

Higuchi, Kei; Kitamura, Atsushi; Okura, Takashi; Deguchi, Yoshiharu

2015-04-01

Memantine is clinically used for the treatment of patients with Alzheimer's disease and is highly distributed to the brain. The aim of this study is to characterize memantine transport at the blood-brain barrier (BBB) using hCMEC/D3 cells, a human BBB model. The initial uptake velocity of memantine in hCMEC/D3 cells was concentration-dependent, and was reduced by metabolic inhibitors, but was independent of extracellular sodium ion and membrane potential. Intracellular alkalization and intracellular acidification markedly reduced and enhanced the uptake, respectively. The uptake was strongly inhibited by quinidine, pyrilamine and verapamil, and was moderately inhibited by TEA (substrate of OCTs and OCTNs) and l-carnitine (substrate of OCTN2), but was not inhibited by MPP(+) (substrate of OCTs and PMAT) or ergothioneine (substrate of OCTN1). Although relatively abundant expression of OCTN2 gene has been observed in hCMEC/D3 cells, knockdown of OCTN2 with siRNA did not decrease memantine uptake. Memantine and diphenhydramine each showed inhibition of the other's uptake in a competitive manner. Thus, proton-coupled organic cation antiporter(s) appears to be involved in the transport of memantine in hCMEC/D3 cells, at least in part. Our results indicate that the in vivo BBB permeability of memantine in humans can be predicted from the in vitro uptake clearance in hCMEC/D3 cells. Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection.

PubMed

Li, Fang; Wang, Yueyun; Yu, Lan; Cao, Shengbo; Wang, Ke; Yuan, Jiaolong; Wang, Chong; Wang, Kunlun; Cui, Min; Fu, Zhen F

2015-05-01

Japanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage. In vitro studies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB. Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infection per se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-γ) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN-γ could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Oleocanthal Enhances Amyloid-β Clearance from the Brains of TgSwDI Mice and in Vitro across a Human Blood-Brain Barrier Model

PubMed Central

2015-01-01

Numerous clinical and preclinical studies have suggested several health promoting effects for the dietary consumption of extra-virgin olive oil (EVOO) that could protect and decrease the risk of developing Alzheimer’s disease (AD). Moreover, recent studies have linked this protective effect to oleocanthal, a phenolic secoiridoid component of EVOO. This protective effect of oleocanthal against AD has been related to its ability to prevent amyloid-β (Aβ) and tau aggregation in vitro, and enhance Aβ clearance from the brains of wild type mice in vivo; however, its effect in a mouse model of AD is not known. In the current study, we investigated the effect of oleocanthal on pathological hallmarks of AD in TgSwDI, an animal model of AD. Mice treatment for 4 weeks with oleocanthal significantly decreased amyloid load in the hippocampal parenchyma and microvessels. This reduction was associated with enhanced cerebral clearance of Aβ across the blood-brain barrier (BBB). Further mechanistic studies demonstrated oleocanthal to increase the expression of important amyloid clearance proteins at the BBB including P-glycoprotein and LRP1, and to activate the ApoE-dependent amyloid clearance pathway in the mice brains. The anti-inflammatory effect of oleocanthal in the brains of these mice was also obvious where it was able to reduce astrocytes activation and IL-1β levels. Finally, we could recapitulate the observed protective effect of oleocanthal in an in vitro human-based model, which could argue against species difference in response to oleocanthal. In conclusion, findings from in vivo and in vitro studies provide further support for the protective effect of oleocanthal against the progression of AD. PMID:26348065

The use of TMZ embedded hydrogels for the treatment of orthotopic human glioma xenografts.

PubMed

Adhikari, Bandita; Li, Jie; Brandel, Michael G; Futalan, Diahnn; Akers, Johnny; Deming, Timothy; Chen, Clark C; Carter, Bob S

2017-11-01

The current treatment of glioblastoma multiforme (GBM) is limited by the restricted arsenal of agents which effectively cross the blood brain barrier (BBB). For example, only a fraction of temozolomide (TMZ) administered systemically is available for therapeutic effect because of the BBB and the instability of TMZ under physiologic conditions. A novel approach to overcome this obstacle is to bypass the BBB and locally deliver chemotherapeutic agents directly to the tumor mass. We have explored the loading of TMZ into a novel hydrogel matrix, which can be delivered in liquid form and then solidifies in situ and releases chemotherapy as the matrix dissolves. Here, we tested the effect of amphiphilic diblock copolypeptide hydrogels (DCHs) of 180-poly-lysine and 20-poly-leucine (K 180 L 20 ) on TMZ using Glioblastoma models. In both the in vitro model, which involved treatment of a human glioblastoma GSC line suspended as neurospheres, and in vivo using an orthotopic glioma xenograft mouse model, we found that K 180 L 20 could safely enhance the efficacy of TMZ. This technique may offer the opportunity to 'coat' the inner lining of the cavity following glioma resection with a slow-release TMZ and potentially decrease recurrence. Future studies in larger animals are needed to delineate this effect. Copyright © 2017. Published by Elsevier Ltd.

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro

PubMed Central

Üllen, Andreas; Fauler, Günter; Bernhart, Eva; Nusshold, Christoph; Reicher, Helga; Leis, Hans-Jörg; Malle, Ernst; Sattler, Wolfgang

2012-01-01

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase–hydrogen peroxide–chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood–brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting. PMID:22982051

Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease

PubMed Central

Nikolskaia, Olga V.; de A. Lima, Ana Paula C.; Kim, Yuri V.; Lonsdale-Eccles, John D.; Fukuma, Toshihide; Scharfstein, Julio; Grab, Dennis J.

2006-01-01

In this study we investigated why bloodstream forms of Trypanosoma brucei gambiense cross human brain microvascular endothelial cells (BMECs), a human blood-brain barrier (BBB) model system, at much greater efficiency than do T. b. brucei. After noting that T. b. gambiense displayed higher levels of cathepsin L–like cysteine proteases, we investigated whether these enzymes contribute to parasite crossing. First, we found that T. b. gambiense crossing of human BMECs was abrogated by N-methylpiperazine-urea-Phe-homopheylalanine-vinylsulfone-benzene (K11777), an irreversible inhibitor of cathepsin L–like cysteine proteases. Affinity labeling and immunochemical studies characterized brucipain as the K11777-sensitive cysteine protease expressed at higher levels by T. b. gambiense. K11777-treated T. b. gambiense failed to elicit calcium fluxes in BMECs, suggesting that generation of activation signals for the BBB is critically dependant on brucipain activity. Strikingly, crossing of T. b. brucei across the BBB was enhanced upon incubation with brucipain-rich supernatants derived from T. b. gambiense. The effects of the conditioned medium, which correlated with ability to evoke calcium fluxes, were canceled by K11777, but not by the cathepsin B inhibitor CA074. Collectively, these in vitro studies implicate brucipain as a critical driver of T. b. gambiense transendothelial migration of the human BBB. PMID:16998589

The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier

PubMed Central

Na Pombejra, Sarisa; Salemi, Michelle; Phinney, Brett S.; Gelli, Angie

2017-01-01

Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans, the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of CnMPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by CnMPR1 (i.e., Sc strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, Sc-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of Sc strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of Sc. Additionally, the co-localization of AnxA2 and Sc suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of Sc is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain microvascular endothelial cells and thereby engages AnxA2 in order to facilitate fungal transcytosis of the BBB. PMID:28713781

The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier.

PubMed

Na Pombejra, Sarisa; Salemi, Michelle; Phinney, Brett S; Gelli, Angie

2017-01-01

Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans , the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of Cn MPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by Cn MPR1 (i.e., Sc strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, Sc-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of Sc strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of Sc. Additionally, the co-localization of AnxA2 and Sc suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of Sc is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain microvascular endothelial cells and thereby engages AnxA2 in order to facilitate fungal transcytosis of the BBB.

MMP-9 gene silencing by a Quantum Dot-siRNA nanoplex delivery to maintain the integrity of the blood brain barrier

PubMed Central

Bonoiu, Adela; Mahajan, Supriya D.; Ye, Ling; Kumar, Rajiv; Ding, Hong; Yong, Ken-Tye; Roy, Indrajit; Aalinkeel, Ravikumar; Nair, Bindukumar; Reynolds, Jessica L; Sykes, Donald E; Imperiale, Marco A; Bergey, Earl J.; Schwartz, Stanley A.; Prasad, Paras N.

2009-01-01

The matrix-degrading metalloproteinases (MMPs), particularly MMP-9, are involved in the neuroinflammation processes leading to disrupting of the blood brain barrier (BBB), thereby exacerbating neurological diseases such as HIV-1 AIDS dementia and cerebral ischemia. Nanoparticles have been proposed to act as non-viral gene delivery vectors and have great potential for therapeutic applications in several disease states. In this study, we evaluated the specificity and efficiency of quantum dot (QD) complexed with MMP-9-siRNA (nanoplex) in downregulating the expression of MMP-9 gene in brain microvascular endothelial cells (BMVEC) that constitute the BBB. We hypothesize that silencing MMP-9 gene expression in BMVECs and other cells such as leukocytes may help prevent breakdown of the BBB and inhibit subsequent invasion of the central nervous system (CNS) by infected and inflammatory cells. Our results show that silencing of MMP-9 gene expression resulted in the upregulation of extracellular matrix (ECM) proteins like collagen I, IV, V and a decrease in endothelial permeability, as reflected by reduction of transendothelial resistance across the BBB in a well validated in-vitro BBB model. MMP-9 gene silencing also resulted in an increase in expression of the gene tissue inhibitor of metalloproteinase-1 (TIMP-1). This indicates the importance of a balance between the levels of MMP-9 and its natural inhibitor TIMP-1 in maintaining the basement membrane integrity. These studies promise the application of a novel nanoparticle based siRNA delivery system in modulating the MMP-9 activity in BMVECs and other MMP-9 producing cells. This will prevent neuroinflammation and maintain the integrity of the BBB. PMID:19477169

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

PubMed Central

Díaz-Coránguez, Mónica; Segovia, José; López-Ornelas, Adolfo; Puerta-Guardo, Henry; Ludert, Juan; Chávez, Bibiana; Meraz-Cruz, Noemi; González-Mariscal, Lorenza

2013-01-01

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2. PMID:23637756

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier

PubMed Central

Baghirov, Habib; Karaman, Didem; Viitala, Tapani; Duchanoy, Alain; Lou, Yan-Ru; Mamaeva, Veronika; Pryazhnikov, Evgeny; Khiroug, Leonard; de Lange Davies, Catharina; Sahlgren, Cecilia; Rosenholm, Jessica M.

2016-01-01

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport. PMID:27547955

Copper transport mediated by nanocarrier systems in a blood-brain barrier in vitro model.

PubMed

Fehse, Susanne; Nowag, Sabrina; Quadir, Mohiuddin; Kim, Kwang Sik; Haag, Rainer; Multhaup, Gerd

2014-05-12

Copper (Cu) is a cofactor of various metalloenzymes and has a role in neurodegenerative diseases with disturbed Cu homeostasis, for example, in Alzheimer's disease (AD) and Menkes disease. To address Cu imbalances, we synthesized two different dendritic nanoparticles (NP) for the transport of Cu(II) ions across the blood-brain barrier (BBB). The synthesized NPs show low toxicity and high water solubility and can stabilize high amounts of Cu(II). The Cu(II)-laden NPs crossed cellular membranes and increased the cellular Cu level. A human brain microvascular endothelial cell (HBMEC) model was established to investigate the permeability of the NPs through the BBB. By comparing the permeability × surface area product (PSe) of reference substances with those of NPs, we observed that NPs crossed the BBB model two times more effectively than (14)C-sucrose and sodium fluorescein (NaFl) and up to 60× better than Evans Blue labeled albumin (EBA). Our results clearly indicate that NPs cross the BBB model effectively. Furthermore, Cu was shielded by the NPs, which decreased the Cu toxicity. The novel design of the core-shell NP enabled the complexation of Cu(II) in the outer shell and therefore facilitated the pH-dependent release of Cu in contrast to core-multishell NPs, where the Cu(II) ions are encapsulated in the core. This allows a release of Cu into the cytoplasm. In addition, by using a cellular detection system based on a metal response element with green fluorescent protein (MRE-GFP), we demonstrated that Cu could also be released intracellularly from NPs and is accessible for biological processes. Our results indicate that NPs are potential candidates to rebalance metal-ion homeostasis in disease conditions affecting brain and neuronal systems.

Tumour necrosis factor-α-mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin-6.

PubMed

Rochfort, Keith D; Collins, Laura E; McLoughlin, Alisha; Cummins, Philip M

2016-02-01

The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/mL, 0-24 h) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species, down-regulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signalling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signalling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been widely reported. Using human brain microvascular endothelial cells (HBMvEC), we show that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of endothelial IL-6 production and signalling. We demonstrate that the TNF-α-dependent generation of reactive oxygen species (ROS), down-regulation of interendothelial junctions, and concomitant elevation of HBMvEC permeability, could be significantly attenuated by using either an IL-6 neutralizing antibody or an IL-6-specific siRNA. These findings provide insight into the complex nature of proinflammatory cytokine injury at the BBB microvascular endothelium interface. © 2015 International Society for Neurochemistry.

Enhancing Anticancer Effect of Gefitinib across the Blood–Brain Barrier Model Using Liposomes Modified with One α-Helical Cell-Penetrating Peptide or Glutathione and Tween 80

PubMed Central

Lin, Kuan-Hung; Hong, Shu-Ting; Wang, Hsiang-Tsui; Lo, Yu-Li; Lin, Anya Maan-Yuh; Yang, James Chih-Hsin

2016-01-01

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been demonstrated to effectively treat the patients of extracranial non-small cell lung cancer (NSCLC). However, these patients often develop brain metastasis (BM) during their disease course. The major obstacle to treat BM is the limited penetration of anticancer drugs across the blood–brain barrier (BBB). In the present study, we utilized gefitinib-loaded liposomes with different modifications to improve gefitinib delivery across the in vitro BBB model of bEnd.3 cells. Gefitinib was encapsulated in small unilamellar liposomes modified with glutathione (GSH) and Tween 80 (SUV-G+T; one ligand plus one surfactant) or RF (SUV-RF; one α-helical cell-penetrating peptide). GSH, Tween 80, and RF were tested by the sulforhodamine B (SRB) assay to find their non-cytotoxic concentrations on bEnd.3 cells. The enhancement on gefitinib across the BBB was evaluated by cytotoxicity assay on human lung adenocarcinoma PC9 cells under the bEnd.3 cells grown on the transwell inserts. Our findings showed that gefitinib incorporated in SUV-G+T or SUV-RF across the bEnd.3 cells significantly reduced the viability of PC9 cells more than that of free gefitinib. Furthermore, SUV-RF showed no cytotoxicity on bEnd.3 cells and did not affect the transendothelial electrical resistance (TEER) and transendothelial permeability of sodium fluorescein across the BBB model. Moreover, flow cytometry and confocal laser scanning microscopy were employed to evaluate the endocytosis pathways of SUV-RF. The results indicated that the uptake into bEnd.3 cells was mainly through adsorptive-mediated mechanism via electrostatic interaction and partially through clathrin-mediated endocytosis. In conclusion, cell penetrating peptide-conjugated SUV-RF shed light on improving drug transport across the BBB via modulating the transcytosis pathway(s). PMID:27916828

Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen.

PubMed

Santiago-Tirado, Felipe H; Onken, Michael D; Cooper, John A; Klein, Robyn S; Doering, Tamara L

2017-01-31

The blood-brain barrier (BBB) protects the central nervous system (CNS) by restricting the passage of molecules and microorganisms. Despite this barrier, however, the fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that is estimated to kill over 600,000 people annually. Cryptococcal infection begins in the lung, and experimental evidence suggests that host phagocytes play a role in subsequent dissemination, although this role remains ill defined. Additionally, the disparate experimental approaches that have been used to probe various potential routes of BBB transit make it impossible to assess their relative contributions, confounding any integrated understanding of cryptococcal brain entry. Here we used an in vitro model BBB to show that a "Trojan horse" mechanism contributes significantly to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. We also, for the first time, directly imaged C. neoformans-containing phagocytes crossing the BBB, showing that they do so via transendothelial pores. Finally, we found that Trojan horse crossing enables CNS entry of fungal mutants that cannot otherwise traverse the BBB, and we demonstrate additional intercellular interactions that may contribute to brain entry. Our work elucidates the mechanism of cryptococcal brain invasion and offers approaches to study other neuropathogens. The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain. Copyright © 2017 Santiago-Tirado et al.

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

PubMed Central

Saint-Laurent, Olivia; Bourbonnière, Lyne; Larouche, Sandra; Larochelle, Catherine; Michel, Laure; Charabati, Marc; Abadier, Michael; Zandee, Stephanie; Haghayegh Jahromi, Neda; Gowing, Elizabeth; Pittet, Camille; Lyck, Ruth; Engelhardt, Britta

2017-01-01

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood–brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35–55–induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity. PMID:28069965

Mesenchymal Stem Cells Regulate Blood Brain Barrier Integrity in Traumatic Brain Injury Through Production of the Soluble Factor TIMP3

PubMed Central

Menge, Tyler; Zhao, Yuhai; Zhao, Jing; Wataha, Kathryn; Geber, Michael; Zhang, Jianhu; Letourneau, Phillip; Redell, John; Shen, Li; Wang, Jing; Peng, Zhalong; Xue, Hasen; Kozar, Rosemary; Cox, Charles S.; Khakoo, Aarif Y.; Holcomb, John B.; Dash, Pramod K.; Pati, Shibani

2013-01-01

Mesenchymal stem cells (MCSs) have been shown to have therapeutic potential in multiple disease states associated with vascular instability including traumatic brain injury (TBI). In the present study, Tissue Inhibitor of Matrix Metalloproteinase-3 (TIMP3) is identified as the soluble factor produced by MSCs that can recapitulate the beneficial effects of MSCs on endothelial function and blood brain barrier (BBB) compromise in TBI. Attenuation of TIMP3 expression in MSCs completely abrogates the effect of MSCs on BBB permeability and stability, while intravenous administration of rTIMP3 alone can inhibit BBB permeability in TBI. Our results demonstrate that MSCs increase circulating levels of soluble TIMP3, which inhibits VEGF-A induced breakdown of endothelial AJs in vitro and in vivo. These findings elucidate a clear molecular mechanism for the effects of MSCs on the BBB in TBI, and directly demonstrate a role for TIMP3 in regulation of BBB integrity. PMID:23175708

Investigation of the metabolism of substance P at the blood-brain barrier using LC-MS/MS.

PubMed

Chappa, Arvind K; Cooper, Joshua D; Audus, Kenneth L; Lunte, Susan M

2007-03-12

Substance P (SP) has been associated with pain and depression as well as neurodegenerative diseases. Many of these diverse actions of SP can potentially be attributed to SP metabolites generated at the blood-brain barrier (BBB). In this study, the metabolism of SP was investigated using an in vitro model of the BBB and LC-MS/MS. Substance P metabolism was found to be non-saturable in the concentration range of 100 nM to 10 microM, with approximately 70% of the peptide remaining intact after 5 h. The major metabolites of SP were identified by MS as 3-11 and 5-11. Two previously unreported metabolites, 5-11 and 6-11, were also found in our studies. Several additional minor SP metabolites, including 1-9 and 2-11, were also identified. A profile of the SP metabolites generated by the BBB over time was obtained. The results from the present study provide a better understanding of the role of the blood-brain barrier in the pharmacology of SP.

INVESTIGATION OF THE METABOLISM OF SUBSTANCE P AT THE BLOOD-BRAIN BARRIER USING LC-MS/MS

PubMed Central

Chappa, Arvind K.; Cooper, Joshua D.; Audus, Kenneth L.; Lunte, Susan M.

2007-01-01

Substance P (SP) has been associated with pain and depression as well as neurodegenerative diseases. Many of these diverse actions of SP can potentially be attributed to SP metabolites generated at the blood-brain barrier (BBB). In this study, the metabolism of SP was investigated using an in vitro model of the BBB and LC-MS/MS. Substance P metabolism was found to be non-saturable in the concentration range of 100 nM to 10 μM, with approximately 70% of the peptide remaining intact after 5 h. The major metabolites of SP were identified by MS as 3-11 and 5-11. Two previously unreported metabolites, 5-11 and 6-11, were also found in our studies. Several additional minor SP metabolites, including 1-9 and 2-11, were also identified. A profile of the SP metabolites generated by the BBB over time was obtained. The results from the present study provide us a better understanding of the role of the blood-brain barrier in the pharmacology of SP. PMID:17118606

Low-Pressure Burst-Mode Focused Ultrasound Wave Reconstruction and Mapping for Blood-Brain Barrier Opening: A Preclinical Examination

PubMed Central

Xia, Jingjing; Tsui, Po-Hsiang; Liu, Hao-Li

2016-01-01

Burst-mode focused ultrasound (FUS) exposure has been shown to induce transient blood-brain barrier (BBB) opening for potential CNS drug delivery. FUS-BBB opening requires imaging guidance during the intervention, yet current imaging technology only enables postoperative outcome confirmation. In this study, we propose an approach to visualize short-burst low-pressure focal beam distribution that allows to be applied in FUS-BBB opening intervention on small animals. A backscattered acoustic-wave reconstruction method based on synchronization among focused ultrasound emission, diagnostic ultrasound receiving and passively beamformed processing were developed. We observed that focal beam could be successfully visualized for in vitro FUS exposure with 0.5–2 MHz without involvement of microbubbles. The detectable level of FUS exposure was 0.467 MPa in pressure and 0.05 ms in burst length. The signal intensity (SI) of the reconstructions was linearly correlated with the FUS exposure level both in-vitro (r2 = 0.9878) and in-vivo (r2 = 0.9943), and SI level of the reconstructed focal beam also correlated with the success and level of BBB-opening. The proposed approach provides a feasible way to perform real-time and closed-loop control of FUS-based brain drug delivery. PMID:27295608

Low-Pressure Burst-Mode Focused Ultrasound Wave Reconstruction and Mapping for Blood-Brain Barrier Opening: A Preclinical Examination

NASA Astrophysics Data System (ADS)

Xia, Jingjing; Tsui, Po-Hsiang; Liu, Hao-Li

2016-06-01

Burst-mode focused ultrasound (FUS) exposure has been shown to induce transient blood-brain barrier (BBB) opening for potential CNS drug delivery. FUS-BBB opening requires imaging guidance during the intervention, yet current imaging technology only enables postoperative outcome confirmation. In this study, we propose an approach to visualize short-burst low-pressure focal beam distribution that allows to be applied in FUS-BBB opening intervention on small animals. A backscattered acoustic-wave reconstruction method based on synchronization among focused ultrasound emission, diagnostic ultrasound receiving and passively beamformed processing were developed. We observed that focal beam could be successfully visualized for in vitro FUS exposure with 0.5-2 MHz without involvement of microbubbles. The detectable level of FUS exposure was 0.467 MPa in pressure and 0.05 ms in burst length. The signal intensity (SI) of the reconstructions was linearly correlated with the FUS exposure level both in-vitro (r2 = 0.9878) and in-vivo (r2 = 0.9943), and SI level of the reconstructed focal beam also correlated with the success and level of BBB-opening. The proposed approach provides a feasible way to perform real-time and closed-loop control of FUS-based brain drug delivery.

Postarrest stalling rather than crawling favors CD8+ over CD4+ T‐cell migration across the blood–brain barrier under flow in vitro

PubMed Central

Rudolph, Henriette; Klopstein, Armelle; Gruber, Isabelle; Blatti, Claudia; Lyck, Ruth

2016-01-01

Although CD8+ T cells have been implied in the pathogenesis of multiple sclerosis (MS), the molecular mechanisms mediating CD8+ T‐cell migration across the blood–brain barrier (BBB) into the central nervous system (CNS) are ill defined. Using in vitro live cell imaging, we directly compared the multistep extravasation of activated CD4+ and CD8+ T cells across primary mouse brain microvascular endothelial cells (pMBMECs) as a model for the BBB under physiological flow. Significantly higher numbers of CD8+ than CD4+ T cells arrested on pMBMECs under noninflammatory and inflammatory conditions. While CD4+ T cells polarized and crawled prior to their diapedesis, the majority of CD8+ T cells stalled and readily crossed the pMBMEC monolayer preferentially via a transcellular route. T‐cell arrest and crawling were independent of G‐protein‐coupled receptor signaling. Rather, absence of endothelial ICAM‐1 and ICAM‐2 abolished increased arrest of CD8+ over CD4+ T cells and abrogated T‐cell crawling, leading to the efficient reduction of CD4+, but to a lesser degree of CD8+, T‐cell diapedesis across ICAM‐1null/ICAM‐2−/− pMBMECs. Thus, cellular and molecular mechanisms mediating the multistep extravasation of activated CD8+ T cells across the BBB are distinguishable from those involved for CD4+ T cells. PMID:27338806

Crocus sativus Extract Tightens the Blood-Brain Barrier, Reduces Amyloid β Load and Related Toxicity in 5XFAD Mice.

PubMed

Batarseh, Yazan S; Bharate, Sonali S; Kumar, Vikas; Kumar, Ajay; Vishwakarma, Ram A; Bharate, Sandip B; Kaddoumi, Amal

2017-08-16

Crocus sativus, commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes as an aphrodisiac, antispasmodic, and expectorant. Previous evidence suggested that Crocus sativus is linked to improving cognitive function in Alzheimer's disease (AD) patients. The aim of this study was to in vitro and in vivo investigate the mechanism(s) by which Crocus sativus exerts its positive effect against AD. The effect of Crocus sativus extract on Aβ load and related toxicity was evaluated. In vitro results showed that Crocus sativus extract increases the tightness of a cell-based blood-brain barrier (BBB) model and enhances transport of Aβ. Further in vivo studies confirmed the effect of Crocus sativus extract (50 mg/kg/day, added to mice diet) on the BBB tightness and function that was associated with reduced Aβ load and related pathological changes in 5XFAD mice used as an AD model. Reduced Aβ load could be explained, at least in part, by Crocus sativus extract effect to enhance Aβ clearance pathways including BBB clearance, enzymatic degradation and ApoE clearance pathway. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuroinflammation associated with Aβ pathology in the brains of 5XFAD mice. Crocin, a major active constituent of Crocus sativus and known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo in 5XFAD mice. Crocin (10 mg/kg/day) was able to reduce Aβ load but to a lesser extent when compared to Crocus sativus extract. Collectively, findings from this study support the positive effect of Crocus sativus against AD by reducing Aβ pathological manifestations.

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro.

PubMed

Ullen, Andreas; Fauler, Günter; Bernhart, Eva; Nusshold, Christoph; Reicher, Helga; Leis, Hans-Jörg; Malle, Ernst; Sattler, Wolfgang

2012-11-01

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood-brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting. Copyright © 2012 Elsevier Inc. All rights reserved.

Multifunctional targeting vinorelbine plus tetrandrine liposomes for treating brain glioma along with eliminating glioma stem cells

PubMed Central

Li, Xue-tao; Tang, Wei; Jiang, Ying; Wang, Xiao-min; Wang, Yan-hong; Cheng, Lan; Meng, Xian-sheng

2016-01-01

Malignant brain glioma is the most lethal and aggressive type of cancer. Surgery and radiotherapy cannot eliminate all glioma stem cells (GSCs) and blood–brain barrier (BBB) restricts the movement of antitumor drugs from blood to brain, thus leading to the poor prognosis with high recurrence rate. In the present study, the targeting conjugates of cholesterol polyethylene glycol polyethylenimine (CHOL-PEG2000-PEI) and D-a-tocopheryl polyethylene glycol 1000 succinate vapreotide (TPGS1000-VAP) were newly synthesized for transporting drugs across the BBB and targeting glioma cells and GSCs. The multifunctional targeting vinorelbine plus tetrandrine liposomes were constructed by modifying the targeting conjugates. The studies were undertaken on BBB model, glioma cells, GSCs, and glioma-bearing mice. In vitro results showed that multifunctional targeting drugs-loaded liposomes with suitable physicochemical property could enhance the transport drugs across the BBB, increase the intracellular uptake, inhibit glioma cells and GSCs, penetrate and destruct the GSCs spheroids, and induce apoptosis via activating related apoptotic proteins. In vivo results demonstrated that multifunctional targeting drugs-loaded liposomes could significantly accumulate into brain tumor location, show the specificity to tumor sites, and result in a robust overall antitumor efficacy in glioma-bearing mice. These data suggested that the multifunctional targeting vinorelbine plus tetrandrine liposomes could offer a promising strategy for treating brain glioma. PMID:27029055

Penetration of blood-brain barrier and antitumor activity and nerve repair in glioma by doxorubicin-loaded monosialoganglioside micelles system.

PubMed

Zou, Dan; Wang, Wei; Lei, Daoxi; Yin, Ying; Ren, Peng; Chen, Jinju; Yin, Tieying; Wang, Bochu; Wang, Guixue; Wang, Yazhou

2017-01-01

For the treatment of glioma and other central nervous system diseases, one of the biggest challenges is that most therapeutic drugs cannot be delivered to the brain tumor tissue due to the blood-brain barrier (BBB). The goal of this study was to construct a nanodelivery vehicle system with capabilities to overcome the BBB for central nervous system administration. Doxorubicin as a model drug encapsulated in ganglioside GM1 micelles was able to achieve up to 9.33% loading efficiency and 97.05% encapsulation efficiency by orthogonal experimental design. The in vitro study demonstrated a slow and sustainable drug release in physiological conditions. In the cellular uptake studies, mixed micelles could effectively transport into both human umbilical vein endothelial cells and C6 cells. Furthermore, biodistribution imaging of mice showed that the DiR/GM1 mixed micelles were accumulated sustainably and distributed centrally in the brain. Experiments on zebrafish confirmed that drug-loaded GM1 micelles can overcome the BBB and enter the brain. Among all the treatment groups, the median survival time of C6-bearing rats after administering DOX/GM1 micelles was significantly prolonged. In conclusion, the ganglioside nanomicelles developed in this work can not only penetrate BBB effectively but also repair nerves and kill tumor cells at the same time.

Electromagnetic interference in the permeability of saquinavir across the blood-brain barrier using nanoparticulate carriers.

PubMed

Kuo, Yung-Chih; Kuo, Chan-Ying

2008-03-03

Transport of antiretroviral agents across the blood-brain barrier (BBB) is of key importance to the treatment for the acquired immunodeficiency syndrome (AIDS). In this study, impact of exposure to electromagnetic field (EMF) on the permeability of saquinavir (SQV) across BBB was investigated. The in vitro BBB model was based on human brain-microvascular endothelial cells (HBMEC), and the concentration of SQV in receiver chamber of the transport system was evaluated. Polybutylcyanoacrylate (PBCA), methylmethacrylate-sulfopropylmethacrylate (MMA-SPM), and solid lipid nanoparticle (SLN) were employed as carriers for the delivery systems. Cytotoxicity of SLN decreased as content of cacao butter increased. Power of 5mV was apposite for the study on HBMEC without obvious apoptosis. Square wave produced greater permeability than sine and triangle waves. The carrier order on permeability of SQV across HBMEC monolayer under exposure to EMF was SLN>PBCA>MMA-SPM. Also, a larger frequency, modulation or depth of amplitude modulation (AM), or modulation or deviation of frequency modulation (FM) yielded a greater permeability. Besides, enhancement of permeability by AM wave was more significant than that by FM wave. Transport behavior of SQV across BBB was strongly influenced by the combination of nanoparticulate PBCA, MMA-SPM, and SLN with EMF exposure. This combination would be beneficial to the clinical application to the therapy of AIDS and other brain-related diseases.

Blood-brain barrier-on-a-chip: Microphysiological systems that capture the complexity of the blood-central nervous system interface.

PubMed

Phan, Duc Tt; Bender, R Hugh F; Andrejecsk, Jillian W; Sobrino, Agua; Hachey, Stephanie J; George, Steven C; Hughes, Christopher Cw

2017-11-01

The blood-brain barrier is a dynamic and highly organized structure that strictly regulates the molecules allowed to cross the brain vasculature into the central nervous system. The blood-brain barrier pathology has been associated with a number of central nervous system diseases, including vascular malformations, stroke/vascular dementia, Alzheimer's disease, multiple sclerosis, and various neurological tumors including glioblastoma multiforme. There is a compelling need for representative models of this critical interface. Current research relies heavily on animal models (mostly mice) or on two-dimensional (2D) in vitro models, neither of which fully capture the complexities of the human blood-brain barrier. Physiological differences between humans and mice make translation to the clinic problematic, while monolayer cultures cannot capture the inherently three-dimensional (3D) nature of the blood-brain barrier, which includes close association of the abluminal side of the endothelium with astrocyte foot-processes and pericytes. Here we discuss the central nervous system diseases associated with blood-brain barrier pathology, recent advances in the development of novel 3D blood-brain barrier -on-a-chip systems that better mimic the physiological complexity and structure of human blood-brain barrier, and provide an outlook on how these blood-brain barrier-on-a-chip systems can be used for central nervous system disease modeling. Impact statement The field of microphysiological systems is rapidly evolving as new technologies are introduced and our understanding of organ physiology develops. In this review, we focus on Blood-Brain Barrier (BBB) models, with a particular emphasis on how they relate to neurological disorders such as Alzheimer's disease, multiple sclerosis, stroke, cancer, and vascular malformations. We emphasize the importance of capturing the three-dimensional nature of the brain and the unique architecture of the BBB - something that until recently had not been well modeled by in vitro systems. Our hope is that this review will provide a launch pad for new ideas and methodologies that can provide us with truly physiological BBB models capable of yielding new insights into the function of this critical interface.

Predicting A Drug'S Membrane Permeability: Evolution of a Computational Model Validated with in Vitro Permeability Assay Data

DOE PAGES

Carpenter, Timothy S.; McNerney, M. Windy; Be, Nicholas A.; ...

2016-02-16

Membrane permeability is a key property to consider in drug design, especially when the drugs in question need to cross the blood-brain barrier (BBB). A comprehensive in vivo assessment of the BBB permeability of a drug takes considerable time and financial resources. A current, simplified in vitro model to investigate drug permeability is a Parallel Artificial Membrane Permeability Assay (PAMPA) that generally provides higher throughput and initial quantification of a drug's passive permeability. Computational methods can also be used to predict drug permeability. Our methods are highly advantageous as they do not require the synthesis of the desired drug, andmore » can be implemented rapidly using high-performance computing. In this study, we have used umbrella sampling Molecular Dynamics (MD) methods to assess the passive permeability of a range of compounds through a lipid bilayer. Furthermore, the permeability of these compounds was comprehensively quantified using the PAMPA assay to calibrate and validate the MD methodology. And after demonstrating a firm correlation between the two approaches, we then implemented our MD method to quantitatively predict the most permeable potential drug from a series of potential scaffolds. This permeability was then confirmed by the in vitro PAMPA methodology. Therefore, in this work we have illustrated the potential that these computational methods hold as useful tools to help predict a drug's permeability in a faster and more cost-effective manner. Release number: LLNL-ABS-677757.« less

Predicting A Drug'S Membrane Permeability: Evolution of a Computational Model Validated with in Vitro Permeability Assay Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carpenter, Timothy S.; McNerney, M. Windy; Be, Nicholas A.

Membrane permeability is a key property to consider in drug design, especially when the drugs in question need to cross the blood-brain barrier (BBB). A comprehensive in vivo assessment of the BBB permeability of a drug takes considerable time and financial resources. A current, simplified in vitro model to investigate drug permeability is a Parallel Artificial Membrane Permeability Assay (PAMPA) that generally provides higher throughput and initial quantification of a drug's passive permeability. Computational methods can also be used to predict drug permeability. Our methods are highly advantageous as they do not require the synthesis of the desired drug, andmore » can be implemented rapidly using high-performance computing. In this study, we have used umbrella sampling Molecular Dynamics (MD) methods to assess the passive permeability of a range of compounds through a lipid bilayer. Furthermore, the permeability of these compounds was comprehensively quantified using the PAMPA assay to calibrate and validate the MD methodology. And after demonstrating a firm correlation between the two approaches, we then implemented our MD method to quantitatively predict the most permeable potential drug from a series of potential scaffolds. This permeability was then confirmed by the in vitro PAMPA methodology. Therefore, in this work we have illustrated the potential that these computational methods hold as useful tools to help predict a drug's permeability in a faster and more cost-effective manner. Release number: LLNL-ABS-677757.« less

Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood-Brain Barrier under Inflammatory Conditions In Vitro.

PubMed

De Laere, Maxime; Sousa, Carmelita; Meena, Megha; Buckinx, Roeland; Timmermans, Jean-Pierre; Berneman, Zwi; Cools, Nathalie

2017-01-01

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood-brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.

Paramagnetic perfluorocarbon-filled albumin-(Gd-DTPA) microbubbles for the induction of focused-ultrasound-induced blood-brain barrier opening and concurrent MR and ultrasound imaging

NASA Astrophysics Data System (ADS)

Liao, Ai-Ho; Liu, Hao-Li; Su, Chia-Hao; Hua, Mu-Yi; Yang, Hung-Wei; Weng, Yu-Ting; Hsu, Po-Hung; Huang, Sheng-Min; Wu, Shih-Yen; Wang, Hsin-Ell; Yen, Tzu-Chen; Li, Pai-Chi

2012-05-01

This paper presents new albumin-shelled Gd-DTPA microbubbles (MBs) that can concurrently serve as a dual-modality contrast agent for ultrasound (US) imaging and magnetic resonance (MR) imaging to assist blood-brain barrier (BBB) opening and detect intracerebral hemorrhage (ICH) during focused ultrasound brain drug delivery. Perfluorocarbon-filled albumin-(Gd-DTPA) MBs were prepared with a mean diameter of 2320 nm and concentration of 2.903×109 MBs ml-1 using albumin-(Gd-DTPA) and by sonication with perfluorocarbon (C3F8) gas. The albumin-(Gd-DTPA) MBs were then centrifuged and the procedure was repeated until the free Gd3+ ions were eliminated (which were detected by the xylenol orange sodium salt solution). The albumin-(Gd-DTPA) MBs were also characterized and evaluated both in vitro and in vivo by US and MR imaging. Focused US was used with the albumin-(Gd-DTPA) MBs to induce disruption of the BBB in 18 rats. BBB disruption was confirmed with contrast-enhanced T1-weighted turbo-spin-echo sequence MR imaging. Heavy T2*-weighted 3D fast low-angle shot sequence MR imaging was used to detect ICH. In vitro US imaging experiments showed that albumin-(Gd-DTPA) MBs can significantly enhance the US contrast in T1-, T2- and T2*-weighted MR images. The r1 and r2 relaxivities for Gd-DTPA were 7.69 and 21.35 s-1mM-1, respectively, indicating that the MBs represent a positive contrast agent in T1-weighted images. In vivo MR imaging experiments on 18 rats showed that focused US combined with albumin-(Gd-DTPA) MBs can be used to both induce disruption of the BBB and detect ICH. To compare the signal intensity change between pure BBB opening and BBB opening accompanying ICH, albumin-(Gd-DTPA) MB imaging can provide a ratio of 5.14 with significant difference (p = 0.026), whereas Gd-DTPA imaging only provides a ratio of 2.13 and without significant difference (p = 0.108). The results indicate that albumin-(Gd-DTPA) MBs have potential as a US/MR dual-modality contrast agent for BBB opening and differentiating focused-US-induced BBB opening from ICH, and can monitor the focused ultrasound brain drug delivery process.

Paramagnetic perfluorocarbon-filled albumin-(Gd-DTPA) microbubbles for the induction of focused-ultrasound-induced blood-brain barrier opening and concurrent MR and ultrasound imaging.

PubMed

Liao, Ai-Ho; Liu, Hao-Li; Su, Chia-Hao; Hua, Mu-Yi; Yang, Hung-Wei; Weng, Yu-Ting; Hsu, Po-Hung; Huang, Sheng-Min; Wu, Shih-Yen; Wang, Hsin-Ell; Yen, Tzu-Chen; Li, Pai-Chi

2012-05-07

This paper presents new albumin-shelled Gd-DTPA microbubbles (MBs) that can concurrently serve as a dual-modality contrast agent for ultrasound (US) imaging and magnetic resonance (MR) imaging to assist blood-brain barrier (BBB) opening and detect intracerebral hemorrhage (ICH) during focused ultrasound brain drug delivery. Perfluorocarbon-filled albumin-(Gd-DTPA) MBs were prepared with a mean diameter of 2320 nm and concentration of 2.903×10(9) MBs ml(-1) using albumin-(Gd-DTPA) and by sonication with perfluorocarbon (C(3)F(8)) gas. The albumin-(Gd-DTPA) MBs were then centrifuged and the procedure was repeated until the free Gd(3+) ions were eliminated (which were detected by the xylenol orange sodium salt solution). The albumin-(Gd-DTPA) MBs were also characterized and evaluated both in vitro and in vivo by US and MR imaging. Focused US was used with the albumin-(Gd-DTPA) MBs to induce disruption of the BBB in 18 rats. BBB disruption was confirmed with contrast-enhanced T(1)-weighted turbo-spin-echo sequence MR imaging. Heavy T(2)*-weighted 3D fast low-angle shot sequence MR imaging was used to detect ICH. In vitro US imaging experiments showed that albumin-(Gd-DTPA) MBs can significantly enhance the US contrast in T(1)-, T(2)- and T(2)*-weighted MR images. The r(1) and r(2) relaxivities for Gd-DTPA were 7.69 and 21.35 s(-1)mM(-1), respectively, indicating that the MBs represent a positive contrast agent in T(1)-weighted images. In vivo MR imaging experiments on 18 rats showed that focused US combined with albumin-(Gd-DTPA) MBs can be used to both induce disruption of the BBB and detect ICH. To compare the signal intensity change between pure BBB opening and BBB opening accompanying ICH, albumin-(Gd-DTPA) MB imaging can provide a ratio of 5.14 with significant difference (p = 0.026), whereas Gd-DTPA imaging only provides a ratio of 2.13 and without significant difference (p = 0.108). The results indicate that albumin-(Gd-DTPA) MBs have potential as a US/MR dual-modality contrast agent for BBB opening and differentiating focused-US-induced BBB opening from ICH, and can monitor the focused ultrasound brain drug delivery process.

Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells).

PubMed

Lee, Kyeong-Eun; Kang, Young-Sook

2017-05-10

L -Citrulline is a neutral amino acid and a major precursor of L -arginine in the nitric oxide (NO) cycle. Recently it has been reported that L -citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L -citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L -citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB. The uptake study of [ 14 C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b 0,+ , and CAT1 small interfering RNA study were performed in TR-BBB cells. The uptake of [ 14 C] L -citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis-Menten constant of 30.9 ± 1.0 μM (Km 1 ) and 1.69 ± 0.43 mM (Km 2 ). The uptake of [ 14 C] L -citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [ 14 C] L -citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B 0 , B 0,+ and harmaline, the inhibitor of system b 0,+ . Gabapentin and L -dopa as the substrates of LAT1 competitively inhibited the uptake of [ 14 C] L -citrulline. IC 50 values for L -dopa, gabapentin, L -phenylalanine and L -arginine were 501 μM, 223 μM, 68.9 μM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b 0,+ in TR-BBB cells. In the studies of LAT1, system b 0,+ and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [ 14 C] L -citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells. Our results suggest that transport of L -citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.

Tau depletion prevents progressive blood-brain barrier damage in a mouse model of tauopathy.

PubMed

Blair, Laura J; Frauen, Haley D; Zhang, Bo; Nordhues, Bryce A; Bijan, Sara; Lin, Yen-Chi; Zamudio, Frank; Hernandez, Lidice D; Sabbagh, Jonathan J; Selenica, Maj-Linda B; Dickey, Chad A

2015-01-31

The blood-brain barrier (BBB) is damaged in tauopathies, including progressive supranuclear palsy (PSP) and Alzheimer's disease (AD), which is thought to contribute to pathogenesis later in the disease course. In AD, BBB dysfunction has been associated with amyloid beta (Aß) pathology, but the role of tau in this process is not well characterized. Since increased BBB permeability is found in tauopathies without Aß pathology, like PSP, we suspected that tau accumulation alone could not only be sufficient, but even more important than Aß for BBB damage. Longitudinal evaluation of brain tissue from the tetracycline-regulatable rTg4510 tau transgenic mouse model showed progressive IgG, T cell and red blood cell infiltration. The Evans blue (EB) dye that is excluded from the brain when the BBB is intact also permeated the brains of rTg4510 mice following peripheral administration, indicative of a bonafide BBB defect, but this was only evident later in life. Thus, despite the marked brain atrophy and inflammation that occurs earlier in this model, BBB integrity is maintained. Interestingly, BBB dysfunction emerged at the same time that perivascular tau emerged around major hippocampal blood vessels. However, when tau expression was suppressed using doxycycline, BBB integrity was preserved, suggesting that the BBB can be stabilized in a tauopathic brain by reducing tau levels. For the first time, these data demonstrate that tau alone can initiate breakdown of the BBB, but the BBB is remarkably resilient, maintaining its integrity in the face of marked brain atrophy, neuroinflammation and toxic tau accumulation. Moreover, the BBB can recover integrity when tau levels are reduced. Thus, late stage interventions targeting tau may slow the vascular contributions to cognitive impairment and dementia that occur in tauopathies.

Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier.

PubMed

Iwanaga, Kazunori; Yoneda, Shinji; Hamahata, Yukimi; Miyazaki, Makoto; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kakemi, Masawo

2011-01-01

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.

A nanostructure of functional targeting epirubicin liposomes dually modified with aminophenyl glucose and cyclic pentapeptide used for brain glioblastoma treatment

PubMed Central

Zhang, Cheng-Xiang; Zhao, Wei-Yu; Liu, Lei; Ju, Rui-Jun; Mu, Li-Min; Zhao, Yao; Zeng, Fan; Xie, Hong-Jun; Yan, Yan; Lu, Wan-Liang

2015-01-01

The objectives of the present study were to develop functional targeting epirubicin liposomes for transferring drugs across the blood-brain barrier (BBB), treating glioblastoma, and disabling neovascularization. The studies were performed on glioblastoma cells in vitro and on glioblastoma-bearing mice. The results showed that the constructed liposomes had a high encapsulation efficiency for drugs (>95%), suitable particle size (109 nm), and less leakage in the blood component-containing system; were significantly able to be transported across the BBB; and exhibited efficacies in killing glioblastoma cells and in destroying glioblastoma neovasculature in vitro and in glioblastoma-bearing mice. The action mechanisms of functional targeting epirubicin liposomes correlated with the following features: the long circulation in the blood system, the ability to be transported across the BBB via glucose transporter-1, and the targeting effects on glioblastoma cells and on the endothelial cells of the glioblastoma neovasculature via the integrin β3 receptor. In conclusion, functional targeting epirubicin liposomes could be used as a potential therapy for treating brain glioblastoma and disabling neovascularization in brain glioblastomas. PMID:26418720

Methotrexate loaded polyether-copolyester dendrimers for the treatment of gliomas: enhanced efficacy and intratumoral transport capability.

PubMed

Dhanikula, Renu Singh; Argaw, Anteneh; Bouchard, Jean-Francois; Hildgen, Patrice

2008-01-01

Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the blood-brain barrier (BBB), drug resistance, and poor penetration into the tumor tissue. In an attempt to overcome these hurdles, polyether-copolyester (PEPE) dendrimers were evaluated as drug carriers for the treatment of gliomas. Dendrimers were conjugated to d-glucosamine as the ligand for enhancing BBB permeability and tumor targeting. The efficacy of methotrexate (MTX)-loaded dendrimers was established against U87 MG and U 343 MGa cells. Permeability of rhodamine-labeled dendrimers and MTX-loaded dendrimers across the in vitro BBB model and their distribution into avascular human glioma tumor spheroids was also studied. Glucosylated dendrimers were found to be endocytosed in significantly higher amounts than nonglucosylated dendrimers by both the cell lines. IC 50 of MTX after loading in dendrimers was lower than that of the free MTX, suggesting that loading MTX in PEPE dendrimers increased its potency. Similar higher activity of MTX-loaded glucosylated and nonglucosylated dendrimers was found in the reduction of tumor spheroid size. These MTX-loaded dendrimers were able to kill even MTX-resistant cells highlighting their ability to overcome MTX resistance. In addition, the amount of MTX-transported across BBB was three to five times more after loading in the dendrimers. Glucosylation further increased the cumulative permeation of dendrimers across BBB and hence increased the amount of MTX available across it. Glucosylated dendrimers distributed through out the avascular tumor spheroids within 6 h, while nonglucosylated dendrimers could do so in 12 h. The results show that glucosamine can be used as an effective ligand not only for targeting glial tumors but also for enhanced permeability across BBB. Thus, glucosylated PEPE dendrimers can serve as potential delivery system for the treatment of gliomas.

Glutaric aciduria type I and methylmalonic aciduria: simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood-brain barrier and the choroid plexus.

PubMed

Sauer, Sven W; Opp, Silvana; Mahringer, Anne; Kamiński, Marcin M; Thiel, Christian; Okun, Jürgen G; Fricker, Gert; Morath, Marina A; Kölker, Stefan

2010-06-01

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs - glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) - across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood-brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na(+)-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na(+)-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na(+)-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus. Copyright 2010 Elsevier B.V. All rights reserved.

Activation of VEGF/Flk-1-ERK Pathway Induced Blood-Brain Barrier Injury After Microwave Exposure.

PubMed

Wang, Li-Feng; Li, Xiang; Gao, Ya-Bing; Wang, Shui-Ming; Zhao, Li; Dong, Ji; Yao, Bin-Wei; Xu, Xin-Ping; Chang, Gong-Min; Zhou, Hong-Mei; Hu, Xiang-Jun; Peng, Rui-Yun

2015-08-01

Microwaves have been suggested to induce neuronal injury and increase permeability of the blood-brain barrier (BBB), but the mechanism remains unknown. The role of the vascular endothelial growth factor (VEGF)/Flk-1-Raf/MAPK kinase (MEK)/extracellular-regulated protein kinase (ERK) pathway in structural and functional injury of the blood-brain barrier (BBB) following microwave exposure was examined. An in vitro BBB model composed of the ECV304 cell line and primary rat cerebral astrocytes was exposed to microwave radiation (50 mW/cm(2), 5 min). The structure was observed by scanning electron microscopy (SEM) and the permeability was assessed by measuring transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) transmission. Activity and expression of VEGF/Flk-1-ERK pathway components and occludin also were examined. Our results showed that microwave radiation caused intercellular tight junctions to broaden and fracture with decreased TEER values and increased HRP permeability. After microwave exposure, activation of the VEGF/Flk-1-ERK pathway and Tyr phosphorylation of occludin were observed, along with down-regulated expression and interaction of occludin with zonula occludens-1 (ZO-1). After Flk-1 (SU5416) and MEK1/2 (U0126) inhibitors were used, the structure and function of the BBB were recovered. The increase in expression of ERK signal transduction molecules was muted, while the expression and the activity of occludin were accelerated, as well as the interactions of occludin with p-ERK and ZO-1 following microwave radiation. Thus, microwave radiation may induce BBB damage by activating the VEGF/Flk-1-ERK pathway, enhancing Tyr phosphorylation of occludin, while partially inhibiting expression and interaction of occludin with ZO-1.

Assessment of blood-brain barrier penetration: in silico, in vitro and in vivo.

PubMed

Feng, Meihua Rose

2002-12-01

The amount of drug achieved and maintained in the brain after systemic administration is determined by the agent's permeability at blood-brain barrier (BBB), potential involvement of transport systems, and the distribution, metabolism and elimination properties. Passive diffusion permeability may be predicted by an in silico method based on a molecule's structure property. In vitro cell culture is another useful tool for the assessment of passive permeability and BBB transports (e.g. PGP, MRP). In situ or in vivo techniques like carotid artery single injection or perfusion, brain microdialysis, autoradiography, and others are used at various stages of drug discovery and development to estimate CNS penetration and PK/PD correlation. Each technique has its own application with specific advantages and limitations.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.

PubMed

Miyata, Yoshinari; Li, Xiaokai; Lee, Hsiu-Fang; Jinwal, Umesh K; Srinivasan, Sharan R; Seguin, Sandlin P; Young, Zapporah T; Brodsky, Jeffrey L; Dickey, Chad A; Sun, Duxin; Gestwicki, Jason E

2013-06-19

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model.

PubMed

Luo, Su; Cao, Qing; Ma, Ke; Wang, Zhaofei; Liu, Guangjin; Lu, Chengping; Liu, Yongjie

2017-10-19

Streptococcus agalactiae is a pathogen causing meningitis in animals and humans. However, little is known about the entry of S. agalactiae into brain tissue. In this study, we developed a BALB/c mouse model based on the intravenous injection of β-galactosidase-positive Escherichia coli M5 as an indicator of blood-brain barrier (BBB) opening. Under physiological conditions, the BBB is impermeable to E. coli M5. In pathological conditions caused by S. agalactiae, E. coli M5 is capable of penetrating the brain through a disrupted BBB. The level of BBB opening can be assessed by quantitative measurement of E. coli M5 loads per gram of brain tissue. Further, we used the model to evaluate the role of S. agalactiae hyaluronidase in BBB opening. The inactivation of hylB gene encoding a hyaluronidase, HylB, resulted in significantly decreased E. coli M5 colonization, and the intravenous injection of purified HylB protein induced BBB opening in a dose-dependent manner. This finding verified the direct role of HylB in BBB invasion and traversal, and further demonstrated the practicability of the in vivo mouse model established in this study. This model will help to understand the S. agalactiae-host interactions that are involved in this bacterial traversal of the BBB and to develop efficacious strategies to prevent central nervous system infections.

Anesthesia-Induced Hypothermia Attenuates Early-Phase Blood-Brain Barrier Disruption but Not Infarct Volume following Cerebral Ischemia.

PubMed

Liu, Yu-Cheng; Lee, Yu-Da; Wang, Hwai-Lee; Liao, Kate Hsiurong; Chen, Kuen-Bao; Poon, Kin-Shing; Pan, Yu-Ling; Lai, Ted Weita

2017-01-01

Blood-brain barrier (BBB) disruption is thought to facilitate the development of cerebral infarction after a stroke. In a typical stroke model (such as the one used in this study), the early phase of BBB disruption reaches a peak 6 h post-ischemia and largely recovers after 8-24 h, whereas the late phase of BBB disruption begins 48-58 h post-ischemia. Because cerebral infarct develops within 24 h after the onset of ischemia, and several therapeutic agents have been shown to reduce the infarct volume when administered at 6 h post-ischemia, we hypothesized that attenuating BBB disruption at its peak (6 h post-ischemia) can also decrease the infarct volume measured at 24 h. We used a mouse stroke model obtained by combining 120 min of distal middle cerebral arterial occlusion (dMCAo) with ipsilateral common carotid arterial occlusion (CCAo). This model produced the most reliable BBB disruption and cerebral infarction compared to other models characterized by a shorter duration of ischemia or obtained with dMCAO or CCAo alone. The BBB permeability was measured by quantifying Evans blue dye (EBD) extravasation, as this tracer has been shown to be more sensitive for the detection of early-phase BBB disruption compared to other intravascular tracers that are more appropriate for detecting late-phase BBB disruption. We showed that a 1 h-long treatment with isoflurane-anesthesia induced marked hypothermia and attenuated the peak of BBB disruption when administered 6 h after the onset of dMCAo/CCAo-induced ischemia. We also demonstrated that the inhibitory effect of isoflurane was hypothermia-dependent because the same treatment had no effect on ischemic BBB disruption when the mouse body temperature was maintained at 37°C. Importantly, inhibiting the peak of BBB disruption by hypothermia had no effect on the volume of brain infarct 24 h post-ischemia. In conclusion, inhibiting the peak of BBB disruption is not an effective neuroprotective strategy, especially in comparison to the inhibitors of the neuronal death signaling cascade; these, in fact, can attenuate the infarct volume measured at 24 h post-ischemia when administered at 6 h in our same stroke model.

Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration.

PubMed

Yang, Zhen-Zhen; Zhang, Yan-Qing; Wang, Zhan-Zhang; Wu, Kai; Lou, Jin-Ning; Qi, Xian-Rong

2013-08-16

Alzheimer's disease (AD) is a common progressive neurodegenerative disorder associated with cholinergic neurons degeneration. The blood-brain barrier (BBB) not only provides protection for the brain but also hinders the treatment and diagnosis of this neurological disease, because the drugs must cross BBB to reach the lesions. The present work was aimed at formulating rivastigmine liposomes (Lp) and cell-penetrating peptide (CPP) modified liposomes (CPP-Lp) to improve rivastigmine distribution in brain and proceed to enhance pharmacodynamics by intranasal (IN) administration and minimize side effects. The results revealed that Lp especially the CPP-Lp can enhance the permeability across the BBB by murine brain microvascular endothelial cells model in vitro. IN administration of rivastigmine solution and rivastigmine liposomes demonstrated the capacity to improve rivastigmine distribution and adequate retention in CNS regions especially in hippocampus and cortex, which were the regions most affected by AD, than that of IV administration. Importantly, the lagging but intense inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were relative to the extended release, absorption and retention. In addition, there was very mild nasal toxicity of liposomal formulations. The data suggest that rivastigmine liposomes especially CPP-Lp improve the brain delivery and enhance pharmacodynamics which respect to BBB penetration and nasal olfactory pathway into brain after IN administration, and simultaneously decrease the hepatic first pass metabolism and gastrointestinal adverse effects. Copyright © 2013 Elsevier B.V. All rights reserved.

Delivery of Dual Drug Loaded Lipid Based Nanoparticles across the Blood-Brain Barrier Impart Enhanced Neuroprotection in a Rotenone Induced Mouse Model of Parkinson's Disease.

PubMed

Kundu, Paromita; Das, Manasi; Tripathy, Kalpalata; Sahoo, Sanjeeb K

2016-12-21

Parkinson's disease (PD) is the most widespread form of dementia where there is an age related degeneration of dopaminergic neurons in the substantia nigra region of the brain. Accumulation of α-synuclein (αS) protein aggregate, mitochondrial dysfunction, oxidative stress, and neuronal cell death are the pathological hallmarks of PD. In this context, amalgamation of curcumin and piperine having profound cognitive properties, and antioxidant activity seems beneficial. However, the blood-brain barrier (BBB) is the major impediment for delivery of neurotherapeutics to the brain. The present study involves formulation of curcumin and piperine coloaded glyceryl monooleate (GMO) nanoparticles coated with various surfactants with a view to enhance the bioavailability of curcumin and penetration of both drugs to the brain tissue crossing the BBB and to enhance the anti-parkinsonism effect of both drugs in a single platform. In vitro results demonstrated augmented inhibition of αS protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative stress, and apoptosis, and activation of autophagic pathway by dual drug loaded NPs compared to native counterpart. Further, in vivo studies revealed that our formulated dual drug loaded NPs were able to cross BBB, rescued the rotenone induced motor coordination impairment, and restrained dopaminergic neuronal degeneration in a PD mouse model.

Freeze-drying of HI-6-loaded recombinant human serum albumin nanoparticles for improved storage stability.

PubMed

Dadparvar, Miriam; Wagner, Sylvia; Wien, Sascha; Worek, Franz; von Briesen, Hagen; Kreuter, Jörg

2014-10-01

Severe intoxications with organophosphates require the immediate administration of atropine in combination with acetyl cholinesterase (AChE) reactivators such as HI-6. Although this therapy regimen enables the treatment of peripheral symptoms, the blood-brain barrier (BBB) restricts the access of the hydrophilic antidotes to the central nervous system which could lead to a fatal respiratory arrest. Therefore, HI-6-loaded albumin nanoparticles were previously developed to enhance the transport across this barrier and were able to reactivate organophosphate-(OP)-inhibited AChE in an in vitro BBB model. Since HI-6 is known to be moisture-sensitive, the feasibility of freeze-drying of the HI-6-loaded nanoparticles was investigated in the present study using different cryo- and lyoprotectants at different concentrations. Trehalose and sucrose (3%, w/v)-containing formulations were superior to mannitol concerning the physicochemical parameters of the nanoparticles whereas trehalose-containing samples were subject of a prolonged storage stability study at temperatures between -20°C and +40°C for predetermined time intervals. Shelf-life computations of the freeze-dried HI-6 nanoparticle formulations revealed a shelf-life time of 18 months when stored at -20°C. The formulations' efficacy was proven in vitro by reactivation of OP-inhibited AChE after transport over a porcine brain capillary endothelial cell layer model. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of blood-brain barrier and blood-cerebrospinal fluid barrier permeability of 2-phenoxy-indan-1-one derivatives using in vitro cell models.

PubMed

Hu, Hai-Hong; Bian, Yi-Cong; Liu, Yao; Sheng, Rong; Jiang, Hui-Di; Yu, Lu-Shan; Hu, Yong-Zhou; Zeng, Su

2014-01-02

2-Phenoxy-indan-1-one derivatives (PIOs) are a series of novel central-acting cholinesterase inhibitors for the treatment of Alzheimer's disease (AD). The adequate distribution of PIOs to the central nervous system (CNS) is essential for its effectiveness. However, articles related with their permeability in terms of CNS penetration across the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) have not been found. This study was undertaken to evaluate the in vitro BBB and BCSFB transport of PIOs using Madin-Darby canine kidney (MDCK), MDCK-MDR1 and Z310 cell line models. As a result, the transepithelial transport of PIOs did not differ between MDCK and MDCK-MDR1, and the result suggested that PIOs were not substrates for P-gp, which means that multidrug resistance (MDR) function would not affect PIOs absorption and brain distribution. High permeability of PIOs in Z310 was found and it suggested that PIOs had high brain uptake potential. The experiment also showed that PIOs had inhibitory effects on the MDR1-mediated transport of Rhodamine123 with an IC50 value of 40-54 μM. And we suggested that 5,6-dimethoxy-1-indanone might be the pharmacophoric moiety of PIOs that interacts with the binding site of P-gp. Copyright © 2013 Elsevier B.V. All rights reserved.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption.

PubMed

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Mardor, Yael; Miklavcic, Damijan

2016-03-01

Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r(2) = 0.79; p < 0.008, r(2) = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup.

Development of magnetic resonance imaging based detection methods for beta amyloids via sialic acid-functionalized magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Kouyoumdjian, Hovig

The development of a non-invasive method for the detection of Alzheimer's disease is of high current interest, which can be critical in early diagnosis and in guiding preventive treatment of the disease. The aggregates of beta amyloids are a pathological hallmark of Alzheimer's disease. Carbohydrates such as sialic acid terminated gangliosides have been shown to play significant roles in initiation of amyloid aggregation. Herein, we report a biomimetic approach using sialic acid coated iron oxide superparamagnetic nanoparticles for in vitro detection in addition to the assessment of the in vivo mouse-BBB (Blood brain barrier) crossing of the BSA (bovine serum albumin)-modified ones. The sialic acid functionalized dextran nanoparticles were shown to bind with beta amyloids through several techniques including ELISA (enzyme linked immunosorbent assay), MRI (magnetic resonance imaging), TEM (transmission electron microscopy), gel electrophoresis and tyrosine fluorescence assay. The superparamagnetic nature of the nanoparticles allowed easy detection of the beta amyloids in mouse brains in both in vitro and ex vivo model by magnetic resonance imaging. Furthermore, the sialic acid nanoparticles greatly reduced beta amyloid induced cytotoxicity to SH-SY5Y neuroblastoma cells, highlighting the potential of the glyconanoparticles for detection and imaging of beta amyloids. Sialic acid functionalized BSA (bovine serum albumin) nanoparticles also showed significant binding to beta amyloids, through ELISA and ex vivo mouse brain MRI experiments. Alternatively, the BBB crossing was demonstrated by several techniques such as confocal microscopy, endocytosis, exocytosis assays and were affirmed by nanoparticles transcytosis assays through bEnd.3 endothelial cells. Finally, the BBB crossing was confirmed by analyzing the MRI signal of nanoparticle-injected CD-1 mice.

Functionalized liposomes and phytosomes loading Annona muricata L. aqueous extract: Potential nanoshuttles for brain-delivery of phenolic compounds.

PubMed

Mancini, Simona; Nardo, Luca; Gregori, Maria; Ribeiro, Inês; Mantegazza, Francesco; Delerue-Matos, Cristina; Masserini, Massimo; Grosso, Clara

2018-03-15

Multi-target drugs have gained significant recognition for the treatment of multifactorial diseases such as depression. Under a screening study of multi-potent medicinal plants with claimed antidepressant-like activity, the phenolic-rich Annona muricata aqueous extract (AE) emerged as a moderate monoamine oxidase A (hMAO-A) inhibitor and a strong hydrogen peroxide (H 2 O 2 ) scavenger. In order to protect this extract from gastrointestinal biotransformation and to improve its permeability across the blood-brain barrier (BBB), four phospholipid nanoformulations of liposomes and phytosomes functionalized with a peptide ligand promoting BBB crossing were produced. AE and nanoformulations were characterized by HPLC-DAD-ESI-MS n , HPLC-DAD, spectrophotometric, fluorescence and dynamic light scattering methods. Cytotoxicity and permeability studies were carried out using an in vitro transwell model of the BBB, composed of immortalized human microvascular endothelial cells (hCMEC/D3), and in vitro hMAO-A inhibition and H 2 O 2 scavenging activities were performed with all samples. The encapsulation/binding of AE was more efficient with phytosomes, while liposomes were more stable, displaying a slower extract release over time. In general, phytosomes were less toxic than liposomes in hCMEC/D3 cells and, when present, cholesterol improved the permeability across the cell monolayer of all tested nanoformulations. All nanoformulations conserved the antioxidant potential of AE, while phosphatidylcholine interfered with MAO-A inhibition assay. Overall, phytosome formulations registered the best performance in terms of binding efficiency, enzyme inhibition and scavenging activity, thus representing a promising multipotent phenolic-rich nanoshuttle for future in vivo depression treatment. Copyright © 2018 Elsevier GmbH. All rights reserved.

Integrating in Silico and in Vitro Approaches To Predict Drug Accessibility to the Central Nervous System.

PubMed

Zhang, Yan-Yan; Liu, Houfu; Summerfield, Scott G; Luscombe, Christopher N; Sahi, Jasminder

2016-05-02

Estimation of uptake across the blood-brain barrier (BBB) is key to designing central nervous system (CNS) therapeutics. In silico approaches ranging from physicochemical rules to quantitative structure-activity relationship (QSAR) models are utilized to predict potential for CNS penetration of new chemical entities. However, there are still gaps in our knowledge of (1) the relationship between marketed human drug derived CNS-accessible chemical space and preclinical neuropharmacokinetic (neuroPK) data, (2) interpretability of the selected physicochemical descriptors, and (3) correlation of the in vitro human P-glycoprotein (P-gp) efflux ratio (ER) and in vivo rodent unbound brain-to-blood ratio (Kp,uu), as these are assays routinely used to predict clinical CNS exposure, during drug discovery. To close these gaps, we explored the CNS druglike property boundaries of 920 market oral drugs (315 CNS and 605 non-CNS) and 846 compounds (54 CNS drugs and 792 proprietary GlaxoSmithKline compounds) with available rat Kp,uu data. The exact permeability coefficient (Pexact) and P-gp ER were determined for 176 compounds from the rat Kp,uu data set. Receiver operating characteristic curves were performed to evaluate the predictive power of human P-gp ER for rat Kp,uu. Our data demonstrates that simple physicochemical rules (most acidic pKa ≥ 9.5 and TPSA < 100) in combination with P-gp ER < 1.5 provide mechanistic insights for filtering BBB permeable compounds. For comparison, six classification modeling methods were investigated using multiple sets of in silico molecular descriptors. We present a random forest model with excellent predictive power (∼0.75 overall accuracy) using the rat neuroPK data set. We also observed good concordance between the structural interpretation results and physicochemical descriptor importance from the Kp,uu classification QSAR model. In summary, we propose a novel, hybrid in silico/in vitro approach and an in silico screening model for the effective development of chemical series with the potential to achieve optimal CNS exposure.

A method to predict different mechanisms for blood-brain barrier permeability of CNS activity compounds in Chinese herbs using support vector machine.

PubMed

Jiang, Ludi; Chen, Jiahua; He, Yusu; Zhang, Yanling; Li, Gongyu

2016-02-01

The blood-brain barrier (BBB), a highly selective barrier between central nervous system (CNS) and the blood stream, restricts and regulates the penetration of compounds from the blood into the brain. Drugs that affect the CNS interact with the BBB prior to their target site, so the prediction research on BBB permeability is a fundamental and significant research direction in neuropharmacology. In this study, we combed through the available data and then with the help of support vector machine (SVM), we established an experiment process for discovering potential CNS compounds and investigating the mechanisms of BBB permeability of them to advance the research in this field four types of prediction models, referring to CNS activity, BBB permeability, passive diffusion and efflux transport, were obtained in the experiment process. The first two models were used to discover compounds which may have CNS activity and also cross the BBB at the same time; the latter two were used to elucidate the mechanism of BBB permeability of those compounds. Three optimization parameter methods, Grid Search, Genetic Algorithm (GA), and Particle Swarm Optimization (PSO), were used to optimize the SVM models. Then, four optimal models were selected with excellent evaluation indexes (the accuracy, sensitivity and specificity of each model were all above 85%). Furthermore, discrimination models were utilized to study the BBB properties of the known CNS activity compounds in Chinese herbs and this may guide the CNS drug development. With the relatively systematic and quick approach, the application rationality of traditional Chinese medicines for treating nervous system disease in the clinical practice will be improved.

Region-specific disruption of the blood-brain barrier following repeated inflammatory dural stimulation in a rat model of chronic trigeminal allodynia

PubMed Central

Fried, Nathan T; Maxwell, Christina R; Elliott, Melanie B; Oshinsky, Michael L

2017-01-01

Background The blood-brain barrier (BBB) has been hypothesized to play a role in migraine since the late 1970s. Despite this, limited investigation of the BBB in migraine has been conducted. We used the inflammatory soup rat model of trigeminal allodynia, which closely mimics chronic migraine, to determine the impact of repeated dural inflammatory stimulation on BBB permeability. Methods The sodium fluorescein BBB permeability assay was used in multiple brain regions (trigeminal nucleus caudalis (TNC), periaqueductal grey, frontal cortex, sub-cortex, and cortex directly below the area of dural activation) during the episodic and chronic stages of repeated inflammatory dural stimulation. Glial activation was assessed in the TNC via GFAP and OX42 immunoreactivity. Minocycline was tested for its ability to prevent BBB disruption and trigeminal sensitivity. Results No astrocyte or microglial activation was found during the episodic stage, but BBB permeability and trigeminal sensitivity were increased. Astrocyte and microglial activation, BBB permeability, and trigeminal sensitivity were increased during the chronic stage. These changes were only found in the TNC. Minocycline treatment prevented BBB permeability modulation and trigeminal sensitivity during the episodic and chronic stages. Discussion Modulation of BBB permeability occurs centrally within the TNC following repeated dural inflammatory stimulation and may play a role in migraine. PMID:28457145

Increased miR-21-3p in injured brain microvascular endothelial cells following traumatic brain injury aggravates blood-brain barrier damage by promoting cellular apoptosis and inflammation through targeting MAT2B.

PubMed

Ge, Xintong; Li, Wenzhu; Huang, Shan; Yin, Zhenyu; Yang, Mengchen; Han, Zhenying; Han, Zhaoli; Chen, Fanglian; Wang, Haichen; Lei, Ping; Zhang, Jian-Ning

2018-04-26

Our recent papers have reported that increased miR-21-5p in brain following traumatic brain injury (TBI) could improve the neurological outcome through alleviating blood-brain barrier (BBB) damage. miR-21-3p is another mature miRNA derived from pre-miR-21 after Dicer Procession other than miR-21-5p. Its roles in various diseases, such as tumors and myocardial disease aroused great interest for research in recent years. To further explore the function and underlying mechanism of miR-21, especially miR-21-3p in regulating the pathological development of BBB damage after TBI, we designed this research and focused on studying the impact of miR-21-3p on apoptosis and inflammation in brain microvascular endothelial cells (BMVECs), the major cellular component of BBB. We performed controlled cortical impact on mouse brain, and employed the oxygen glucose deprivation/reoxygenation (OGD)-treated bEnd.3 cells injury model. We found that miR-21-3p level in BMVECs from injured cerebral cortex of controlled cortical impact (CCI) mice, and bEnd.3 cells with OGD treatment were both increased after injury. For in-vitro experiments, downregulation on miR-21-3p level by transfecting miR-21-3p antagomir in cultured cells alleviated OGD-induced BBB damage, characterized by decreased BBB leakage and increased expression of tight junction proteins. Besides, miR-21-3p antagomir could suppress cell death by anti-apoptosis, and control inflammatory response by inhibiting the activity of NF-κB signaling. Using luciferase reporter assay and a MAT2B-silenced shRNA vector, we further proved that miR-21-3p exerted above functions through targeting MAT2B. In addition, in-vivo experiments also confirmed that intracerebroventricular infusion of miR-21-3p antagomir could alleviate BBB leakage after TBI. It reduced Evans Blue extravasation and promoted the expression of tight junction proteins, thus contributed to improve the neurological outcome of CCI mice. Taken together, increased miR-21-3p in BMVECs after TBI was bad for restoration of injured BBB. Downregulation on miR-21-3p level in injured brain could be a promising therapeutic strategy for BBB damage after TBI.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption

PubMed Central

Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Miklavcic, Damijan

2016-01-01

Background Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Material and methods Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Results Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r2 = 0.79; p < 0.008, r2 = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. Conclusions The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup. PMID:27069447

Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers.

PubMed

Lin, Mei-Na; Shang, De-Shu; Sun, Wei; Li, Bo; Xu, Xin; Fang, Wen-Gang; Zhao, Wei-Dong; Cao, Liu; Chen, Yu-Hua

2013-06-04

Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

B7-H1 shapes T-cell-mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity.

PubMed

Klotz, Luisa; Kuzmanov, Ivan; Hucke, Stephanie; Gross, Catharina C; Posevitz, Vilmos; Dreykluft, Angela; Schulte-Mecklenbeck, Andreas; Janoschka, Claudia; Lindner, Maren; Herold, Martin; Schwab, Nicholas; Ludwig-Portugall, Isis; Kurts, Christian; Meuth, Sven G; Kuhlmann, Tanja; Wiendl, Heinz

2016-10-11

Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood-brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4 + T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity.

Synthesis and Evaluation of Neuroprotective Selenoflavanones

PubMed Central

Choi, Yong-Sung; Kim, Dong-Myung; Kim, Yoon-Jung; Yang, Sai; Lee, Kyung-Tae; Ryu, Jong Hoon; Jeong, Jin-Hyun

2015-01-01

The physicochemical properties and antioxidant activity of a molecule could be improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones showed improved physicochemical properties, suggestive of the ability to pass through the blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse model were significantly reduced by the selenoflavanone treatments. PMID:26690420

Cytokine-mediated dysregulation of zonula occludens-1 properties in human brain microvascular endothelium.

PubMed

Rochfort, Keith D; Cummins, Philip M

2015-07-01

Zonula occludens-1 (ZO-1) is essential to the proper assembly of interendothelial junction complexes that control blood-brain barrier (BBB) integrity. The goal of the current paper was to improve our understanding of how proinflammatory cytokines modulate ZO-1 properties within the human BBB microvascular endothelium. In this respect, we investigated the effects of TNF-α and IL-6 on ZO-1 using human brain microvascular endothelial cells (HBMvECs). Following treatment of HBMvECs with either cytokine (0-100 ng/ml, 18 h), we observed significantly decreased ZO-1 expression and ZO-1:occludin co-association, in parallel with increased ZO-1 phosphorylation (pTyr and pThr). All effects were dose-dependent. Either cytokine also caused extensive cell-cell border delocalization of ZO-1 in parallel with elevated HBMvEC permeability. Furthermore, pre-treatment of HBMvECs with antioxidants (superoxide dismutase, catalase, apocynin, N-acetylcysteine), or employing targeted inhibition of NADPH oxidase activation (NSC23766, gp91/p47 siRNA), were all found to comparably attenuate the cytokine-dependent decrease in ZO-1 protein expression. In summary, we present an in vitro model of how different proinflammatory cytokines can dysregulate ZO-1 properties in HBMvECs. A causal role for NADPH oxidase activation and oxidant signalling is also confirmed. Our findings add mechanistic depth to current in vivo models of BBB injury manifesting ZO-1 dysregulation. Copyright © 2015 Elsevier Inc. All rights reserved.

Cryptococcal transmigration across a model brain blood-barrier: evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265.

PubMed

Sorrell, Tania C; Juillard, Pierre-Georges; Djordjevic, Julianne T; Kaufman-Francis, Keren; Dietmann, Anelia; Milonig, Alban; Combes, Valery; Grau, Georges E R

2016-01-01

Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-γ stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Microbiome-host systems interactions: protective effects of propionate upon the blood-brain barrier.

PubMed

Hoyles, Lesley; Snelling, Tom; Umlai, Umm-Kulthum; Nicholson, Jeremy K; Carding, Simon R; Glen, Robert C; McArthur, Simon

2018-03-21

Gut microbiota composition and function are symbiotically linked with host health and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognised mechanisms exist by which the microbiome influences the gut-brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesised interactions between circulating gut-derived microbial metabolites, and the blood-brain barrier (BBB) also contribute to the gut-brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed. After demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 μM) on BBB properties in vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signalling. Together, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut-brain axis that warrants further attention.

Α-aryl-N-alkyl nitrones, as potential agents for stroke treatment: synthesis, theoretical calculations, antioxidant, anti-inflammatory, neuroprotective, and brain-blood barrier permeability properties.

PubMed

Chioua, Mourad; Sucunza, David; Soriano, Elena; Hadjipavlou-Litina, Dimitra; Alcázar, Alberto; Ayuso, Irene; Oset-Gasque, María Jesús; González, María Pilar; Monjas, Leticia; Rodríguez-Franco, María Isabel; Marco-Contelles, José; Samadi, Abdelouahid

2012-01-12

We report the synthesis, theoretical calculations, the antioxidant, anti-inflammatory, and neuroprotective properties, and the ability to cross the blood-brain barrier (BBB) of (Z)-α-aryl and heteroaryl-N-alkyl nitrones as potential agents for stroke treatment. The majority of nitrones compete with DMSO for hydroxyl radicals, and most of them are potent lipoxygenase inhibitors. Cell viability-related (MTT assay) studies clearly showed that nitrones 1-3 and 10 give rise to significant neuroprotection. When compounds 1-11 were tested for necrotic cell death (LDH release test) nitrones 1-3, 6, 7, and 9 proved to be neuroprotective agents. In vitro evaluation of the BBB penetration of selected nitrones 1, 2, 10, and 11 using the PAMPA-BBB assay showed that all of them cross the BBB. Permeable quinoline nitrones 2 and 3 show potent combined antioxidant and neuroprotective properties and, therefore, can be considered as new lead compounds for further development in specific tests for potential stroke treatment.

Decreased levels of active uPA and KLK8 assessed by [111 In]MICA-401 binding correlate with the seizure burden in an animal model of temporal lobe epilepsy.

PubMed

Missault, Stephan; Peeters, Lore; Amhaoul, Halima; Thomae, David; Van Eetveldt, Annemie; Favier, Barbara; Thakur, Anagha; Van Soom, Jeroen; Pitkänen, Asla; Augustyns, Koen; Joossens, Jurgen; Staelens, Steven; Dedeurwaerdere, Stefanie

2017-09-01

Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [ 111 In]MICA-401. As the first step in exploring the applicability of [ 111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [ 111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). In the KASE model, in vitro autoradiography with [ 111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. At 7 days post-SE, in vitro autoradiography revealed significantly decreased [ 111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [ 111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [ 111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. Strong association of reduced [ 111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [ 111 In]MICA-401 currently limits its application in vivo. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Pharmacokinetics of pericyte involvement in small-molecular drug transport across the blood-brain barrier.

PubMed

Mihajlica, Nebojsa; Betsholtz, Christer; Hammarlund-Udenaes, Margareta

2018-06-19

Pericytes are perivascular cells that play important roles in the regulation of the blood-brain barrier (BBB) properties. Pericyte-deficiency causes compromised BBB integrity and increase in permeability to different macromolecules mainly by upregulated transcytosis. The aim of the present study was to investigate pericyte involvement in the extent of small-molecular drug transport across the BBB. This was performed with five compounds: diazepam, digoxin, levofloxacin, oxycodone and paliperidone. Compounds were administered at low doses via subcutaneous injections as a cassette (simultaneously) to pericyte-deficient Pdgfb ret/ret mice and corresponding WT controls. Total drug partitioning across the BBB was calculated as the ratio of total drug exposures in brain tissue and plasma (K p,brain ). In addition, equilibrium dialysis experiments were performed to estimate unbound drug fractions in brain (f u,brain ) and plasma (f u,plasma ). This enabled estimation of unbound drug partitioning coefficients (K p,uu,brain ). The results indicated slight tendencies towards increase of total brain exposures in Pdgfb ret/ret mice as reflected in K p,brain values, which were within the 2-fold limit. Part of these differences could be explained by differences in plasma protein binding. No difference was found in brain tissue binding. The combined in vivo and in vitro data resulted in no differences in BBB transport in pericyte-deficiency, as described by similar K p,uu,brain values in Pdgfb ret/ret and control mice. In conclusion, these findings imply no influence of pericytes on the extent of BBB transport of small-molecular drugs, and suggest preserved BBB features relevant for handling of this type of molecules irrespective of pericyte presence at the brain endothelium. Copyright © 2018. Published by Elsevier B.V.

Neutral amino acid transport across brain microvessel endothelial cell monolayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Audus, K.L.; Borchardt, R.T.

1986-03-01

Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional,more » and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.« less

N-Acetylcysteine amide protects against methamphetamine-induced oxidative stress and neurotoxicity in immortalized human brain endothelial cells.

PubMed

Zhang, Xinsheng; Banerjee, Atrayee; Banks, William A; Ercal, Nuran

2009-06-12

Oxidative stress plays an important role in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Methamphetamine (METH) is an amphetamine analog that causes degeneration of the dopaminergic system in mammals and subsequent oxidative stress. In our present study, we have used immortalized human brain microvascular endothelial (HBMVEC) cells to test whether N-acetylcysteine amide (NACA), a novel antioxidant, prevents METH-induced oxidative stress in vitro. Our studies showed that NACA protects against METH-induced oxidative stress in HBMVEC cells. NACA significantly protected the integrity of our blood brain barrier (BBB) model, as shown by permeability and trans-endothelial electrical resistance (TEER) studies. NACA also significantly increased the levels of intracellular glutathione (GSH) and glutathione peroxidase (GPx). Malondialdehyde (MDA) levels increased dramatically after METH exposure, but this increase was almost completely prevented when the cells were treated with NACA. Generation of reactive oxygen species (ROS) also increased after METH exposure, but was reduced to control levels with NACA treatment, as measured by dichlorofluorescin (DCF). These results suggest that NACA protects the BBB integrity in vitro, which could prevent oxidative stress-induced damage; therefore, the effectiveness of this antioxidant should be evaluated for the treatment of neurodegenerative diseases in the future.

Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier.

PubMed

Vatine, Gad D; Al-Ahmad, Abraham; Barriga, Bianca K; Svendsen, Soshana; Salim, Ariel; Garcia, Leslie; Garcia, Veronica J; Ho, Ritchie; Yucer, Nur; Qian, Tongcheng; Lim, Ryan G; Wu, Jie; Thompson, Leslie M; Spivia, Weston R; Chen, Zhaohui; Van Eyk, Jennifer; Palecek, Sean P; Refetoff, Samuel; Shusta, Eric V; Svendsen, Clive N

2017-06-01

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy. Copyright © 2017 Elsevier Inc. All rights reserved.

A Genetic Algorithm Based Support Vector Machine Model for Blood-Brain Barrier Penetration Prediction

PubMed Central

Zhang, Daqing; Xiao, Jianfeng; Zhou, Nannan; Luo, Xiaomin; Jiang, Hualiang; Chen, Kaixian

2015-01-01

Blood-brain barrier (BBB) is a highly complex physical barrier determining what substances are allowed to enter the brain. Support vector machine (SVM) is a kernel-based machine learning method that is widely used in QSAR study. For a successful SVM model, the kernel parameters for SVM and feature subset selection are the most important factors affecting prediction accuracy. In most studies, they are treated as two independent problems, but it has been proven that they could affect each other. We designed and implemented genetic algorithm (GA) to optimize kernel parameters and feature subset selection for SVM regression and applied it to the BBB penetration prediction. The results show that our GA/SVM model is more accurate than other currently available log BB models. Therefore, to optimize both SVM parameters and feature subset simultaneously with genetic algorithm is a better approach than other methods that treat the two problems separately. Analysis of our log BB model suggests that carboxylic acid group, polar surface area (PSA)/hydrogen-bonding ability, lipophilicity, and molecular charge play important role in BBB penetration. Among those properties relevant to BBB penetration, lipophilicity could enhance the BBB penetration while all the others are negatively correlated with BBB penetration. PMID:26504797

Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

PubMed

Pelisch, Nicolas; Dan, Takashi; Ichimura, Atsuhiko; Sekiguchi, Hiroki; Vaughan, Douglas E; van Ypersele de Strihou, Charles; Miyata, Toshio

2015-01-01

Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

Modeling the role of microglia during neurovascular development (WC10)

EPA Science Inventory

Microglia, resident brain macrophages, have important roles in blood-brain barrier (BBB) development and during focal BBB disruption. We reconstructed these complex dynamics utilizing computational and molecular methods: 1) constructing a systems map of BBB development from known...

[Repair of spinal cord injury with rats' umbilical cord MSCs].

PubMed

Zhu, Yuhai; Feng, Shiqing; Wang, Xue

2009-12-01

To study the growth characteristics of umbilical cord MSCs (UCMSCs) in vitro and its effect on the nerve regeneration after spinal cord injury (SCI). UCMSCs isolated from pregnant rats umbilical cord were cultured and purified in vitro. Sixty female Wistar rats weighing (300 +/- 10) g were randomized into three groups (n=20 per group). UCMSCs group (group A) in which UCMSCs suspension injection was conducted; DMEM control group (group B) in which 10% DMEM injection was conducted; sham group (group C) in which the animal received laminectomy only. Establish acute SCI model (T10) by Impactor model-II device in group A and group B. The recovery of the lower extremity was observed using BBB locomotor scoring system, neurofilament 200 (NF-200) immunofluorescence staining was performed to detect the neural regeneration, and then the corticospinal tract (CST) was observed using the biotinylated dextran amine (BDA) tracing. Cultured UCMSCs were spindle-shaped fibrocyte-like adherent growth, swirling or parallelly. The USMSCs expressed CD29, but not CD31, CD45, and HLA-DR. The BBB score was higher in group A than group B 4, 5, and 6 weeks after operation, and there was a significant difference between two groups (P < 0.05). The BBB scores at different time points were significantly lower in groups A and B than that in group C (P < 0.05). UCMSCs was proved to survive and assemble around the injured place by frozen section of the cords 6 weeks after injury. NF-200 positive response area in groups A, B, and C was (11,943 +/- 856), (7,986 +/- 627), and (13,117 +/- 945) pixels, respectively, suggesting there was a significant difference between groups A, C and group B (P < 0.05), and no significant difference was evident between group A and group C (P > 0.05). BDA anterograde tracing 10 weeks after operation demonstrated that more regenerated nerve fibers went through injured area in group A, but just quite few nerve fibers in group B went through the injuried cavity. The ratios of regenerative axons amount to T5 axons in group A and group B were smaller than that of group C (P < 0.05). UCMSCs can proliferate rapidly in vitro, survive and differentiate to neurons after being grafted into injured spinal cord. The transplantation of UCMSCs is effective in promoting functional recovery and axonal regeneration after SCI.

MRI measurements of Blood-Brain Barrier function in dementia: A review of recent studies.

PubMed

Raja, Rajikha; Rosenberg, Gary A; Caprihan, Arvind

2018-05-15

Blood-brain barrier (BBB) separates the systemic circulation and the brain, regulating transport of most molecules to protect the brain microenvironment. Multiple structural and functional components preserve the integrity of the BBB. Several imaging modalities are available to study disruption of the BBB. However, the subtle changes in BBB leakage that occurs in vascular cognitive impairment and Alzheimer's disease have been less well studied. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is the most widely adopted non-invasive imaging technique for evaluating BBB breakdown. It is used as a significant marker for a wide variety of diseases with large permeability leaks, such as brain tumors and multiple sclerosis, to more subtle disruption in chronic vascular disease and dementia. DCE-MRI analysis of BBB includes both model-free parameters and quantitative parameters using pharmacokinetic modelling. We review MRI studies of BBB breakdown in dementia. The challenges in measuring subtle BBB changes and the state of the art techniques are initially examined. Subsequently, a systematic review comparing methodologies from recent in-vivo MRI studies is presented. Various factors related to subtle BBB permeability measurement such as DCE-MRI acquisition parameters, arterial input assessment, T 1 mapping and data analysis methods are reviewed with the focus on finding the optimal technique. Finally, the reported BBB permeability values in dementia are compared across different studies and across various brain regions. We conclude that reliable measurement of low-level BBB permeability across sites remains a difficult problem and a standardization of the methodology for both data acquisition and quantitative analysis is required. This article is part of the Special Issue entitled 'Cerebral Ischemia'. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developing Enhanced Blood–Brain Barrier Permeability Models: Integrating External Bio-Assay Data in QSAR Modeling

PubMed Central

Wang, Wenyi; Kim, Marlene T.; Sedykh, Alexander

2015-01-01

Purpose Experimental Blood–Brain Barrier (BBB) permeability models for drug molecules are expensive and time-consuming. As alternative methods, several traditional Quantitative Structure-Activity Relationship (QSAR) models have been developed previously. In this study, we aimed to improve the predictivity of traditional QSAR BBB permeability models by employing relevant public bio-assay data in the modeling process. Methods We compiled a BBB permeability database consisting of 439 unique compounds from various resources. The database was split into a modeling set of 341 compounds and a validation set of 98 compounds. Consensus QSAR modeling workflow was employed on the modeling set to develop various QSAR models. A five-fold cross-validation approach was used to validate the developed models, and the resulting models were used to predict the external validation set compounds. Furthermore, we used previously published membrane transporter models to generate relevant transporter profiles for target compounds. The transporter profiles were used as additional biological descriptors to develop hybrid QSAR BBB models. Results The consensus QSAR models have R2=0.638 for fivefold cross-validation and R2=0.504 for external validation. The consensus model developed by pooling chemical and transporter descriptors showed better predictivity (R2=0.646 for five-fold cross-validation and R2=0.526 for external validation). Moreover, several external bio-assays that correlate with BBB permeability were identified using our automatic profiling tool. Conclusions The BBB permeability models developed in this study can be useful for early evaluation of new compounds (e.g., new drug candidates). The combination of chemical and biological descriptors shows a promising direction to improve the current traditional QSAR models. PMID:25862462

Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.

PubMed

Chaitanya, Ganta V; Cromer, Walter E; Wells, Shannon R; Jennings, Merilyn H; Couraud, P Olivier; Romero, Ignacio A; Weksler, Babette; Erdreich-Epstein, Anat; Mathis, J Michael; Minagar, Alireza; Alexander, J Steven

2011-11-23

The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs). © 2011 Chaitanya et al; licensee BioMed Central Ltd.

Ginsenoside Rg1 nanoparticle penetrating the blood-brain barrier to improve the cerebral function of diabetic rats complicated with cerebral infarction.

PubMed

Shen, Junyi; Zhao, Zhiming; Shang, Wei; Liu, Chunli; Zhang, Beibei; Zhao, Lingjie; Cai, Hui

2017-01-01

Diabetic cerebral infarction is with poorer prognosis and high rates of mortality. Ginsenoside Rg1 (Rg1) has a wide variety of therapeutic values for central nervous system (CNS) diseases for the neuron protective effects. However, the blood-brain barrier (BBB) restricts Rg1 in reaching the CNS. In this study, we investigated the therapeutic effects of Rg1 nanoparticle (PHRO, fabricated with γ-PGA, L-PAE (H), Rg1, and OX26 antibody), targeting transferrin receptor, on the diabetes rats complicated with diabetic cerebral infarction in vitro and in vivo. Dynamic light scattering analysis shows the average particle size of PHRO was 79±18 nm and the polydispersity index =0.18. The transmission electron microscope images showed that all NPs were spherical in shape with diameters of 89±23 nm. PHRO released Rg1 with sustained release manner and could promote the migration of cerebrovascular endothelial cells and tube formation and even penetrated the BBB in vitro. PHRO could penetrate the BBB with high concentration in brain tissue to reduce the cerebral infarction volume and promote neuronal recovery in vivo. PHRO was promising to be a clinical treatment of diabetes mellitus with cerebral infarction.

Ginsenoside Rg1 nanoparticle penetrating the blood–brain barrier to improve the cerebral function of diabetic rats complicated with cerebral infarction

PubMed Central

Shen, Junyi; Zhao, Zhiming; Shang, Wei; Liu, Chunli; Zhang, Beibei; Zhao, Lingjie; Cai, Hui

2017-01-01

Diabetic cerebral infarction is with poorer prognosis and high rates of mortality. Ginsenoside Rg1 (Rg1) has a wide variety of therapeutic values for central nervous system (CNS) diseases for the neuron protective effects. However, the blood–brain barrier (BBB) restricts Rg1 in reaching the CNS. In this study, we investigated the therapeutic effects of Rg1 nanoparticle (PHRO, fabricated with γ-PGA, L-PAE (H), Rg1, and OX26 antibody), targeting transferrin receptor, on the diabetes rats complicated with diabetic cerebral infarction in vitro and in vivo. Dynamic light scattering analysis shows the average particle size of PHRO was 79±18 nm and the polydispersity index =0.18. The transmission electron microscope images showed that all NPs were spherical in shape with diameters of 89±23 nm. PHRO released Rg1 with sustained release manner and could promote the migration of cerebrovascular endothelial cells and tube formation and even penetrated the BBB in vitro. PHRO could penetrate the BBB with high concentration in brain tissue to reduce the cerebral infarction volume and promote neuronal recovery in vivo. PHRO was promising to be a clinical treatment of diabetes mellitus with cerebral infarction. PMID:28919749

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport.

PubMed

Veszelka, Szilvia; Tóth, András; Walter, Fruzsina R; Tóth, Andrea E; Gróf, Ilona; Mészáros, Mária; Bocsik, Alexandra; Hellinger, Éva; Vastag, Monika; Rákhely, Gábor; Deli, Mária A

2018-01-01

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

Antiamyloidogenic Activity of Aβ42-Binding Peptoid in Modulating Amyloid Oligomerization.

PubMed

Zhao, Zijian; Zhu, Ling; Li, Haiyun; Cheng, Peng; Peng, Jiaxi; Yin, Yudan; Yang, Yang; Wang, Chen; Hu, Zhiyuan; Yang, Yanlian

2017-01-01

The oligomerization and aggregation of amyloid β (Aβ) play central role in the pathogenesis of Alzheimer's disease (AD). Molecular binding agents for modulating the formation of Aβ oligomers and fibrils have promising application potential in AD therapies. By screening a peptoid library using surface plasmon resonance imaging, amyloid inhibitory peptoid 1 (AIP1) that has high affinity to Aβ42 is identified. AIP1 is demonstrated to inhibit Aβ42 oligomerization and fibrillation and to rescue Aβ42-induced cytotoxicity through decreasing the content of Aβ42 oligomers that is related to cell membrane permeability. Molecular docking suggests that the binding sites of AIP1 may be at the N-terminus of Aβ42. The blood-brain barrier (BBB) permeability of AIP1 using an in vitro BBB model is also revealed. This work provides a strategy for the design and development of peptoid-based antiamyloidogenic agents. The obtained amyloid inhibitory peptoid shows prospects in the therapeutic application in AD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel hits for acetylcholinesterase inhibition derived by docking-based screening on ZINC database.

PubMed

Doytchinova, Irini; Atanasova, Mariyana; Valkova, Iva; Stavrakov, Georgi; Philipova, Irena; Zhivkova, Zvetanka; Zheleva-Dimitrova, Dimitrina; Konstantinov, Spiro; Dimitrov, Ivan

2018-12-01

The inhibition of the enzyme acetylcholinesterase (AChE) increases the levels of the neurotransmitter acetylcholine and symptomatically improves the affected cognitive function. In the present study, we searched for novel AChE inhibitors by docking-based virtual screening of the standard lead-like set of ZINC database containing more than 6 million small molecules using GOLD software. The top 10 best-scored hits were tested in vitro for AChE affinity, neurotoxicity, GIT and BBB permeability. The main pharmacokinetic parameters like volume of distribution, free fraction in plasma, total clearance, and half-life were predicted by previously derived models. Nine of the compounds bind to the enzyme with affinities from 0.517 to 0.735 µM, eight of them are non-toxic. All hits permeate GIT and BBB and bind extensively to plasma proteins. Most of them are low-clearance compounds. In total, seven of the 10 hits are promising for further lead optimisation. These are structures with ZINC IDs: 00220177, 44455618, 66142300, 71804814, 72065926, 96007907, and 97159977.

Development of magneto-plasmonic nanoparticles for multimodal image-guided therapy to the brain.

PubMed

Tomitaka, Asahi; Arami, Hamed; Raymond, Andrea; Yndart, Adriana; Kaushik, Ajeet; Jayant, Rahul Dev; Takemura, Yasushi; Cai, Yong; Toborek, Michal; Nair, Madhavan

2017-01-05

Magneto-plasmonic nanoparticles are one of the emerging multi-functional materials in the field of nanomedicine. Their potential for targeting and multi-modal imaging is highly attractive. In this study, magnetic core/gold shell (MNP@Au) magneto-plasmonic nanoparticles were synthesized by citrate reduction of Au ions on magnetic nanoparticle seeds. Hydrodynamic size and optical properties of magneto-plasmonic nanoparticles synthesized with the variation of Au ions and reducing agent concentrations were evaluated. The synthesized magneto-plasmonic nanoparticles exhibited superparamagnetic properties, and their magnetic properties contributed to the concentration-dependent contrast in magnetic resonance imaging (MRI). The imaging contrast from the gold shell part of the magneto-plasmonic nanoparticles was also confirmed by X-ray computed tomography (CT). The transmigration study of the magneto-plasmonic nanoparticles using an in vitro blood-brain barrier (BBB) model proved enhanced transmigration efficiency without disrupting the integrity of the BBB, and showed potential to be used for brain diseases and neurological disorders.

Enhanced Delivery of Gold Nanoparticles with Therapeutic Potential for Targeting Human Brain Tumors

NASA Astrophysics Data System (ADS)

Etame, Arnold B.

The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti-cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non-viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti-cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport-permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP-based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size-dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport-permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport-permissive BBB. Given the significant delivery limitations through the non-transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB.

Adaptation of the Basso-Beattie-Bresnahan locomotor rating scale for use in a clinical model of spinal cord injury in dogs.

PubMed

Song, Rachel B; Basso, D Michele; da Costa, Ronaldo C; Fisher, Lesley C; Mo, Xiaokui; Moore, Sarah A

2016-08-01

Naturally occurring acute spinal cord injury (SCI) in pet dogs provides an important clinical animal model through which to confirm and extend findings from rodent studies; however, validated quantitative outcome measures for dogs are limited. We adapted the Basso Beattie Bresnahan (BBB) scale for use in a clinical dog model of acute thoracolumbar SCI. Based on observation of normal dogs, modifications were made to account for species differences in locomotion. Assessments of paw and tail position, and trunk stability were modified to produce a 19 point scale suitable for use in dogs, termed the canine BBB scale (cBBB). Pet dogs with naturally occurring acute SCI were assigned cBBB scores at 3, 10 and 30days after laminectomy. Scores assigned via the cBBB were stable across testing sessions in normal dogs but increased significantly between days 3 and 30 in SCI-affected dogs (p=0.0003). The scale was highly responsive to changes in locomotor recovery over a 30day period, with a standardized response mean of 1.34. Concurrent validity was good, with strong correlations observed between the cBBB and two other locomotor scales, the OSCIS (r=0.94; p<0.001) and the MFS (r=0.85; p<0.0001). cBBB scores inversely correlated with other assessments of recovery including mechanical sensory threshold (r=-0.68; p<0.0001) and coefficient of variation of stride length (r=-0.49; p<0.0001). These results support the use of the cBBB to assess locomotor recovery in canine clinical translational models of SCI. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of multidrug resistance protein (MRP-1) as an active efflux transporter on blood-brain barrier (BBB) permeability.

PubMed

Lingineni, Karthik; Belekar, Vilas; Tangadpalliwar, Sujit R; Garg, Prabha

2017-05-01

Drugs acting on central nervous system (CNS) may take longer duration to reach the market as these compounds have a higher attrition rate in clinical trials due to the complexity of the brain, side effects, and poor blood-brain barrier (BBB) permeability compared to non-CNS-acting compounds. The roles of active efflux transporters with BBB are still unclear. The aim of the present work was to develop a predictive model for BBB permeability that includes the MRP-1 transporter, which is considered as an active efflux transporter. A support vector machine model was developed for the classification of MRP-1 substrates and non-substrates, which was validated with an external data set and Y-randomization method. An artificial neural network model has been developed to evaluate the role of MRP-1 on BBB permeation. A total of nine descriptors were selected, which included molecular weight, topological polar surface area, ClogP, number of hydrogen bond donors, number of hydrogen bond acceptors, number of rotatable bonds, P-gp, BCRP, and MRP-1 substrate probabilities for model development. We identified 5 molecules that fulfilled all criteria required for passive permeation of BBB, but they all have a low logBB value, which suggested that the molecules were effluxed by the MRP-1 transporter.

Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.

PubMed

Li, Tengfei; Bourgeois, Jean-Pierre; Celli, Susanna; Glacial, Fabienne; Le Sourd, Anne-Marie; Mecheri, Salah; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Rougeon, François; Lafaye, Pierre

2012-10-01

Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

Serum-borne bioactivity caused by pulmonary multiwalled carbon nanotubes induces neuroinflammation via blood-brain barrier impairment.

PubMed

Aragon, Mario J; Topper, Lauren; Tyler, Christina R; Sanchez, Bethany; Zychowski, Katherine; Young, Tamara; Herbert, Guy; Hall, Pamela; Erdely, Aaron; Eye, Tracy; Bishop, Lindsey; Saunders, Samantha A; Muldoon, Pretal P; Ottens, Andrew K; Campen, Matthew J

2017-03-07

Pulmonary exposure to multiwalled carbon nanotubes (MWCNTs) causes indirect systemic inflammation through unknown pathways. MWCNTs translocate only minimally from the lungs into the systemic circulation, suggesting that extrapulmonary toxicity may be caused indirectly by lung-derived factors entering the circulation. To assess a role for MWCNT-induced circulating factors in driving neuroinflammatory outcomes, mice were acutely exposed to MWCNTs (10 or 40 µg/mouse) via oropharyngeal aspiration. At 4 h after MWCNT exposure, broad disruption of the blood-brain barrier (BBB) was observed across the capillary bed with the small molecule fluorescein, concomitant with reactive astrocytosis. However, pronounced BBB permeation was noted, with frank albumin leakage around larger vessels (>10 µm), overlain by a dose-dependent astroglial scar-like formation and recruitment of phagocytic microglia. As affirmed by elevated inflammatory marker transcription, MWCNT-induced BBB disruption and neuroinflammation were abrogated by pretreatment with the rho kinase inhibitor fasudil. Serum from MWCNT-exposed mice induced expression of adhesion molecules in primary murine cerebrovascular endothelial cells and, in a wound-healing in vitro assay, impaired cell motility and cytokinesis. Serum thrombospondin-1 level was significantly increased after MWCNT exposure, and mice lacking the endogenous receptor CD36 were protected from the neuroinflammatory and BBB permeability effects of MWCNTs. In conclusion, acute pulmonary exposure to MWCNTs causes neuroinflammatory responses that are dependent on the disruption of BBB integrity.

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease.

PubMed

Di Pardo, Alba; Castaldo, Salvatore; Capocci, Luca; Amico, Enrico; Vittorio, Maglione

2017-11-08

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In vitro characterization of transport and metabolism of the alkaloids: vincamine, vinpocetine and eburnamonine.

PubMed

Fandy, Tamer E; Abdallah, Inas; Khayat, Maan; Colby, David A; Hassan, Hazem E

2016-02-01

Vincamine, vinpocetine and eburnamonine are alkaloids known for their neuroprotective attributes, enhancement of cerebrovascular blood flow and antitumor effect of their derivatives. However, the relative metabolic stability of these alkaloids and their extrusion by the drug efflux transporters expressed at the blood-brain barrier (BBB) are not clear. In this study, we developed rapid and sensitive methods for the detection of these alkaloids and investigated their relative metabolic stability and their interaction with drug efflux transporters. UPLC methods were developed to analyze metabolic in vitro samples. Intrinsic clearance was determined using rat liver microsomal enzymes. Drug-stimulated transporter activity was estimated by measuring inorganic phosphate released from ATP spectrophotometrically. The UPLC methods quantification level ranged from 0.02 to 0.025 µg/mL, indicating high sensitivity. The intrinsic clearance of eburnamonine was significantly less than both vincamine and vinpocetine. Different concentrations of the three drugs (4, 20 and 100 µM) induced minimal stimulation of the ATPase activity of the Bcrp and Pgp membrane transporters. The developed simple, sensitive and reliable UPLC analysis methods can be utilized in future in vitro and in vivo studies. The three alkaloids demonstrated minimal interaction with the drug efflux transporters Pgp and Bcrp, concordant with the ability of these alkaloids to cross the BBB. The relative metabolic stability of eburnamonine compared to the other alkaloids suggests the use of eburnamonine or its derivatives as lead compounds for the development of antitumor and nootropic agents that need to cross the BBB and produce their pharmacological effects in the CNS.

Enhancement of transport of curcumin to brain in mice by poly( n-butylcyanoacrylate) nanoparticle

NASA Astrophysics Data System (ADS)

Sun, Min; Gao, Yan; Guo, Chenyu; Cao, Fengliang; Song, Zhimei; Xi, Yanwei; Yu, Aihua; Li, Aiguo; Zhai, Guangxi

2010-10-01

Curcumin, a widely used coloring agent and spice in food, has a potential in blocking brain tumor formation and curing Alzheimer's disease. Due to the specific properties of blood-brain barrier (BBB), only traces of curcumin were transported across BBB. The aim of the present study was to design and characterize curcumin loaded polybutylcyanoacrylate nanoparticles (PBCN) coated with polysorbate 80, and to evaluate the effect of PBCN as a delivery system on carrying curcumin across BBB. Curcumin loaded nanoparticles were prepared by an anionic polymerization method, and they presented in a core-shell spherical shape under transmission electron microscopy, with an average diameter of 152.0 nm. The average drug loading was 21.1%. Physicochemical status of curcumin in the nanoparticles was confirmed with differential scanning colorimetry and Fourier transform infrared spectroscopy. The in vitro release behavior of drug from the nanoparticles was fitted to a double phase kinetics model. The studies of pharmacokinetic and bio-distribution to brain were conducted in mice after intravenous administration of the nanoparticle formulation at the dose of 5 mg/kg and curcumin solution at the dose of 10 mg/kg via the tail vein. The results showed that in plasma, the area under concentration-time curve (AUC0-∞) for curcumin loaded nanoparticles was greater than that for the control solution, moreover, the mean residence time of curcumin loaded nanoparticles was 14-fold that of the control solution. In brain, AUC0-∞ for curcumin loaded nanoparticles was 2.53-fold that for the control solution. In conclusion, the present study demonstrated that PBCN could enhance the transport of curcumin to brain and have a potential as a delivery system to cross the BBB.

Na+/H+ Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation.

PubMed

Beydoun, Rami; Hamood, Mohamed A; Gomez Zubieta, Daniela M; Kondapalli, Kalyan C

2017-03-10

Iron is essential for brain function, with loss of iron homeostasis in the brain linked to neurological diseases ranging from rare syndromes to more common disorders, such as Parkinson's and Alzheimer's diseases. Iron entry into the brain is regulated by the blood-brain barrier (BBB). Molecular mechanisms regulating this transport are poorly understood. Using an in vitro model of the BBB, we identify NHE9, an endosomal cation/proton exchanger, as a novel regulator of this system. Human brain microvascular endothelial cells (hBMVECs) that constitute the BBB receive brain iron status information via paracrine signals from ensheathing astrocytes. In hBMVECs, we show that NHE9 expression is up-regulated very early in a physiological response invoked by paracrine signals from iron-starved astrocytes. Ectopic expression of NHE9 in hBMVECs without external cues induced up-regulation of the transferrin receptor (TfR) and down-regulation of ferritin, leading to an increase in iron uptake. Mechanistically, we demonstrate that NHE9 localizes to recycling endosomes in hBMVECs where it raises the endosomal pH. The ensuing alkalization of the endosomal lumen increased translocation of TfRs to the hBMVEC membrane. TfRs on the membrane were previously shown to facilitate both recycling-dependent and -independent iron uptake. We propose that NHE9 regulates TfR-dependent, recycling-independent iron uptake in hBMVECs by fine-tuning the endosomal pH in response to paracrine signals and is therefore an important regulator in iron mobilization pathway at the BBB. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Photoacoustic micro-imaging of focused ultrasound induced blood-brain-barrier opening in a rat model

NASA Astrophysics Data System (ADS)

Wang, Po-Hsun; Hsu, Po-Hung; Liu, Hao-Li; Wang, Churng-Ren Chris; Li, Meng-Lin

2010-02-01

Blood brain barrier (BBB) prevents most of the drug from transmitting into the brain tissue and decreases the treatment performance for brain disease. One of the methods to overcome the difficulty of drug delivery is to locally increase the permeability of BBB with high-intensity focused ultrasound. In this study, we have investigated the feasibility of photoacoustic microscopy of focused-ultrasound induced BBB opening in a rat model in vivo with gold nanorods (AuNRs) as a contrast agent. This study takes advantage of the strong near-infrared absorption of AuNRs and their extravasation tendency from BBB opening foci due to their nano-scale size. Before the experiments, craniotomy was performed on rats to provide a path for focused ultrasound beam. Localized BBB opening at the depth of about 3 mm from left cortex of rat brains was achieved by delivering 1.5 MHz focused ultrasound energy into brain tissue in the presence of microbubbles. PEGylated AuNRs with a peak optical absorption at ~800 nm were then intravenously administered. Pre-scan prior to BBB disruption and AuNR injection was taken to mark the signal background. After injection, the distribution of AuNRs in rat brains was monitored up to 2 hours. Experimental results show that imaging AuNRs reveals BBB disruption area in left brains while there are no changes observed in the right brains. From our results, photoacoustic imaging plus AuNRs shows the promise as a novel monitoring strategy in identifying the location and variation of focused-ultrasound BBB-opening in a rat model.

Gold-nanorod contrast-enhanced photoacoustic micro-imaging of focused-ultrasound induced blood-brain-barrier opening in a rat model

NASA Astrophysics Data System (ADS)

Wang, Po-Hsun; Liu, Hao-Li; Hsu, Po-Hung; Lin, Chia-Yu; Chris Wang, Churng-Ren; Chen, Pin-Yuan; Wei, Kuo-Chen; Yen, Tzu-Chen; Li, Meng-Lin

2012-06-01

In this study, we develop a novel photoacoustic imaging technique based on gold nanorods (AuNRs) for quantitatively monitoring focused-ultrasound (FUS) induced blood-brain barrier (BBB) opening in a rat model in vivo. This study takes advantage of the strong near-infrared absorption (peak at ~800 nm) of AuNRs and the extravasation tendency from BBB opening foci due to their nano-scale size to passively label the BBB disruption area. Experimental results show that AuNR contrast-enhanced photoacoustic microscopy (PAM) successfully reveals the spatial distribution and temporal response of BBB disruption area in the rat brains. The quantitative measurement of contrast enhancement has potential to estimate the local concentration of AuNRs and even the dosage of therapeutic molecules when AuNRs are further used as nano-carrier for drug delivery or photothermal therapy. The photoacoustic results also provide complementary information to MRI, being helpful to discover more details about FUS induced BBB opening in small animal models.

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells

PubMed Central

Bleau, Christian; Filliol, Aveline; Samson, Michel

2015-01-01

ABSTRACT Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. IMPORTANCE Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion. PMID:26202229

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells.

PubMed

Bleau, Christian; Filliol, Aveline; Samson, Michel; Lamontagne, Lucie

2015-10-01

Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Sialic acid (SA)-modified selenium nanoparticles coated with a high blood-brain barrier permeability peptide-B6 peptide for potential use in Alzheimer's disease.

PubMed

Yin, Tiantian; Yang, Licong; Liu, Yanan; Zhou, Xianbo; Sun, Jing; Liu, Jie

2015-10-01

The blood-brain barrier (BBB) is a formidable gatekeeper toward exogenous substances, playing an important role in brain homeostasis and maintaining a healthy microenvironment for complex neuronal activities. However, it also greatly hinders drug permeability into the brain and limits the management of brain diseases. The development of new drugs that show improved transport across the BBB represents a promising strategy for Alzheimer's disease (AD) intervention. Whereas, previous study of receptor-mediated endogenous BBB transport systems has focused on a strategy of using transferrin to facilitate brain drug delivery system, a system that still suffers from limitations including synthesis procedure, stability and immunological response. In the present study, we synthetised sialic acid (SA)-modified selenium (Se) nanoparticles conjugated with an alternative peptide-B6 peptide (B6-SA-SeNPs, a synthetic selenoprotein analogue), which shows high permeability across the BBB and has the potential to serve as a novel nanomedicine for disease modification in AD. Laser-scanning confocal microscopy, flow cytometry analysis and inductively coupled plasma-atomic emission spectroscopy ICP-AES revealed high cellular uptake of B6-SA-SeNPs by cerebral endothelial cells (bEnd.3). The transport efficiency of B6-SA-SeNPs was evaluated in a Transwell experiment based on in vitro BBB model. It provided direct evidence for B6-SA-SeNPs crossing the BBB and being absorbed by PC12 cells. Moreover, inhibitory effects of B6-SA-SeNPs on amyloid-β peptide (Aβ) fibrillation could be demonstrated in PC12 cells and bEnd3 cells. B6-SA-SeNPs could not only effectively inhibit Aβ aggregation but could disaggregate preformed Aβ fibrils into non-toxic amorphous oligomers. These results suggested that B6-SA-SeNPs may provide a promising platform, particularly for the application of nanoparticles in the treatment of brain diseases. Alzheimer's disease (AD) is the world's most common form of dementia characterized by intracellular neurofibrillary tangles in the brain. Over the past decades, the blood-brain barrier (BBB) limits access of therapeutic or diagnostic agents into the brain, which greatly hinders the development of new drugs for treating AD. In this work, we evaluated the efficiency of B6-SA-SeNPs across BBB and investigated the interactions between B6-SA-SeNPs and amyloid-β peptide (Aβ). We confirm that B6-SA-SeNPs could provide a promising platform because of its high brain delivery efficiency, anti-amyloid properties and anti-oxidant properties, which may serve as a novel nanomedicine for the application in the treatment of brain diseases. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Insulin differentially affects the distribution kinetics of amyloid beta 40 and 42 in plasma and brain.

PubMed

Swaminathan, Suresh Kumar; Ahlschwede, Kristen M; Sarma, Vidur; Curran, Geoffry L; Omtri, Rajesh S; Decklever, Teresa; Lowe, Val J; Poduslo, Joseph F; Kandimalla, Karunya K

2018-05-01

Impaired brain clearance of amyloid-beta peptides (Aβ) 40 and 42 across the blood-brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer's disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aβ accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aβ accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aβ transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aβ40 increased, but Aβ42 clearance reduced; the plasma-to-brain influx of Aβ40 increased, and that of Aβ42 reduced; and the clearance of intracerebrally injected Aβ40 decreased, whereas Aβ42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aβ40 increased and that of Aβ42 reduced; the abluminal-to-luminal permeability of Aβ40 decreased, whereas Aβ42 permeability increased. Moreover, Aβ cellular trafficking machinery was altered. In summary, Aβ40 and Aβ42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

PubMed Central

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

PubMed

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Therapeutics for Brain Metastases, v3.

PubMed

Steeg, Patricia S; Zimmer, Alexandra; Gril, Brunilde

2016-12-15

The role of blood-brain barrier (BBB) permeability in the efficacy of brain metastasis therapeutics is debated. Both BBB-permeable and BBB-impermeable compounds were compared in a melanoma brain metastasis model using imaging through a cranial window. Only the BBB-permeable compound inhibited both the ∼30% permeable metastases and the ∼70% impermeable metastases. Clin Cancer Res; 22(24); 5953-5. ©2016 AACRSee related article by Osswald et al., p. 6078. ©2016 American Association for Cancer Research.

Rapid and efficient crossing blood-brain barrier: Hydrophobic drug delivery system based on propionylated amylose helix nanoclusters.

PubMed

Gao, Wei; Liu, Yongchun; Jing, Guixia; Li, Ke; Zhao, Yuan; Sha, Baoyong; Wang, Qiang; Wu, Daocheng

2017-01-01

A novel strategy of rapid transport across the blood-brain barrier (BBB) via phosphatidylethanolamine-triggered release is developed through both molecular dynamics (MD) simulation and experiments. Hydrophobic drugs, namely, propofol, iodine, and 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide, were loaded with propionylated amylose helix (HLPAH) nanoclusters to form PLPAH, ILPAH, and DLPAH nanoclusters, respectively. These clusters were subjected to MD simulation, structure measurement, in vitro triggered study, in vivo DLPAH imaging, and analysis of PLPAH sedative effects on rabbits. Results indicated that HLPAH nanoclusters were initially located on the BBB, and the helix was unfolded to release the loaded hydrophobic drugs. The released drugs crossed the BBB and performed their functions in the central nervous system (CNS) through concentration gradient and hydrophobicity. This mechanism of HLPAH across the BBB featured high membrane permeability and specificity, rapid onset, short maintenance, rapid recovery, and lower dosage of drugs. Hence, this novel strategy is very meaningful for the development of CNS drug carriers and the proposed system could be used to improve the therapeutic effects of CNS diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Bayesian approach to in silico blood-brain barrier penetration modeling.

PubMed

Martins, Ines Filipa; Teixeira, Ana L; Pinheiro, Luis; Falcao, Andre O

2012-06-25

The human blood-brain barrier (BBB) is a membrane that protects the central nervous system (CNS) by restricting the passage of solutes. The development of any new drug must take into account its existence whether for designing new molecules that target components of the CNS or, on the other hand, to find new substances that should not penetrate the barrier. Several studies in the literature have attempted to predict BBB penetration, so far with limited success and few, if any, application to real world drug discovery and development programs. Part of the reason is due to the fact that only about 2% of small molecules can cross the BBB, and the available data sets are not representative of that reality, being generally biased with an over-representation of molecules that show an ability to permeate the BBB (BBB positives). To circumvent this limitation, the current study aims to devise and use a new approach based on Bayesian statistics, coupled with state-of-the-art machine learning methods to produce a robust model capable of being applied in real-world drug research scenarios. The data set used, gathered from the literature, totals 1970 curated molecules, one of the largest for similar studies. Random Forests and Support Vector Machines were tested in various configurations against several chemical descriptor set combinations. Models were tested in a 5-fold cross-validation process, and the best one tested over an independent validation set. The best fitted model produced an overall accuracy of 95%, with a mean square contingency coefficient (ϕ) of 0.74, and showing an overall capacity for predicting BBB positives of 83% and 96% for determining BBB negatives. This model was adapted into a Web based tool made available for the whole community at http://b3pp.lasige.di.fc.ul.pt.

Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

PubMed Central

2013-01-01

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741. PMID:23668665

Prediction of blood-brain partitioning: a model based on molecular electronegativity distance vector descriptors.

PubMed

Zhang, Yong-Hong; Xia, Zhi-Ning; Qin, Li-Tang; Liu, Shu-Shen

2010-09-01

The objective of this paper is to build a reliable model based on the molecular electronegativity distance vector (MEDV) descriptors for predicting the blood-brain barrier (BBB) permeability and to reveal the effects of the molecular structural segments on the BBB permeability. Using 70 structurally diverse compounds, the partial least squares regression (PLSR) models between the BBB permeability and the MEDV descriptors were developed and validated by the variable selection and modeling based on prediction (VSMP) technique. The estimation ability, stability, and predictive power of a model are evaluated by the estimated correlation coefficient (r), leave-one-out (LOO) cross-validation correlation coefficient (q), and predictive correlation coefficient (R(p)). It has been found that PLSR model has good quality, r=0.9202, q=0.7956, and R(p)=0.6649 for M1 model based on the training set of 57 samples. To search the most important structural factors affecting the BBB permeability of compounds, we performed the values of the variable importance in projection (VIP) analysis for MEDV descriptors. It was found that some structural fragments in compounds, such as -CH(3), -CH(2)-, =CH-, =C, triple bond C-, -CH<, =C<, =N-, -NH-, =O, and -OH, are the most important factors affecting the BBB permeability. (c) 2010. Published by Elsevier Inc.

Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

PubMed

Berghoff, Stefan A; Düking, Tim; Spieth, Lena; Winchenbach, Jan; Stumpf, Sina K; Gerndt, Nina; Kusch, Kathrin; Ruhwedel, Torben; Möbius, Wiebke; Saher, Gesine

2017-12-01

In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.

Exploration of a Doxorubicin-Polymer Conjugate in Lipid-Polymer Hybrid Nanoparticle Drug Delivery

NASA Astrophysics Data System (ADS)

Lough, Emily

Nanoparticle (NP) drug delivery is a major focus in the research community because of its potential to use existing drugs in safer and more effective ways. Chemotherapy encapsulation in NPs shields the drug from the rest of the body while it is within the NP, with less systemic exposure leading to fewer off-target effects of the drug. However, passive loading of drugs into NPs is a suboptimal method, often leading to burst release upon administration. This work explores the impact of incorporating the drug-polymer conjugate doxorubicin-poly (lactic-co-glycolic) acid (Dox-PLGA) into a lipid-polymer hybrid nanoparticle (LPN). The primary difference in using a drug-polymer conjugate for NP drug delivery is the drug's release kinetics. Dox-PLGA LPNs showed a more sustained and prolonged release profile over 28 days compared to LPNs with passively loaded, unconjugated doxorubicin. This sustained release translates to cytotoxicity; when systemic circulation was simulated using dialysis, Dox-PLGA LPNs retained their cytotoxicity at a higher level than the passively loaded LPNs. The in vivo implication of preserving cytotoxic potency through a slower release profile is that the majority of Dox delivered via Dox-PLGA LPNs will be kept within the LPN until it reaches the tumor. This will result in fewer systemic side effects and more effective treatments given the higher drug concentration at the tumor site. An intriguing clinical application of this drug delivery approach lies in using Dox-PLGA LPNs to cross the blood-brain barrier (BBB). The incorporation of Dox-PLGA is hypothesized to have a protective effect on the BBB as its slow release profile will prevent drug from harming the BBB. Using induced pluripotent stem cells differentiated to human brain microvascular endothelial cells that comprise the BBB, the Dox-PLGA LPNs were shown to be less destructive to the BBB than their passively loaded counterparts. Dox-PLGA LPNs showed superior cytotoxicity against plated tumor cells than the passively loaded Dox LPNs after passing through an in vitro transwell BBB model. Dox-PLGA LPNs and drug-polymer conjugates are exciting alternatives to passively loaded NPs and show strong clinical promise of a treatment that is more potent with fewer side effects and less frequent administration.

Transcranial MRI-guided FUS-induced BBB opening in the rat brain

NASA Astrophysics Data System (ADS)

Treat, Lisa H.; McDannold, Nathan J.; Hynynen, Kullervo

2004-05-01

The blood-brain barrier (BBB) has been a major limitation in treating diseases of the brain because therapeutic agents are either unable to penetrate or have dose-limiting side effects in diffuse opening of the BBB. A previous study demonstrated that focused ultrasound (FUS) can locally open the BBB in a rabbit model when a piece of skull is removed and that magnetic resonance imaging (MRI) can be used to guide and monitor the procedure. This study examined whether the same desired effect of local BBB disruption can be achieved by applying FUS through an intact skull in a rat model. Twenty-eight Sprague-Dawley rats were anesthetized, shaved, and sonicated at four focal locations in the brain, using a 1.5-MHz focused transducer. Contrast-enhanced MR images were obtained before and after sonication. The images indicated contrast agent penetration at the focal coordinates following Optison-enhanced sonication. This study demonstrated that the distortion of the ultrasound beam by the rat skull was not significant enough to inhibit focal BBB opening. Subsequent experiments using MRI-guided FUS to aid in targeted drug delivery to brain tumors in a rodent model could thus be performed more efficiently without cranial surgery. [Research funded by NIH Grant No. CA76550.

Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation

NASA Astrophysics Data System (ADS)

Ding, Hong; Sagar, Vidya; Agudelo, Marisela; Pilakka-Kanthikeel, Sudheesh; Subba Rao Atluri, Venkata; Raymond, Andrea; Samikkannu, Thangavel; Nair, Madhavan P.

2014-02-01

The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ˜10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ˜130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation.

PubMed

Ding, Hong; Sagar, Vidya; Agudelo, Marisela; Pilakka-Kanthikeel, Sudheesh; Atluri, Venkata Subba Rao; Raymond, Andrea; Samikkannu, Thangavel; Nair, Madhavan P

2014-02-07

The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ∼10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ∼130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

PubMed

Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

2017-04-01

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Tissue-engineered microenvironment systems for modeling human vasculature.

PubMed

Tourovskaia, Anna; Fauver, Mark; Kramer, Gregory; Simonson, Sara; Neumann, Thomas

2014-09-01

The high attrition rate of drug candidates late in the development process has led to an increasing demand for test assays that predict clinical outcome better than conventional 2D cell culture systems and animal models. Government agencies, the military, and the pharmaceutical industry have started initiatives for the development of novel in-vitro systems that recapitulate functional units of human tissues and organs. There is growing evidence that 3D cell arrangement, co-culture of different cell types, and physico-chemical cues lead to improved predictive power. A key element of all tissue microenvironments is the vasculature. Beyond transporting blood the microvasculature assumes important organ-specific functions. It is also involved in pathologic conditions, such as inflammation, tumor growth, metastasis, and degenerative diseases. To provide a tool for modeling this important feature of human tissue microenvironments, we developed a microfluidic chip for creating tissue-engineered microenvironment systems (TEMS) composed of tubular cell structures. Our chip design encompasses a small chamber that is filled with an extracellular matrix (ECM) surrounding one or more tubular channels. Endothelial cells (ECs) seeded into the channels adhere to the ECM walls and grow into perfusable tubular tissue structures that are fluidically connected to upstream and downstream fluid channels in the chip. Using these chips we created models of angiogenesis, the blood-brain barrier (BBB), and tumor-cell extravasation. Our angiogenesis model recapitulates true angiogenesis, in which sprouting occurs from a "parent" vessel in response to a gradient of growth factors. Our BBB model is composed of a microvessel generated from brain-specific ECs within an ECM populated with astrocytes and pericytes. Our tumor-cell extravasation model can be utilized to visualize and measure tumor-cell migration through vessel walls into the surrounding matrix. The described technology can be used to create TEMS that recapitulate structural, functional, and physico-chemical elements of vascularized human tissue microenvironments in vitro. © 2014 by the Society for Experimental Biology and Medicine.

Identification of Reversible Disruption of the Human Blood-Brain Barrier Following Acute Ischemia.

PubMed

Simpkins, Alexis N; Dias, Christian; Leigh, Richard

2016-09-01

Animal models of acute cerebral ischemia have demonstrated that diffuse blood-brain barrier (BBB) disruption can be reversible after early reperfusion. However, irreversible, focal BBB disruption in humans is associated with hemorrhagic transformation in patients receiving intravenous thrombolytic therapy. The goal of this study was to use a magnetic resonance imaging biomarker of BBB permeability to differentiate these 2 forms of BBB disruption. Acute stroke patients imaged with magnetic resonance imaging before, 2 hours after, and 24 hours after treatment with intravenous tissue-type plasminogen activator were included. The average BBB permeability of the acute ischemic region before and 2 hours after treatment was calculated using a T2* perfusion-weighted source images. Change in average permeability was compared with percent reperfusion using linear regression. Focal regions of maximal BBB permeability from the pretreatment magnetic resonance imaging were compared with the occurrence of parenchymal hematoma (PH) formation on the 24-hour magnetic resonance imaging scan using logistic regression. Signals indicating reversible BBB permeability were detected in 18/36 patients. Change in average BBB permeability correlated inversely with percent reperfusion (P=0.006), indicating that early reperfusion is associated with decreased BBB permeability, whereas sustained ischemia is associated with increased BBB disruption. Focal regions of maximal BBB permeability were significantly associated with subsequent formation of PH (P=0.013). This study demonstrates that diffuse, mild BBB disruption in the acutely ischemic human brain is reversible with reperfusion. This study also confirms prior findings that focal severe BBB disruption confers an increased risk of hemorrhagic transformation in patients treated with intravenous tissue-type plasminogen activator. © 2016 American Heart Association, Inc.

Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood–Brain Barrier Damage

PubMed Central

Qie, Xiaojuan; Wen, Di; Guo, Hongyan; Xu, Guanjie; Liu, Shuai; Shen, Qianchao; Liu, Yi; Zhang, Wenfang; Cong, Bin; Ma, Chunling

2017-01-01

Methamphetamine (METH) abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood–brain barrier (BBB). Herein, we explored the potential mechanism of endoplasmic reticulum (ER) stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3) cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS) and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo) and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption. PMID:28959203

Blood-brain barrier specific permeability assay reveals N-methylated tyramine derivatives in standardised leaf extracts and herbal products of Ginkgo biloba.

PubMed

Könczöl, Árpád; Rendes, Kata; Dékány, Miklós; Müller, Judit; Riethmüller, Eszter; Balogh, György Tibor

2016-11-30

The linkage between the central nervous system availability and neuropharmacological activity of the constituents of Ginkgo biloba L. extracts (GBE) is still incomplete. In this study, the in vitro blood-brain barrier (BBB) permeability profile of the standardised GBE was investigated by the parallel artificial membrane permeability assay (PAMPA). Biomarkers, such as terpene trilactones, flavonoid aglycones and ginkgotoxin exerted moderate or good BBB-permeability potential (BBB+), while glycosides and biflavones were predicted as unable to pass the BBB. N-methyltyramine (NMT) and N,N-dimethyltyramine or hordenine (Hor) were identified among BBB+ compounds, while subsequent direct HRMS analysis revealed tyramine (Tyr) and N,N,N-trimethyltyramine or candicine (Can) in GBE as trace constituents. Distribution of Tyr, NMT, Hor and Can was determined by a validated ion-exchange mechanism-based liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method in G. biloba samples, such as herbal drugs and dietary supplements. The total content of the four tyramine derivatives in various GBEs ranged from 7.3 up to 6357μg/g dry extract with NMT and Hor as most abundant ones. Considering the pharmacological activities and the revealed fluctuation in the concentration of the analysed adrenergic protoalkaloids, the presented rapid LC-ESI-MS method is proposed for monitoring of the levels of Tyr, NMT, Hor and Can in G. biloba products. Copyright © 2016 Elsevier B.V. All rights reserved.

Dual Targeting of Amyloid-beta Clearance and Neuroinflammation as a Novel Therapeutic Approach against Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Batarseh, Yazan S.

Amyloid-beta (Abeta) cascade hypothesis suggests that Alzheimer's disease (AD) is related to an imbalance between the production and clearance of Abeta peptide. Sporadic AD has been related to faulty clearance of Abeta. Accumulation of Abeta oligomers (Abetao) has been linked to several downstream toxic effects including neuroinflammation, synaptic loss, and cellular death. Abeta transport across the blood-brain barrier (BBB) is one of the primary pathways for reducing Abeta load in the brain, which work hand in hand with other parenchymal mechanisms to reduce Abeta levels including intra and extracellular degradation by a family of Abeta degrading enzymes. Established AD drugs, such as the cholinesterase inhibitor donepezil, have been reported to have several additional non-cholinergic effects that alter Abeta pathology; reduce Abeta load, anti-inflammatory response, and attenuate synaptic loss. However, their limited effect only lead to minor improvements in AD symptoms without improving the prognosis of the disease. The lack of effective medical treatment for AD led to several studies focusing on establishing new therapeutic approaches to reduce Abeta pathology. We aimed to identify and characterize natural products that are capable of enhancing the BBB clearance of Abeta in addition to reducing neuroinflammation. Our first project was to investigate the role of oleocanthal (one of the active ingredients in extra-virgin olive oil; EVOO) on attenuating Abeta toxic effects on neurons and astrocytes. We developed Abeta oligomers (Abetao) induced inflammatory environment by exposing neurons and astrocytes to accumulative doses of Abetao to investigate oleocanthal effect on modulating Abetao pathological changes in neurons and astrocytes. Our findings demonstrated oleocanthal prevented Abetao-induced synaptic proteins, SNAP-25 and PSD-95, down-regulation in neurons, attenuated Abetao-induced inflammation, and restored glutamine transporter (GLT1) and glucose transporter (GLUT1) expressions in astrocytes. Results from this study support the protective effect of the EVOO-derived phenolic secoiridoid oleocanthal against AD pathology. Next, we evaluated the role of EVOO in enhancing donepezil's effect on increasing Abeta clearance and reducing neuroinflammation in AD transgenic model, namely 5XFAD mice. The long-term consumption of EVOO in combination with donepezil is expected to enhance and expand donepezil protective mechanisms against Abeta pathology. EVOO consumption in combination with donepezil treatment significantly reduced Abeta load and related pathology; EVOO consumption with donepezil up-regulated synaptic proteins, enhanced BBB tightness and reduced neuroinflammation associated with Abeta pathology. Long-term consumption of EVOO significantly reduced Abeta pathological manifestations in addition to enhancing and expanding donepezil protective mechanisms against Abeta pathology when given concomitantly. Therefore, EVOO consumption as a medical food combined with donepezil offers an effective therapeutic approach by enhancing the non-cholinergic mechanisms of donepezil and by providing additional mechanisms to attenuate Abeta related pathology in AD patients. In the third project, the effect of Crocus sativus extract on Abeta clearance across the BBB and related pathology were evaluated in vitro and in vivo in wild-type and AD transgenic models. Available studies reported Crocus sativus exerts a positive effect against AD, however, the mechanism(s) for such effect is unknown. Therefore, here, we investigated its effect on enhancing Abeta clearance and reducing neuroinflammation. Findings from in vitro studies demonstrated that Crocus sativus extract increased the tightness and enhanced Abeta transport in our cell-based BBB model. Followed in vivo studies confirmed the effect of Crocus sativus extract on the BBB integrity and function that was associated with reduced Abeta load and related pathology in 5XFAD mice. Furthermore, Crocus sativus extract up-regulated synaptic proteins and reduced neuroinflammation associated with Abeta pathology in the brains of 5XFAD mice. Crocin, one of the major active compounds in Crocus sativus, known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo. Crocin was able to reduce Abeta load and related pathologies but to a lesser extent when compared to Crocus sativus extract, which could be explained, at least in part, by the lack of crocin's ability in enhancing Abeta clearance and reducing neuroinflammation. Findings from this project support the positive effect of Crocus sativus against AD by reducing Abeta pathological manifestations. In conclusion, in this work, the therapeutics potential of oleocanthal, EVOO, and Crocus sativus extracts was in vitro and in vivo evaluated for their effect on Abeta clearance, BBB integrity and function, neuroprotective and neuroinflammation. Oleocanthal, EVOO, and Crocus sativus extract enhanced the clearance of Abeta by inducing its transport across the BBB and enhancing its enzymatic degradation and reduced neuroinflammation, which collectively led to Abeta brain levels reduction associated with inflammation reduction and neuroprotection. Therefore, we suggest that natural products such as EVOO, oleocanthal, and Crocus sativus may have a high potential therapeutic role against AD pathology.

Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment

PubMed Central

Belhadj, Zakia; Zhan, Changyou; Ying, Man; Wei, Xiaoli; Xie, Cao; Yan, Zhiqiang; Lu, Weiyue

2017-01-01

Glioblastoma multiforme (GBM) has been considered to be the most malignant brain tumors. Due to the existence of various barriers including the blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB) greatly hinder the accumulation and deep penetration of chemotherapeutics, the treatment of glioma remains to be the most challenging task in clinic. In order to circumvent these hurdles, we developed a multifunctional liposomal glioma-targeted drug delivery system (c(RGDyK)/pHA-LS) modified with cyclic RGD (c(RGDyK)) and p-hydroxybenzoic acid (pHA) in which c(RGDyK) could target integrin αvβ3 overexpressed on the BBTB and glioma cells and pHA could target dopamine receptors on the BBB. In vitro, c(RGDyK)/pHA-LS could target glioblastoma cells (U87), brain capillary endothelial cells (bEnd.3) and umbilical vein endothelial cells (HUVECs) through a comprehensive pathway. Besides, c(RGDyK)/pHA-LS could also increase the cytotoxicity of doxorubicin encapsulated in liposomes on glioblastoma cells, and was able to penetrate inside the glioma spheroids after traversing the in vitro BBB and BBTB. In vivo, we demonstrated the targeting ability of c(RGDyK)/pHA-LS to intracranial glioma. As expected, c(RGDyK)/pHA-LS/DOX showed a median survival time of 35 days, which was 2.31-, 1.76- and 1.5-fold higher than that of LS/DOX, c(RGDyK)-LS/DOX, and pHA-LS/DOX, respectively. The findings here suggested that the multifunctional glioma-targeted drug delivery system modified with both c(RGDyK) and pHA displayed strong antiglioma efficiency in vitro and in vivo, representing a promising platform for glioma therapy. PMID:28978003

Focused ultrasound delivery of Raman nanoparticles across the blood-brain barrier: Potential for targeting experimental brain tumors

PubMed Central

Diaz, Roberto Jose; McVeigh, Patrick Z.; O’Reilly, Meaghan A.; Burrell, Kelly; Bebenek, Matthew; Smith, Christian; Etame, Arnold; Zadeh, Gelareh; Hynynen, Kullervo; Wilson, Brian C.; Rutka, James T.

2014-01-01

Spectral mapping of nanoparticles with surface enhanced Raman scattering (SERS) capability in the near-infrared range is an emerging molecular imaging technique. We used magnetic resonance image-guided transcranial focused ultrasound (TcMRgFUS) to reversibly disrupt the blood-brain barrier (BBB) adjacent to brain tumor margins in rats. Glioma cells were found to internalize SERS capable nanoparticles of 50 nm or 120 nm physical diameter. Surface coating with anti-epidermal growth factor receptor antibody or non-specific human immunoglobulin G, resulted in enhanced cell uptake of nanoparticles in-vitro compared to nanoparticles with methyl terminated 12-unit polyethylene glycol surface. BBB disruption permitted the delivery of SERS capable spherical 50 or 120 nm gold nanoparticles to the tumor margins. Thus, nanoparticles with SERS imaging capability can be delivered across the BBB non-invasively using TcMRgFUS and have the potential to be used as optical tracking agents at the invasive front of malignant brain tumors. PMID:24374363

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis.

PubMed

Pfeiffer, Friederike; Schäfer, Julia; Lyck, Ruth; Makrides, Victoria; Brunner, Sarah; Schaeren-Wiemers, Nicole; Deutsch, Urban; Engelhardt, Britta

2011-11-01

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

Transcranial Cavitation Detection in Primates during Blood-Brain Barrier Opening – A Performance Assessment Study

PubMed Central

Wu, Shih-Ying; Tung, Yao-Sheng; Marquet, Fabrice; Downs, Matthew Eric; Sanchez, Carlos Sierra; Chen, Cherry Chen; Ferrera, Vincent

2014-01-01

Focused ultrasound (FUS) has been shown promise in treating the brain locally and noninvasively. Transcranial passive cavitation detection (PCD) provides methodology of monitoring the treatment in real time, while the skull effects remain a major challenge for its translation to the clinic. In this study, we investigated the sensitivity, reliability, and limitations of PCD through primate (macaque and human) skulls in vitro. The results were further correlated with the in vivo macaque studies including the transcranial PCD calibration and real-time monitoring of BBB opening, with magnetic resonance imaging assessing the opening and safety. The stable cavitation doses using harmonics (SCDh) and ultraharmonics (SCDu), the inertial cavitation dose (ICD), and the cavitation signal-to-noise ratio (SNR) were quantified based on the PCD signals. Results showed that through the macaque skull the pressure threshold for detecting the SCDh remained the same as without the skull in place, while it increased for the SCDu and ICD; through the human skull, it increased for all cavitation doses. The transcranial PCD was found reliable both in vitro and in vivo when the transcranial cavitation SNR exceeded the 1-dB detection limit through the in vitro macaque (attenuation: 4.92 dB/mm) and human (attenuation: 7.33 dB/mm) skull. In addition, using long pulses enabled reliable PCD monitoring and facilitate BBB opening at low pressures. The in vivo results showed that the SCDh became detectable at pressures as low as 100 kPa; the ICD, at 250 kPa while it could occur at lower pressures; the SCDu, at 700 kPa and was less reliable at lower pressures. Real-time monitoring of PCD was further implemented during BBB opening, with successful and safe opening achieved at 250–600 kPa in both the thalamus and the putamen. In conclusion, this study shows that transcranial PCD in macaques in vitro and in vivo as well as humans in vitro is reliable by improving the cavitation SNR beyond the 1-dB detection limit. PMID:24859660

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

PubMed

Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

2017-03-01

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

Opening of brain blood barrier induced by red light and central analgesic improvement of cobra neurotoxin.

PubMed

Ye, Yong; Li, Yue; Fang, Fei

2014-05-05

Cobra neurotoxin (NT) has central analgesic effects, but it is difficult to pass through brain blood barrier (BBB). A novel method of red light induction is designed to help NT across BBB, which is based on photosensitizer activation by red light to generate reactive oxygen species (ROS) to open BBB. The effects were evaluated on cell models and animals in vivo with illumination by semiconductor laser at 670nm on photosensitizer pheophorbide isolated from silkworm excrement. Brain microvascular endothelial cells and astrocytes were co-cultured to build up BBB cell model. The radioactivity of (125)I-NT was measured in cells and tissues for NT permeation. Three ways of cranial irradiation, nasal cavity and intravascular irradiation were tested with combined injection of (125)I-NT 20μg/kg and pheophorbide 100μg/kg to rats, and organs of rats were separated and determined the radioactivity. Paw pressure test in rats, hot plate and writhing test in mice were applied to appraise the analgesic effects. NT across BBB cell model increased with time of illumination, and reached stable level after 60min. So did ROS in cells. NT mainly distributed in liver and kidney of rats, significantly increased in brain after illumination, and improved analgesic effects. Excitation of pheophorbide at red light produces ROS to open BBB, help NT enter brain, and enhance its central action. This research provides a new method for drug across BBB to improve its central role. Copyright © 2014 Elsevier B.V. All rights reserved.

Effective Drug Delivery in Diffuse Intrinsic Pontine Glioma: A Theoretical Model to Identify Potential Candidates

PubMed Central

El-Khouly, Fatma E.; van Vuurden, Dannis G.; Stroink, Thom; Hulleman, Esther; Kaspers, Gertjan J. L.; Hendrikse, N. Harry; Veldhuijzen van Zanten, Sophie E. M.

2017-01-01

Despite decades of clinical trials for diffuse intrinsic pontine glioma (DIPG), patient survival does not exceed 10% at two years post-diagnosis. Lack of benefit from systemic chemotherapy may be attributed to an intact bloodbrain barrier (BBB). We aim to develop a theoretical model including relevant physicochemical properties in order to review whether applied chemotherapeutics are suitable for passive diffusion through an intact BBB or whether local administration via convection-enhanced delivery (CED) may increase their therapeutic potential. Physicochemical properties (lipophilicity, molecular weight, and charge in physiological environment) of anticancer drugs historically and currently administered to DIPG patients, that affect passive diffusion over the BBB, were included in the model. Subsequently, the likelihood of BBB passage of these drugs was ascertained, as well as their potential for intratumoral administration via CED. As only non-molecularly charged, lipophilic, and relatively small sized drugs are likely to passively diffuse through the BBB, out of 51 drugs modeled, only 8 (15%)—carmustine, lomustine, erlotinib, vismodegib, lenalomide, thalidomide, vorinostat, and mebendazole—are theoretically qualified for systemic administration in DIPG. Local administration via CED might create more therapeutic options, excluding only positively charged drugs and drugs that are either prodrugs and/or only available as oral formulation. A wide variety of drugs have been administered systemically to DIPG patients. Our model shows that only few are likely to penetrate the BBB via passive diffusion, which may partly explain the lack of efficacy. Drug distribution via CED is less dependent on physicochemical properties and may increase the therapeutic options for DIPG. PMID:29164054

Host matrix metalloproteinases in cerebral malaria: new kids on the block against blood–brain barrier integrity?

PubMed Central

2014-01-01

Cerebral malaria (CM) is a life-threatening complication of falciparum malaria, associated with high mortality rates, as well as neurological impairment in surviving patients. Despite disease severity, the etiology of CM remains elusive. Interestingly, although the Plasmodium parasite is sequestered in cerebral microvessels, it does not enter the brain parenchyma: so how does Plasmodium induce neuronal dysfunction? Several independent research groups have suggested a mechanism in which increased blood–brain barrier (BBB) permeability might allow toxic molecules from the parasite or the host to enter the brain. However, the reported severity of BBB damage in CM is variable depending on the model system, ranging from mild impairment to full BBB breakdown. Moreover, the factors responsible for increased BBB permeability are still unknown. Here we review the prevailing theories on CM pathophysiology and discuss new evidence from animal and human CM models implicating BBB damage. Finally, we will review the newly-described role of matrix metalloproteinases (MMPs) and BBB integrity. MMPs comprise a family of proteolytic enzymes involved in modulating inflammatory response, disrupting tight junctions, and degrading sub-endothelial basal lamina. As such, MMPs represent potential innovative drug targets for CM. PMID:24467887

Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers

NASA Astrophysics Data System (ADS)

Anand, Prachi; O'Neil, Alison; Lin, Emily; Douglas, Trevor; Holford, Mandë

2015-08-01

The blood brain barrier (BBB) is often an insurmountable obstacle for a large number of candidate drugs, including peptides, antibiotics, and chemotherapeutic agents. Devising an adroit delivery method to cross the BBB is essential to unlocking widespread application of peptide therapeutics. Presented here is an engineered nanocontainer for delivering peptidic drugs across the BBB encapsulating the analgesic marine snail peptide ziconotide (Prialt®). We developed a bi-functional viral nanocontainer based on the Salmonella typhimurium bacteriophage P22 capsid, genetically incorporating ziconotide in the interior cavity, and chemically attaching cell penetrating HIV-Tat peptide on the exterior of the capsid. Virus like particles (VLPs) of P22 containing ziconotide were successfully transported in several BBB models of rat and human brain microvascular endothelial cells (BMVEC) using a recyclable noncytotoxic endocytic pathway. This work demonstrates proof in principle for developing a possible alternative to intrathecal injection of ziconotide using a tunable VLP drug delivery nanocontainer to cross the BBB.

C-type natriuretic peptide-modified lipid vesicles: fabrication and use for the treatment of brain glioma.

PubMed

Wu, Jia-Shuan; Mu, Li-Min; Bu, Ying-Zi; Liu, Lei; Yan, Yan; Hu, Ying-Jie; Bai, Jing; Zhang, Jing-Ying; Lu, Weiyue; Lu, Wan-Liang

2017-06-20

Chemotherapy of brain glioma faces a major obstacle owing to the inability of drug transport across the blood-brain barrier (BBB). Besides, neovasculatures in brain glioma site result in a rapid infiltration, making complete surgical removal virtually impossible. Herein, we reported a novel kind of C-type natriuretic peptide (CNP) modified vinorelbine lipid vesicles for transferring drug across the BBB, and for treating brain glioma along with disrupting neovasculatures. The studies were performed on brain glioma U87-MG cells in vitro and on glioma-bearing nude mice in vivo. The results showed that the CNP-modified vinorelbine lipid vesicles could transport vinorelbine across the BBB, kill the brain glioma, and destroy neovasculatures effectively. The above mechanisms could be associated with the following aspects, namely, long circulation in the blood; drug transport across the BBB via natriuretic peptide receptor B (NPRB)-mediated transcytosis; elimination of brain glioma cells and disruption of neovasculatures by targeting uptake and cytotoxic injury. Besides, CNP-modified vinorelbine lipid vesicles could induce apoptosis of the glioma cells. The mechanisms could be related to the activations of caspase 8, caspase 3, p53, and reactive oxygen species (ROS), and inhibition of survivin. Hence, CNP-modified lipid vesicles could be used as a carrier material for treating brain glioma and disabling glioma neovasculatures.

Focused ultrasound delivery of Raman nanoparticles across the blood-brain barrier: potential for targeting experimental brain tumors.

PubMed

Diaz, Roberto Jose; McVeigh, Patrick Z; O'Reilly, Meaghan A; Burrell, Kelly; Bebenek, Matthew; Smith, Christian; Etame, Arnold B; Zadeh, Gelareh; Hynynen, Kullervo; Wilson, Brian C; Rutka, James T

2014-07-01

Spectral mapping of nanoparticles with surface enhanced Raman scattering (SERS) capability in the near-infrared range is an emerging molecular imaging technique. We used magnetic resonance image-guided transcranial focused ultrasound (TcMRgFUS) to reversibly disrupt the blood-brain barrier (BBB) adjacent to brain tumor margins in rats. Glioma cells were found to internalize SERS capable nanoparticles of 50nm or 120nm physical diameter. Surface coating with anti-epidermal growth factor receptor antibody or non-specific human immunoglobulin G, resulted in enhanced cell uptake of nanoparticles in-vitro compared to nanoparticles with methyl terminated 12-unit polyethylene glycol surface. BBB disruption permitted the delivery of SERS capable spherical 50 or 120nm gold nanoparticles to the tumor margins. Thus, nanoparticles with SERS imaging capability can be delivered across the BBB non-invasively using TcMRgFUS and have the potential to be used as optical tracking agents at the invasive front of malignant brain tumors. This study demonstrates the use of magnetic resonance image-guided transcranial focused ultrasound to open the BBB and enable spectral mapping of nanoparticles with surface enhanced Raman scattering (SERS)-based molecular imaging for experimental tumor tracking. Copyright © 2014 Elsevier Inc. All rights reserved.

Current State and Future Perspectives in QSAR Models to Predict Blood- Brain Barrier Penetration in Central Nervous System Drug R&D.

PubMed

Morales, Juan F; Montoto, Sebastian Scioli; Fagiolino, Pietro; Ruiz, Maria E

2017-01-01

The Blood-Brain Barrier (BBB) is a physical and biochemical barrier that restricts the entry of certain drugs to the Central Nervous System (CNS), while allowing the passage of others. The ability to predict the permeability of a given molecule through the BBB is a key aspect in CNS drug discovery and development, since neurotherapeutic agents with molecular targets in the CNS should be able to cross the BBB, whereas peripherally acting agents should not, to minimize the risk of CNS adverse effects. In this review we examine and discuss QSAR approaches and current availability of experimental data for the construction of BBB permeability predictive models, focusing on the modeling of the biorelevant parameter unbound partitioning coefficient (Kp,uu). Emphasis is made on two possible strategies to overcome the current limitations of in silico models: considering the prediction of brain penetration as a multifactorial problem, and increasing experimental datasets through accurate and standardized experimental techniques.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

PubMed

Bana, Laura; Minniti, Stefania; Salvati, Elisa; Sesana, Silvia; Zambelli, Vanessa; Cagnotto, Alfredo; Orlando, Antonina; Cazzaniga, Emanuela; Zwart, Rob; Scheper, Wiep; Masserini, Massimo; Re, Francesca

2014-10-01

Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aβ (kD=0.6 μM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aβ aggregation/disaggregation as a potential treatment in an Alzheimer's model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimer's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel method for measuring hydraulic conductivity at the human blood-nerve barrier in vitro.

PubMed

Helton, E Scott; Palladino, Steven; Ubogu, Eroboghene E

2017-01-01

Microvascular barrier permeability to water is an essential biophysical property required for the homeostatic maintenance of unique tissue microenvironments. This is of particular importance in peripheral nerves where strict control of ionic concentrations is needed for axonal signal transduction. Previous studies have associated inflammation, trauma, toxin exposure and metabolic disease with increases in water influx and hydrostatic pressure in peripheral nerves with resultant endoneurial edema that may impair axonal function. The regulation of water permeability across endoneurial microvessels that form the blood-nerve barrier (BNB) is poorly understood. Variations exist in apparatus and methods used to measure hydraulic conductivity. The objective of the study was to develop a simplified hydraulic conductivity system using commercially available components to evaluate the BNB. We determined the mean hydraulic conductivity of cultured confluent primary and immortalized human endoneurial endothelial cell layers as 2.00×10 -7 and 2.17×10 -7 cm/s/cm H₂O respectively, consistent with restrictive microvascular endothelial cells in vitro. We also determined the mean hydraulic conductivity of immortalized human brain microvascular endothelial cell layers, a commonly used blood-brain barrier (BBB) cell line, as 0.20×10 -7 cm/s/cm H₂O, implying a mean 10-fold higher resistance to transendothelial water flux in the brain compared to peripheral nerves. To our knowledge, this is the first reported measurement of human BNB and BBB hydraulic conductivities. This model represents an important tool to further characterize the human BNB and deduce the molecular determinants and signaling mechanisms responsible for BNB hydraulic conductivity in normal and disease states in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of Synthetic Matrix Metalloproteinase Inhibitors on Lipopolysaccharide-Induced Blood-Brain Barrier Opening in Rodents: Differences in Response Based on Strains and Solvents

PubMed Central

Rosenberg, Gary A.; Estrada, Eduardo Y.; Mobashery, Shahriar

2007-01-01

Matrix metalloproteinase inhibitors (MMPIs) reduce blood-brain barrier (BBB) disruption and prevent cell death. Animal models of multiple sclerosis, cerebral ischemia and hemorrhage, and bacterial meningitis respond to treatment with MMPIs. We have used the intracerebral injection of lipopolysaccharide (LPS) in rat, which induces MMP production and results in a delayed opening of the BBB, to screen MMPIs to identify therapeutic agents. We hypothesized that the mouse would respond similarly to LPS and that the mouse/LPS model of BBB damage would be more useful for screening of MMPIs. Therefore, we adapted the rat LPS model to the mouse and compared the response to LPS and treatment with MMPIs. Wistar-Kyoto rats (WKY) and three strains of mice had stereotactic injections of LPS into the caudate. 14C-sucrose was used to measure permeability of the BBB 24 hours after injection. Initially, we tested three broad-spectrum MMPIs in the rat, BB-1101, BB-94, and BB-2293, and a MMP-2 selective inhibitor, IW449; both BB-1101 and BB-94 significantly suppressed LPS-induced BBB damage (p<0.05). In the 3 mouse strains, C57/BL6, C57/BL10, and C57/BL10HIIIR2, LPS significantly opened the BBB in C57/BL6, and it was the only strain that showed a reduction in BBB permeability with BB-94. Treatment with methylprednisolone and several broad spectrum MMPIs, including BB-1101, were ineffective in the C57/BL6. There was a significant reduction in BBB permeability seen with 10% dimethyl sulfoxide (DMSO) alone, which was used to dissolve the selective MMP-2 and -9 inhibitor, SB-3CT. The tetracycline derivative, minocycline, reduced the BBB injury in mouse by blocking the production of MMP-9. Our results show variability in rats and mice to LPS and MMPIs, which most likely is based on genetic make-up. Understanding these differences may provide important clues that could guide selection of MMPIs in treatment of neurological diseases. PMID:17184743

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

PubMed

Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

2017-07-01

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

PubMed Central

Chaturvedi, Mayank; Molino, Yves; Sreedhar, Bojja; Khrestchatisky, Michel; Kaczmarek, Leszek

2014-01-01

Aim The aim of this study was to develop poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for delivery of a protein – tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) – across the blood–brain barrier (BBB) to inhibit deleterious matrix metalloproteinases (MMPs). Materials and methods The NPs were formulated by multiple-emulsion solvent-evaporation, and for enhancing BBB penetration, they were coated with polysorbate 80 (Ps80). We compared Ps80-coated and uncoated NPs for their toxicity, binding, and BBB penetration on primary rat brain capillary endothelial cell cultures and the rat brain endothelial 4 cell line. These studies were followed by in vivo studies for brain delivery of these NPs. Results Results showed that neither Ps80-coated nor uncoated NPs caused significant opening of the BBB, and essentially they were nontoxic. NPs without Ps80 coating had more binding to endothelial cells compared to Ps80-coated NPs. Penetration studies showed that TIMP-1 NPs + Ps80 had 11.21%±1.35% penetration, whereas TIMP-1 alone and TIMP-1 NPs without Ps80 coating did not cross the endothelial monolayer. In vivo studies indicated BBB penetration of intravenously injected TIMP-1 NPs + Ps80. Conclusion The study demonstrated that Ps80 coating of NPs does not cause significant toxic effects to endothelial cells and that it can be used to enhance the delivery of protein across endothelial cell barriers, both in vitro and in vivo. PMID:24531257

Tetramethylpyrazine-2'-O-sodium ferulate attenuates blood-brain barrier disruption and brain oedema after cerebral ischemia/reperfusion.

PubMed

Xu, S-H; Yin, M-S; Liu, B; Chen, M-L; He, G-W; Zhou, P-P; Cui, Y-J; Yang, D; Wu, Y-L

2017-07-01

Disruption of blood-brain barrier (BBB) and subsequent oedema are major causes of the pathogenesis in ischaemic stroke with which the current clinical therapy remains unsatisfied. In this study, we examined the therapeutic effect of tetramethylpyrazine-2'-O-sodium ferulate (TSF)-a novel analogue of tetramethylpyrazine in alleviating BBB breakdown and brain oedema after cerebral ischaemia/reperfusion (I/R). Then, we explored the potential mechanism of the protection on BBB disruption in cerebral I/R rat models. Male Sprague-Dawley rats (250-300 g) were subjected to 120 min middle cerebral artery occlusion (MCAO), followed by 48 h reperfusion. TSF (10.8, 18 and 30 mg kg -1 ) and ozagrel (18 mg kg -1 ) were administrated by intravenous injection immediately for the first time and then received the same dose every 24 h for 2 days. We found that TSF treatment significantly attenuated the cerebral water content, infarction volume and improved neurological outcomes in MCAO rats compared to I/R models. Moreover, we investigated the effect of TSF on the BBB for that cerebral oedema is closely related to the permeability of the BBB. We found that the permeability of BBB was improved significantly in TSF groups compared to I/R model group by Evans blue leakage testing. Furthermore, the expressions of tight junction (TJ) proteins junction adhesion molecule-1 and occludin significantly decreased, but the protein expression of matrix metalloproteinase-9 (MMP-9) and aquaporin 4 (AQP4) increased after cerebral I/R, all of which were alleviated by TSF treatment. In conclusion, TSF significantly reduced BBB permeability and brain oedema, which were correlated with regulating the expression of TJ proteins, MMP-9 and AQP4. These findings provide a novel approach to the treatment of ischaemic stroke.

IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood-Brain Barrier Protection in Rats.

PubMed

Niu, Fei; Song, Xiu-Yun; Hu, Jin-Feng; Zuo, Wei; Kong, Ling-Lei; Wang, Xiao-Feng; Han, Ning; Chen, Nai-Hong

2017-10-01

IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study. Copyright © 2017. Published by Elsevier Inc.

Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

PubMed

Pardridge, William M

2015-02-01

Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

Application of an assay Cascade methodology for a deep preclinical characterization of polymeric nanoparticles as a treatment for gliomas.

PubMed

Fornaguera, Cristina; Lázaro, Miguel Ángel; Brugada-Vilà, Pau; Porcar, Irene; Morera, Ingrid; Guerra-Rebollo, Marta; Garrido, Cristina; Rubio, Núria; Blanco, Jerónimo; Cascante, Anna; Borrós, Salvador

2018-11-01

Glioblastoma multiforme (GBM) is the most devastating primary brain tumor due to its infiltrating and diffuse growth characteristics, a situation compounded by the lack of effective treatments. Currently, many efforts are being devoted to find novel formulations to treat this disease, specifically in the nanomedicine field. However, due to the lack of comprehensive characterization that leads to insufficient data on reproducibility, only a reduced number of nanomedicines have reached clinical phases. In this context, the aim of the present study was to use a cascade of assays that evaluate from physical-chemical and structural properties to biological characteristics, both in vitro and in vivo, and also to check the performance of nanoparticles for glioma therapy. An amphiphilic block copolymer, composed of polyester and poly(ethylene glycol; PEG) blocks, has been synthesized. Using a mixture of this copolymer and a polymer containing an active targeting moiety to the Blood Brain Barrier (BBB; Seq12 peptide), biocompatible and biodegradable polymeric nanoparticles have been prepared and extensively characterized. In vitro studies demonstrated that nanoparticles are safe for normal cells but cytotoxic for cancer cells. In vivo studies in mice demonstrated the ability of the Seq12 peptide to cross the BBB. Finally, in vivo efficacy studies using a human tumor model in SCID mice resulted in a significant 50% life-span increase, as compared with non-treated animals. Altogether, this assay cascade provided extensive pre-clinical characterization of our polymeric nanoparticles, now ready for clinical evaluation.

Peroxisome proliferator-activated receptors and Alzheimer's disease: hitting the blood-brain barrier.

PubMed

Zolezzi, Juan M; Inestrosa, Nibaldo C

2013-12-01

The blood-brain barrier (BBB) is often affected in several neurodegenerative disorders, such as Alzheimer's disease (AD). Integrity and proper functionality of the neurovascular unit are recognized to be critical for maintenance of the BBB. Research has traditionally focused on structural integrity more than functionality, and BBB alteration has usually been explained more as a consequence than a cause. However, ongoing evidence suggests that at the early stages, the BBB of a diseased brain often shows distinct expression patterns of specific carriers such as members of the ATP-binding cassette (ABC) transport protein family, which alter BBB traffic. In AD, amyloid-β (Aβ) deposits are a pathological hallmark and, as recently highlighted by Cramer et al. (2012), Aβ clearance is quite fundamental and is a less studied approach. Current knowledge suggests that BBB traffic plays a more important role than previously believed and that pharmacological modulation of the BBB may offer new therapeutic alternatives for AD. Recent investigations carried out in our laboratory indicate that peroxisome proliferator-activated receptor (PPAR) agonists are able to prevent Aβ-induced neurotoxicity in hippocampal neurons and cognitive impairment in a double transgenic mouse model of AD. However, even when enough literature about PPAR agonists and neurodegenerative disorders is available, the problem of how they exert their functions and help to prevent and rescue Aβ-induced neurotoxicity is poorly understood. In this review, along with highlighting the main features of the BBB and its role in AD, we will discuss information regarding the modulation of BBB components, including the possible role of PPAR agonists as BBB traffic modulators.

Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

PubMed

Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

2016-08-26

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. Published by Elsevier Ireland Ltd.

Characteristics of sequential targeting of brain glioma for transferrin-modified cisplatin liposome.

PubMed

Lv, Qing; Li, Li-Min; Han, Min; Tang, Xin-Jiang; Yao, Jin-Na; Ying, Xiao-Ying; Li, Fan-Zhu; Gao, Jian-Qing

2013-02-28

Methods on how to improve the sequential targeting of glioma subsequent to passing of drug through the blood-brain barrier (BBB) have been occasionally reported. However, the characteristics involved are poorly understood. In the present study, cisplatin (Cis) liposome (lipo) was modified with transferrin (Tf) to investigate the characteristics of potential sequential targeting to glioma. In bEnd3/C6 co-culture BBB models, higher transport efficiency across the BBB and cytotoxicity in basal C6 cells induced by Cis-lipo(Tf) than Cis-lipo and Cis-solution, suggest its sequential targeting effect. Interestingly, similar liposomal morphology as that of donor compartment was first demonstrated in the receptor solution of BBB models. Meanwhile, a greater acquisition in the lysosome of bEnd3, distributed sequentially into the nucleus of C6 cells were found for the Cis-lipo(Tf). Pre-incubation of chlorpromazine and Tf inhibited this process, indicating that a clathrin-dependent endocytosis is involved in the transport of Cis-lipo(Tf) across the BBB. Copyright © 2013 Elsevier B.V. All rights reserved.

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease.

PubMed

Argaw, Azeb Tadesse; Asp, Linnea; Zhang, Jingya; Navrazhina, Kristina; Pham, Trinh; Mariani, John N; Mahase, Sean; Dutta, Dipankar J; Seto, Jeremy; Kramer, Elisabeth G; Ferrara, Napoleone; Sofroniew, Michael V; John, Gareth R

2012-07-01

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.

Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability.

PubMed

Madgula, Vamsi L; Avula, Bharathi; Reddy V L, Niranjan; Khan, Ikhlas A; Khan, Shabana I

2007-04-01

Decursin (DE) and decursinol angelate (DA) were isolated from the roots of Angelica gigas (Apiaceae) and purified by HPLC. DE and DA have been reported to exhibit significant neuropharmacological activities, but their intestinal transport and permeability in terms of CNS penetration across the blood-brain barrier (BBB) are unknown. This study was undertaken to evaluate the IN VITRO intestinal and BBB transport of DE and DA using Caco-2 and MDR-MDCK cell monolayer models, respectively. The bidirectional transport of DE and DA across Caco-2 and MDR-MDCK monolayers was examined for 2 hours. Integrity of the monolayer was determined by TEER value and by monitoring the transport of Lucifer yellow (Ly) across the monolayers. Quantitation of DE and DA was performed by HPLC. DE and DA exhibited bidirectional transport with a Papp value in the range of 9.0-12.0x10(-6) cm/sec and 7.2-11.7x10(-6) cm/sec in Caco-2 and MDR-MDCK monolayers, respectively. The TEER values were in the range of 410-440 and 1170-1230 ohm cm2 for Caco-2 and MDR-MDCK monolayers, respectively. Ly measurement, the fluorescent marker of passive paracellular diffusion, resulted in Papp values of 2.5-5.0x10(-6) in Caco-2 and 6.0-8.0x10(-6) cm/sec in MDR-MDCK monolayers, confirming that the monolayer integrity was intact at the end of the experiment. Caco-2:human colonic adenocarcinoma DA:decursinol angelate DE:decursin Ly:Lucifer yellow MDCK:Madin-Darby canine kidney MDR:multidrug resistant Papp:apparent permeability TEER:transepithelial electrical resistance.

Effect of mannose targeting of hydroxyl PAMAM dendrimers on cellular and organ biodistribution in a neonatal brain injury model.

PubMed

Sharma, Anjali; Porterfield, Joshua E; Smith, Elizabeth; Sharma, Rishi; Kannan, Sujatha; Kannan, Rangaramanujam M

2018-06-05

Neurotherapeutics for the treatment of central nervous system (CNS) disorders must overcome challenges relating to the blood-brain barrier (BBB), brain tissue penetration, and the targeting of specific cells. Neuroinflammation mediated by activated microglia is a major hallmark of several neurological disorders, making these cells a desirable therapeutic target. Building on the promise of hydroxyl-terminated generation four polyamidoamine (PAMAM) dendrimers (D4-OH) for penetrating the injured BBB and targeting activated glia, we explored if conjugation of targeting ligands would enhance and modify brain and organ uptake. Since mannose receptors [cluster of differentiation (CD) 206] are typically over-expressed on injured microglia, we conjugated mannose to the surface of multifunctional D4-OH using highly efficient, atom-economical, and orthogonal Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry and evaluated the effect of mannose conjugation on the specific cell uptake of targeted and non-targeted dendrimers both in vitro and in vivo. In vitro results indicate that the conjugation of mannose as a targeting ligand significantly changes the mechanism of dendrimer internalization, giving mannosylated dendrimer a preference for mannose receptor-mediated endocytosis as opposed to non-specific fluid phase endocytosis. We further investigated the brain uptake and biodistribution of targeted and non-targeted fluorescently labeled dendrimers in a maternal intrauterine inflammation-induced cerebral palsy (CP) rabbit model using quantification methods based on fluorescence spectroscopy and confocal microscopy. We found that the conjugation of mannose modified the distribution of D4-OH throughout the body in this neonatal rabbit CP model without lowering the amount of dendrimer delivered to injured glia in the brain, even though significantly higher glial uptake was not observed in this model. Mannose conjugation to the dendrimer modifies the dendrimer's interaction with cells, but does not minimize its inherent inflammation-targeting abilities. Copyright © 2018 Elsevier B.V. All rights reserved.

Hydrogen inhalation ameliorated mast cell mediated brain injury after ICH in mice

PubMed Central

Manaenko, Anatol; Lekic, Tim; Ma, Qingyi; Zhang, John H.; Tang, Jiping

2012-01-01

OBJECTIVE Hydrogen inhalation was neuroprotective in several brain injury models. Its mechanisms are believed to be related to anti-oxidative stress. We investigated the potential neurovascular protective effect of hydrogen inhalation especially effect on mast cell activation in a mouse model of intracerebral hemorrhage (ICH). DESIGN Controlled in vivo laboratory study. SETTING Animal research laboratory SUBJECTS 171, 8 weeks old male CD-1 mice were used. INTERVENTIONS Collagenase-induced ICH model in 8 weeks old, male, CD-1 mice was used. Hydrogen was administrated via spontaneous inhalation. The blood-brain barrier (BBB) permeability and neurological deficits were investigated at 24 and 72 hours after ICH. Mast cell activation was evaluated by Western blot and immuno-staining. The effects of hydrogen inhalation on mast cell activation were confirmed in an autologous blood injection model ICH. MEASURMENT AND MAIN RESULTS At 24 and 72 hours post-ICH, animals showed BBB disruption, brain edema, neurological deficits, accompanied with phosphorylation of Lyn kinase and release of tryptase, indicating mast cell activation. Hydrogen treatment diminished phosphorylation of Lyn kinase and release of tryptase, decreased accumulation and degranulation of mast cells, attenuated BBB disruption and improved neurobehavioral function. CONCLUSION Activation of mast cells following ICH contributed to increase of BBB permeability and brain edema. Hydrogen inhalation preserved BBB disruption by prevention of mast cell activation after ICH. PMID:23388512

Design and synthesis of novel δ opioid receptor agonists with an azatricyclodecane skeleton for improving blood-brain barrier penetration.

PubMed

Watanabe, Yoshikazu; Hayashida, Kohei; Saito, Daisuke; Takahashi, Toshihiro; Sakai, Junichi; Nakata, Eriko; Kanda, Takashi; Iwai, Takashi; Hirayama, Shigeto; Fujii, Hideaki; Yamakawa, Tomio; Nagase, Hiroshi

2017-08-01

We designed and synthesized novel δ opioid receptor (DOR) agonists 3a-i with an azatricyclodecane skeleton, which was a novel structural class of DOR agonists. Among them, 3b exhibited high values of binding affinity and potent agonistic activity for the DOR that were approximately equivalent to those of 2 which bore an oxazatricyclodecane skeleton. In vitro assays using the blood-brain barrier (BBB) permeability test kit supported the idea that 3b achieved an excellent BBB permeability by converting an oxygen atom of 2 to a carbon atom (methylene group) in the core skeleton. As a result, 3b showed potent antinociceptive effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

PAMAM Dendrimers Cross the Blood-Brain Barrier When Administered through the Carotid Artery in C57BL/6J Mice.

PubMed

Srinageshwar, Bhairavi; Peruzzaro, Sarah; Andrews, Melissa; Johnson, Kayla; Hietpas, Allison; Clark, Brittany; McGuire, Crystal; Petersen, Eric; Kippe, Jordyn; Stewart, Andrew; Lossia, Olivia; Al-Gharaibeh, Abeer; Antcliff, Aaron; Culver, Rebecca; Swanson, Douglas; Dunbar, Gary; Sharma, Ajit; Rossignol, Julien

2017-03-14

Drug delivery into the central nervous system (CNS) is challenging due to the blood-brain barrier (BBB) and drug delivery into the brain overcoming the BBB can be achieved using nanoparticles such as dendrimers. The conventional cationic dendrimers used are highly toxic. Therefore, the present study investigates the role of novel mixed surface dendrimers, which have potentially less toxicity and can cross the BBB when administered through the carotid artery in mice. In vitro experiments investigated the uptake of amine dendrimers (G1-NH₂ and G4-NH₂) and novel dendrimers (G1-90/10 and G4-90/10) by primary cortical cultures. In vivo experiments involved transplantation of G4-90/10 into mice through (1) invasive intracranial injections into the striatum; and (2) less invasive carotid injections. The animals were sacrificed 24-h and 1-week post-transplantations and their brains were analyzed. In vivo experiments proved that the G4-90/10 can cross the BBB when injected through the carotid artery and localize within neurons and glial cells. The dendrimers were found to migrate through the corpus callosum 1-week post intracranial injection. Immunohistochemistry showed that the migrating cells are the dendrimer-infected glial cells. Overall, our results suggest that poly-amidoamine (PAMAM) dendrimers may be used as a minimally invasive means to deliver biomolecules for treating neurological diseases or disorders.

Characterization of different bubble formulations for blood-brain barrier opening using a focused ultrasound system with acoustic feedback control.

PubMed

Bing, Chenchen; Hong, Yu; Hernandez, Christopher; Rich, Megan; Cheng, Bingbing; Munaweera, Imalka; Szczepanski, Debra; Xi, Yin; Bolding, Mark; Exner, Agata; Chopra, Rajiv

2018-05-22

Focused ultrasound combined with bubble-based agents serves as a non-invasive way to open the blood-brain barrier (BBB). Passive acoustic detection was well studied recently to monitor the acoustic emissions induced by the bubbles under ultrasound energy, but the ability to perform reliable BBB opening with a real-time feedback control algorithm has not been fully evaluated. This study focuses on characterizing the acoustic emissions of different types of bubbles: Optison, Definity, and a custom-made nanobubble. Their performance on reliable BBB opening under real-time feedback control based on acoustic detection was evaluated both in-vitro and in-vivo. The experiments were conducted using a 0.5 MHz focused ultrasound transducer with in-vivo focal pressure ranges from 0.1-0.7 MPa. Successful feedback control was achieved with all three agents when combining with infusion injection. Localized opening was confirmed with Evans blue dye leakage. Microscopic images were acquired to review the opening effects. Under similar total gas volume, nanobubble showed a more reliable opening effect compared to Optison and Definity (p < 0.05). The conclusions obtained from this study confirm the possibilities of performing stable opening using a feedback control algorithm combined with infusion injection. It also opens another potential research area of BBB opening using sub-micron bubbles.

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models

PubMed Central

2009-01-01

Background Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect. Methods The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours. Results In the four human and the murine glioblastoma cell lines tested, 10 μM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 × 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB. Conclusion These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model. PMID:19619277

Optically enhanced blood-brain-barrier crossing of plasmonic-active nanoparticles in preclinical brain tumor animal models

NASA Astrophysics Data System (ADS)

Yuan, Hsiangkuo; Wilson, Christy M.; Li, Shuqin; Fales, Andrew M.; Liu, Yang; Grant, Gerald; Vo-Dinh, Tuan

2014-02-01

Nanotechnology provides tremendous biomedical opportunities for cancer diagnosis, imaging, and therapy. In contrast to conventional chemotherapeutic agents where their actual target delivery cannot be easily imaged, integrating imaging and therapeutic properties into one platform facilitates the understanding of pharmacokinetic profiles, and enables monitoring of the therapeutic process in each individual. Such a concept dubbed "theranostics" potentiates translational research and improves precision medicine. One particular challenging application of theranostics involves imaging and controlled delivery of nanoplatforms across blood-brain-barrier (BBB) into brain tissues. Typically, the BBB hinders paracellular flux of drug molecules into brain parenchyma. BBB disrupting agents (e.g. mannitol, focused ultrasound), however, suffer from poor spatial confinement. It has been a challenge to design a nanoplatform not only acts as a contrast agent but also improves the BBB permeation. In this study, we demonstrated the feasibility of plasmonic gold nanoparticles as both high-resolution optical contrast agent and focalized tumor BBB permeation-inducing agent. We specifically examined the microscopic distribution of nanoparticles in tumor brain animal models. We observed that most nanoparticles accumulated at the tumor periphery or perivascular spaces. Nanoparticles were present in both endothelial cells and interstitial matrices. This study also demonstrated a novel photothermal-induced BBB permeation. Fine-tuning the irradiating energy induced gentle disruption of the vascular integrity, causing short-term extravasation of nanomaterials but without hemorrhage. We conclude that our gold nanoparticles are a powerful biocompatible contrast agent capable of inducing focal BBB permeation, and therefore envision a strong potential of plasmonic gold nanoparticle in future brain tumor imaging and therapy.

Statins as neuroprotectants: a comparative in vitro study of lipophilicity, blood-brain-barrier penetration, lowering of brain cholesterol, and decrease of neuron cell death.

PubMed

Sierra, Saleta; Ramos, Maria C; Molina, Pilar; Esteo, Cynthia; Vázquez, Jose Antonio; Burgos, Javier S

2011-01-01

There is growing evidence to support the hypothesis that statins may act as neuroprotectants in several neuropathological conditions, including Alzheimer's disease. The mechanisms for neuroprotection are only partially understood, however, and pleiotropic phenomena could be involved. We have made a comparative study of 9 statins (lovastatin, mevastatin, pravastatin, simvastatin, cerivastatin, atorvastatin, fluvastatin, pitavastatin, and rosuvastatin), analyzing several parameters that could be related to neuroprotection, such as chemical structure, lipophilicity, potential blood-brain-barrier penetration (BBB), 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibition, cholesterol modulation in neurons, glia, and human hepatocyte cell lines, and protection against neurodegeneration caused by tau hyperphosphorylation induced by okadaic acid. Our results indicate that monacolin J derivatives (natural and semi-synthetic statins) are the best candidates for the prevention of neurodegenerative conditions due to their higher potential BBB penetration capacity, cholesterol lowering effect on neurons with a satisfactory safety profile, and in vitro protection against cell death caused by okadaic acid in culture. Among the nine statins studied, simvastatin presented the best characteristics for preventing neurodegenerative conditions.

A NASTRAN Vibration Model of the AH-1G Helicopter Airframe. Volume 1

DTIC Science & Technology

1974-06-01

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The blood-brain barrier internalises Cryptococcus neoformans via the EphA2-tyrosine kinase receptor.

PubMed

Aaron, Phylicia A; Jamklang, Mantana; Uhrig, John P; Gelli, Angie

2018-03-01

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis most commonly in populations with impaired immunity. Here, we resolved the transcriptome of the human brain endothelium challenged with C. neoformans to establish whether C. neoformans invades the CNS by co-opting particular signalling pathways as a means to promote its own entry. Among the 5 major pathways targeted by C. neoformans, the EPH-EphrinA1 (EphA2) tyrosine kinase receptor-signalling pathway was examined further. Silencing the EphA2 receptor transcript in a human brain endothelial cell line or blocking EphA2 activity with an antibody or chemical inhibitor prevented transmigration of C. neoformans in an in vitro model of the blood-brain barrier (BBB). In contrast, treating brain endothelial cells with an EphA2 chemical agonist or an EphA2 ligand promoted greater migration of fungal cells across the BBB. C. neoformans activated the EPH-tyrosine kinase pathway through a CD44-dependent phosphorylation of EphA2, promoting clustering and internalisation of EphA2 receptors. Moreover, HEK293T cells expressing EphA2 revealed an association between EphA2 and C. neoformans that boosted internalisation of C. neoformans. Collectively, the results suggest that C. neoformans promotes EphA2 activity via CD44, and this in turn creates a permeable barrier that facilitates the migration of C. neoformans across the BBB. © 2017 John Wiley & Sons Ltd.

Antitumoral Cascade-Targeting Ligand for IL-6 Receptor-Mediated Gene Delivery to Glioma.

PubMed

Wang, Shanshan; Reinhard, Sören; Li, Chengyi; Qian, Min; Jiang, Huiling; Du, Yilin; Lächelt, Ulrich; Lu, Weiyue; Wagner, Ernst; Huang, Rongqin

2017-07-05

The effective treatment of glioma is largely hindered by the poor transfer of drug delivery systems across the blood-brain barrier (BBB) and the difficulty in distinguishing healthy and tumorous cells. In this work, for the first time, an interleukin-6 receptor binding I 6 P 7 peptide was exploited as a cascade-targeting ligand in combination with a succinoyl tetraethylene pentamine (Stp)-histidine oligomer-based nonviral gene delivery system (I 6 P 7 -Stp-His/DNA). The I 6 P 7 peptide provides multiple functions, including the cascade-targeting potential represented by a combined BBB-crossing and subsequent glioma-targeting ability, as well as a direct tumor-inhibiting effect. I 6 P 7 -Stp-His/DNA nanoparticles (NPs) mediated higher gene expression in human glioma U87 cells than in healthy human astrocytes and a deeper penetration into glioma spheroids than scrambled peptide-modified NPs. Transport of I 6 P 7 -modified, but not the control, NPs across the BBB was demonstrated in vitro in a transwell bEnd.3 cell model resulting in transfection of underlying U87 cells and also in vivo in glioma-bearing mice. Intravenous administration of I 6 P 7 -Stp-His/plasmid DNA (pDNA)-encoding inhibitor of growth 4 (pING4) significantly prolonged the survival time of orthotopic U87 glioma-bearing mice. The results denote that I 6 P 7 peptide is a roborant cascade-targeting ligand, and I 6 P 7 -modified NPs might be exploited for efficient glioma therapy. Copyright © 2017. Published by Elsevier Inc.

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model

PubMed Central

Baghirov, Habib; Snipstad, Sofie; Sulheim, Einar; Berg, Sigrid; Hansen, Rune; Thorsen, Frits; Mørch, Yrr; Åslund, Andreas K. O.

2018-01-01

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma. PMID:29338016

Improved Prediction of Blood-Brain Barrier Permeability Through Machine Learning with Combined Use of Molecular Property-Based Descriptors and Fingerprints.

PubMed

Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2018-03-21

Blood-brain barrier (BBB) permeability of a compound determines whether the compound can effectively enter the brain. It is an essential property which must be accounted for in drug discovery with a target in the brain. Several computational methods have been used to predict the BBB permeability. In particular, support vector machine (SVM), which is a kernel-based machine learning method, has been used popularly in this field. For SVM training and prediction, the compounds are characterized by molecular descriptors. Some SVM models were based on the use of molecular property-based descriptors (including 1D, 2D, and 3D descriptors) or fragment-based descriptors (known as the fingerprints of a molecule). The selection of descriptors is critical for the performance of a SVM model. In this study, we aimed to develop a generally applicable new SVM model by combining all of the features of the molecular property-based descriptors and fingerprints to improve the accuracy for the BBB permeability prediction. The results indicate that our SVM model has improved accuracy compared to the currently available models of the BBB permeability prediction.

Noninvasive and Targeted Drug Delivery to the Brain Using Focused Ultrasound

PubMed Central

2013-01-01

Brain diseases are notoriously difficult to treat due to the presence of the blood-brain barrier (BBB). Here, we review the development of focused ultrasound (FUS) as a noninvasive method for BBB disruption, aiding in drug delivery to the brain. FUS can be applied through the skull to a targeted region in the brain. When combined with microbubbles, FUS causes localized and reversible disruption of the BBB. The cellular mechanisms of BBB disruption are presented. Several therapeutic agents have been delivered to the brain resulting in significant improvements in pathology in models of glioblastoma and Alzheimer’s disease. The requirements for clinical translation of FUS will be discussed. PMID:23379618

Focused ultrasound-mediated drug delivery through the blood-brain barrier

PubMed Central

Burgess, Alison; Shah, Kairavi; Hough, Olivia; Hynynen, Kullervo

2015-01-01

Despite recent advances in blood-brain barrier (BBB) research, it remains a significant hurdle for the pharmaceutical treatment of brain diseases. Focused ultrasound (FUS) is one method to transiently increase permeability of the BBB to promote drug delivery to specific brain regions. An introduction to the BBB and a brief overview of the methods which can be used to circumvent the BBB to promote drug delivery is provided. In particular, we discuss the advantages and limitations of FUS technology and the efficacy of FUS-mediated drug delivery in models of disease. MRI for targeting and evaluating FUS treatments, combined with administration of microbubbles, allows for transient, reproducible BBB opening. The integration of a real-time acoustic feedback controller has improved treatment safety. Successful clinical translation of FUS has the potential to transform the treatment of brain disease worldwide without requiring the development of new pharmaceutical agents. PMID:25936845

Magnetic resonance imaging of post-ischemic blood-brain barrier damage with PEGylated iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Liu, Dong-Fang; Qian, Cheng; An, Yan-Li; Chang, Di; Ju, Sheng-Hong; Teng, Gao-Jun

2014-11-01

Blood-brain barrier (BBB) damage during ischemia may induce devastating consequences like cerebral edema and hemorrhagic transformation. This study presents a novel strategy for dynamically imaging of BBB damage with PEGylated supermagnetic iron oxide nanoparticles (SPIONs) as contrast agents. The employment of SPIONs as contrast agents made it possible to dynamically image the BBB permeability alterations and ischemic lesions simultaneously with T2-weighted MRI, and the monitoring could last up to 24 h with a single administration of PEGylated SPIONs in vivo. The ability of the PEGylated SPIONs to highlight BBB damage by MRI was demonstrated by the colocalization of PEGylated SPIONs with Gd-DTPA after intravenous injection of SPION-PEG/Gd-DTPA into a mouse. The immunohistochemical staining also confirmed the leakage of SPION-PEG from cerebral vessels into parenchyma. This study provides a novel and convenient route for imaging BBB alteration in the experimental ischemic stroke model.

Aquaporin-4 Functionality and Virchow-Robin Space Water Dynamics: Physiological Model for Neurovascular Coupling and Glymphatic Flow

PubMed Central

Kwee, Ingrid L.

2017-01-01

The unique properties of brain capillary endothelium, critical in maintaining the blood-brain barrier (BBB) and restricting water permeability across the BBB, have important consequences on fluid hydrodynamics inside the BBB hereto inadequately recognized. Recent studies indicate that the mechanisms underlying brain water dynamics are distinct from systemic tissue water dynamics. Hydrostatic pressure created by the systolic force of the heart, essential for interstitial circulation and lymphatic flow in systemic circulation, is effectively impeded from propagating into the interstitial fluid inside the BBB by the tightly sealed endothelium of brain capillaries. Instead, fluid dynamics inside the BBB is realized by aquaporin-4 (AQP-4), the water channel that connects astrocyte cytoplasm and extracellular (interstitial) fluid. Brain interstitial fluid dynamics, and therefore AQP-4, are now recognized as essential for two unique functions, namely, neurovascular coupling and glymphatic flow, the brain equivalent of systemic lymphatics. PMID:28820467

Aquaporin-4 Functionality and Virchow-Robin Space Water Dynamics: Physiological Model for Neurovascular Coupling and Glymphatic Flow.

PubMed

Nakada, Tsutomu; Kwee, Ingrid L; Igarashi, Hironaka; Suzuki, Yuji

2017-08-18

The unique properties of brain capillary endothelium, critical in maintaining the blood-brain barrier (BBB) and restricting water permeability across the BBB, have important consequences on fluid hydrodynamics inside the BBB hereto inadequately recognized. Recent studies indicate that the mechanisms underlying brain water dynamics are distinct from systemic tissue water dynamics. Hydrostatic pressure created by the systolic force of the heart, essential for interstitial circulation and lymphatic flow in systemic circulation, is effectively impeded from propagating into the interstitial fluid inside the BBB by the tightly sealed endothelium of brain capillaries. Instead, fluid dynamics inside the BBB is realized by aquaporin-4 (AQP-4), the water channel that connects astrocyte cytoplasm and extracellular (interstitial) fluid. Brain interstitial fluid dynamics, and therefore AQP-4, are now recognized as essential for two unique functions, namely, neurovascular coupling and glymphatic flow, the brain equivalent of systemic lymphatics.

Angiopoietin-2 mediates blood-brain barrier impairment and colonization of triple-negative breast cancer cells in brain.

PubMed

Avraham, Hava Karsenty; Jiang, Shuxian; Fu, Yigong; Nakshatri, Harikrishna; Ovadia, Haim; Avraham, Shalom

2014-02-01

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple-negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood-brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO-1 and claudin-5 tight junction (TJ) protein structures. Angiopoietin-2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang-2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang-2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang-2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang-2 may serve as potential therapeutics for brain metastasis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

PubMed Central

Wagner, Sylvia; Zensi, Anja; Wien, Sascha L.; Tschickardt, Sabrina E.; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U.; Kreuter, Jörg; von Briesen, Hagen

2012-01-01

Background The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier. PMID:22396775

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

PubMed

Wagner, Sylvia; Zensi, Anja; Wien, Sascha L; Tschickardt, Sabrina E; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U; Kreuter, Jörg; von Briesen, Hagen

2012-01-01

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

Brain Targeting Delivery Facilitated by Ligand-Functionalized Layered Double Hydroxide Nanoparticles.

PubMed

Chen, Weiyu; Zuo, Huali; Zhang, Enqi; Li, Li; Henrich-Noack, Petra; Cooper, Helen; Qian, Yujin; Xu, Zhi Ping

2018-06-20

A delivery platform with highly selective permeability through the blood-brain barrier (BBB) is essential for brain disease treatment. In this research, we designed and prepared a novel target nanoplatform, that is, layered double hydroxide (LDH) nanoparticle conjugated with targeting peptide-ligand Angiopep-2 (Ang2) or rabies virus glycoprotein (RVG) via intermatrix bovine serum albumin for brain targeting. In vitro studies show that functionalization with the target ligand significantly increases the delivery efficiency of LDH nanoparticles to the brain endothelial (bEnd.3) cells and the transcytosis through the simulated BBB model, that is, bEnd.3 cell-constructed multilayer membrane. In vivo confocal neuroimaging of the rat's blood-retina area dynamically demonstrates that LDH nanoparticles modified with peptide ligands have shown a prolonged retention period within the retina vessel in comparison with the pristine LDH group. Moreover, Ang2-modified LDH nanoparticles are found to more specifically accumulate in the mouse brain than the control and RVG-modified LDH nanoparticles after 2 and 48 h intravenous injection. All these findings strongly suggest that Ang2-modified LDHs can serve as an effective targeting nanoplatform for brain disease treatment.

Phosphorylated recombinant HSP27 protects the brain and attenuates blood-brain barrier disruption following stroke in mice receiving intravenous tissue-plasminogen activator.

PubMed

Shimada, Yoshiaki; Shimura, Hideki; Tanaka, Ryota; Yamashiro, Kazuo; Koike, Masato; Uchiyama, Yasuo; Urabe, Takao; Hattori, Nobutaka

2018-01-01

Loss of integrity of the blood-brain barrier (BBB) in ischemic stroke victims initiates a devastating cascade of events causing brain damage. Maintaining the BBB is important to preserve brain function in ischemic stroke. Unfortunately, recombinant tissue plasminogen activator (tPA), the only effective fibrinolytic treatment at the acute stage of ischemic stroke, also injures the BBB and increases the risk of brain edema and secondary hemorrhagic transformation. Thus, it is important to identify compounds that maintain BBB integrity in the face of ischemic injury in patients with stroke. We previously demonstrated that intravenously injected phosphorylated recombinant heat shock protein 27 (prHSP27) protects the brains of mice with transient middle cerebral artery occlusion (tMCAO), an animal stroke-model. Here, we determined whether prHSP27, in addition to attenuating brain injury, also decreases BBB damage in hyperglycemic tMCAO mice that had received tPA. After induction of hyperglycemia and tMCAO, we examined 4 treatment groups: 1) bovine serum albumin (BSA), 2) prHSP27, 3) tPA, 4) tPA plus prHSP27. We examined the effects of prHSP27 by comparing the BSA and prHSP27 groups and the tPA and tPA plus prHSP27 groups. Twenty-four hours after injection, prHSP27 reduced infarct volume, brain swelling, neurological deficits, the loss of microvessel proteins and endothelial cell walls, and mortality. It also reduced the rates of hemorrhagic transformation, extravasation of endogenous IgG, and MMP-9 activity, signs of BBB damage. Therefore, prHSP27 injection attenuated brain damage and preserved the BBB in tPA-injected, hyperglycemic tMCAO experimental stroke-model mice, in which the BBB is even more severely damaged than in simple tMCAO mice. The attenuation of brain damage and BBB disruption in the presence of tPA suggests the effectiveness of prHSP27 and tPA as a combination therapy. prHSP27 may be a novel therapeutic agent for ischemic stroke patients whose BBBs are injured following tPA injections.

Modulatory effects of perforin gene dosage on pathogen-associated blood-brain barrier (BBB) disruption.

PubMed

Willenbring, Robin C; Jin, Fang; Hinton, David J; Hansen, Mike; Choi, Doo-Sup; Pavelko, Kevin D; Johnson, Aaron J

2016-08-31

CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population. We isolated the effect a single perforin allele has on CNS vascular permeability through the use of perforin-heterozygous (perforin+/-) C57BL/6 mice in the peptide-induced fatal syndrome (PIFS) model of immune-mediated BBB disruption. Seven days following Theiler's murine encephalomyelitis virus (TMEV) CNS infection, neuroinflammation and TMEV viral control were assessed through flow cytometric analysis and quantitative real-time PCR of the viral genome, respectively. Following immune-mediated BBB disruption, gadolinium-enhanced T1-weighted MRI, with 3D volumetric analysis, and confocal microscopy were used to define CNS vascular permeability. Finally, the open field behavior test was used to assess locomotor activity of mice following immune-mediated BBB disruption. Perforin-null mice had negligible CNS vascular permeability. Perforin-WT mice have extensive CNS vascular permeability. Interestingly, perforin-heterozygous mice had an intermediate level of CNS vascular permeability as measured by both gadolinium-enhanced T1-weighted MRI and fibrinogen leakage in the brain parenchyma. Differences in BBB disruption were not a result of increased CNS immune infiltrate. Additionally, TMEV was controlled in a perforin dose-dependent manner. Furthermore, a single perforin allele is sufficient to induce locomotor deficit during immune-mediated BBB disruption. Perforin modulates BBB disruption in a dose-dependent manner. This study demonstrates a potentially advantageous role for decreased perforin expression in reducing BBB disruption. This study also provides insight into the effect SNVs in a single perforin allele could have on functional deficit in neurological disease.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and CNS Homeostasis

PubMed Central

Tran, Khiem A.; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F.; Göthert, Joachim R.; Malik, Asrar B.; Valyi-Nagy, Tibor; Zhao, You-Yang

2015-01-01

Background The blood-brain barrier (BBB) formed by brain endothelial cells (ECs) interconnected by tight junctions (TJs) is essential for the homeostasis of the central nervous system (CNS). Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Methods and Results Using a mouse model with tamoxifen-inducible EC-restricted disruption of ctnnb1 (iCKO), here we show that endothelial β-catenin signaling is essential for maintaining BBB integrity and CNS homeostasis in adult. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and CNS inflammation, and all died postictal. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of specific TJ proteins Claudin-1 and -3 in adult brain ECs. The clinical relevance of the data is indicated by the observation of decreased expression of Claudin-1 and nuclear β-catenin in brain ECs of hemorrhagic lesions of hemorrhagic stroke patients. Conclusion These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity and CNS inflammation. PMID:26538583

Dynamic monitoring of blood-brain barrier integrity using water exchange index (WEI) during mannitol and CO2 challenges in mouse brain.

PubMed

Huang, Shuning; Farrar, Christian T; Dai, Guangping; Kwon, Seon Joo; Bogdanov, Alexei A; Rosen, Bruce R; Kim, Young R

2013-04-01

The integrity of the blood-brain barrier (BBB) is critical to normal brain function. Traditional techniques for the assessment of BBB disruption rely heavily on the spatiotemporal analysis of extravasating contrast agents. However, such methods based on the leakage of relatively large molecules are not suitable for the detection of subtle BBB impairment or for the performance of repeated measurements in a short time frame. Quantification of the water exchange rate constant (WER) across the BBB using strictly intravascular contrast agents could provide a much more sensitive method for the quantification of the BBB integrity. To estimate WER, we have recently devised a powerful new method using a water exchange index (WEI) biomarker and demonstrated BBB disruption in an acute stroke model. Here, we confirm that WEI is sensitive to even very subtle changes in the integrity of the BBB caused by: (i) systemic hypercapnia and (ii) low doses of a hyperosmolar solution. In addition, we have examined the sensitivity and accuracy of WEI as a biomarker of WER using computer simulation. In particular, the dependence of the WEI-WER relation on changes in vascular blood volume, T1 relaxation of cellular magnetization and transcytolemmal water exchange was explored. Simulated WEI was found to vary linearly with WER for typically encountered exchange rate constants (1-4 Hz), regardless of the blood volume. However, for very high WER (>5 Hz), WEI became progressively more insensitive to increasing WER. The incorporation of transcytolemmal water exchange, using a three-compartment tissue model, helped to extend the linear WEI regime to slightly higher WER, but had no significant effect for most physiologically important WERs (WER < 4 Hz). Variation in cellular T1 had no effect on WEI. Using both theoretical and experimental approaches, our study validates the utility of the WEI biomarker for the monitoring of BBB integrity. Copyright © 2012 John Wiley & Sons, Ltd.

Prognostic significance of blood-brain barrier disruption in patients with severe nonpenetrating traumatic brain injury requiring decompressive craniectomy.

PubMed

Ho, Kwok M; Honeybul, Stephen; Yip, Cheng B; Silbert, Benjamin I

2014-09-01

The authors assessed the risk factors and outcomes associated with blood-brain barrier (BBB) disruption in patients with severe, nonpenetrating, traumatic brain injury (TBI) requiring decompressive craniectomy. At 2 major neurotrauma centers in Western Australia, a retrospective cohort study was conducted among 97 adult neurotrauma patients who required an external ventricular drain (EVD) and decompressive craniectomy during 2004-2012. Glasgow Outcome Scale scores were used to assess neurological outcomes. Logistic regression was used to identify factors associated with BBB disruption, defined by a ratio of total CSF protein concentrations to total plasma protein concentration > 0.007 in the earliest CSF specimen collected after TBI. Of the 252 patients who required decompressive craniectomy, 97 (39%) required an EVD to control intracranial pressure, and biochemical evidence of BBB disruption was observed in 43 (44%). Presence of disruption was associated with more severe TBI (median predicted risk for unfavorable outcome 75% vs 63%, respectively; p = 0.001) and with worse outcomes at 6, 12, and 18 months than was absence of BBB disruption (72% vs 37% unfavorable outcomes, respectively; p = 0.015). The only risk factor significantly associated with increased risk for BBB disruption was presence of nonevacuated intracerebral hematoma (> 1 cm diameter) (OR 3.03, 95% CI 1.23-7.50; p = 0.016). Although BBB disruption was associated with more severe TBI and worse long-term outcomes, when combined with the prognostic information contained in the Corticosteroid Randomization after Significant Head Injury (CRASH) prognostic model, it did not seem to add significant prognostic value (area under the receiver operating characteristic curve 0.855 vs 0.864, respectively; p = 0.453). Biochemical evidence of BBB disruption after severe nonpenetrating TBI was common, especially among patients with large intracerebral hematomas. Disruption of the BBB was associated with more severe TBI and worse long-term outcomes, but when combined with the prognostic information contained in the CRASH prognostic model, this information did not add significant prognostic value.

Dynamic Monitoring of Blood-Brain Barrier Integrity using Water Exchange Index (WEI) During Mannitol and CO2 Challenges in Mouse Brain

PubMed Central

Huang, Shuning; Farrar, Christian T.; Dai, Guangping; Kwon, Seon Joo; Bogdanov, Alexei A.; Rosen, Bruce R.; Kim, Young R.

2012-01-01

The integrity of the blood-brain barrier (BBB) is critical to normal brain function. Traditional techniques for assessing BBB disruption rely heavily on the spatiotemporal analysis of extravasating contrast agents. But such methods based on the leakage of relatively large molecules are not suitable to detect subtle BBB impairment or to perform repeated measurements in a short time frame. Quantification of the water exchange rate constant (WER) across the BBB using strictly intravascular contrast agents could provide a much more sensitive method for quantifying the BBB integrity. For estimating the WER, we have recently devised a powerful new method using a water exchange index (WEI) biomarker and demonstrated BBB disruption in an acute stroke model. Here we confirm that the WEI is sensitive to even very subtle changes in the integrity of the BBB caused by (1) systemic hypercapnia and (2) low doses of a hyperosmolar solution. In addition, we have examined the sensitivity and accuracy of the WEI as a biomarker of the WER using computer simulation. In particular, the dependence of the WEI-WER relation on changes in vascular blood volume, T1 relaxation of cellular magnetization, and transcytolemmal water exchange was explored. The simulated WEI was found to vary linearly with the WER for typically encountered exchange rate constants (1–4 Hz) regardless of the blood volume. However, for very high WER (>5 Hz) the WEI became progressively more insensitive to increasing WER. The incorporation of transcytolemmal water exchange, using a three-compartment tissue model, helped to extend the linear WEI regime to slightly higher WER, but had no significant effect for most physiologically important water exchange rate constants (WER<4 Hz). Variation in the cellular T1 had no effect on the WEI. Using both theoretical and experimental approaches, our study validates the utility of the WEI biomarker for monitoring BBB integrity. PMID:23055278

In-vivo imaging of blood-brain barrier permeability using positron emission tomography with 2-amino-[3-11C]isobutyric acid.

PubMed

Okada, Maki; Kikuchi, Tatsuya; Okamura, Toshimitsu; Ikoma, Yoko; Tsuji, Atsushi B; Wakizaka, Hidekatsu; Kamakura, Tomoo; Aoki, Ichio; Zhang, Ming-Rong; Kato, Koichi

2015-12-01

The blood-brain barrier (BBB) limits the entry of some therapeutics into the brain, resulting in reduced efficacy. BBB-opening techniques have been developed to enhance the entry into the brain. However, a noninvasive, highly sensitive and quantitative method for evaluating the changes in BBB permeability induced by such techniques is needed to optimize treatment protocols. We evaluated 2-amino-[3-C]isobutyric acid ([3-C]AIB) as a PET probe to quantify BBB permeability in model rats. BBB opening was induced by a lipopolysaccharide injection or focused ultrasound (FUS) sonication. [3-C]AIB distribution in the brain was evaluated by autoradiography and PET and compared with that of Evans blue, a traditional BBB permeability marker. Kinetics of [3-C]AIB was compared with that of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced MRI. The unidirectional blood-brain transfer constant (Ki) of [3-C]AIB was estimated using the Patlak plot. [3-C]AIB uptake in the lesion area was significantly higher than that in the control area and radioactivity colocalized with Evans blue in both models. [3-C]AIB uptake in the FUS-sonicated region decreased over time after sonication. The ratio of [3-C]AIB accumulation in the FUS-treated to the contralateral side increased during the experimental period, whereas that of the Gd-DTPA intensity reached a maximum at 10 min after injection and decreased thereafter. The [3-C]AIB Ki values were significantly higher in the lesion area than the control area. [3-C]AIB PET is a promising, highly sensitive and quantitative imaging method for assessment of BBB permeability.

Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Haibo; Institute of Toxicology, Binzhou Medical University, Yantai; He, Jie

Curculigoside A has shown protective effects against rat cortical neuron damage in vivo. However, the molecular mechanisms through which Curculigoside A affords this protection are unclear. In the present study, we sought to elucidate the mechanisms of angiogenesis in rat aortic endothelial cells (RAEC), rat aortic smooth muscle cells (RASMC) as well as a rat model of cerebral ischemia and reperfusion injury following treatment with Curculigoside A. We examined the role of Curculigoside A on RAEC and RASMC proliferation, migration, and tube formation in vitro and in a cerebral ischemia and reperfusion injury rat model. We used the recombinant Dickkopfmore » (DKK)-1 protein, a Wnt/β-catenin inhibitor, and the recombinant WIF-1 protein, a Wnt5a antagonist to determine mechanisms. In addition, we measured leakage of the blood–brain barrier (BBB) and tested for angiogenesis associated proteins. Our data suggest that Curculigoside A induces angiogenesis in vitro by increasing proliferation, migration and tube formation in RAEC and RASMC. The increase in Curculigoside A-induced proliferation and tube formation was counteracted by DKK-1 and WIF-1. Curculigoside A increased expression of VEGF, p-VEGFR, p-CREB, Egr-3, VCAM-1, Ang1 and Tie2 while prohibiting BBB leakage in cerebral ischemia and reperfusion injured rats. However, Cyclosporine A, a CREB inhibitor, reduced the expression of p-CREB, Egr-3, VCAM-1, Ang1 and Tie2. These data suggest that Curculigoside A induces cell proliferation and angiogenesis through the Wnt5a/β-catenin and VEGF/CREB/Egr-3/VCAM-1 signaling axis and promotes maturation and stability of new blood vessels via increasing Ang1 and Tie-2 expression. - Highlights: • Curculigoside A induces cell proliferation through Wnt5a/β-catenin pathway. • Curculigoside A induces angiogenesis via VEGF/CREB/Egr-3/VCAM-1 signaling axis. • Curculigoside A promotes blood vessel maturation via Ang1/Tie2 pathway.« less

Specific Binding, Uptake, and Transport of ICAM-1-Targeted Nanocarriers Across Endothelial and Subendothelial Cell Components of the Blood-Brain Barrier

PubMed Central

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-01-01

Purpose The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transporting drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule 1 (ICAM-1), to transport drug carriers into and across BBB models. Methods Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. Results ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. Conclusions CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier. PMID:24558007

Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.

PubMed

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-07-01

The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models. Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.

Role of nitric oxide synthases in early blood-brain barrier disruption following transient focal cerebral ischemia.

PubMed

Jiang, Zheng; Li, Chun; Arrick, Denise M; Yang, Shu; Baluna, Alexandra E; Sun, Hong

2014-01-01

The role of nitric oxide synthases (NOSs) in early blood-brain barrier (BBB) disruption was determined using a new mouse model of transient focal cerebral ischemia. Ischemia was induced by ligating the middle cerebral artery (MCA) at its M2 segment and reperfusion was induced by releasing the ligation. The diameter alteration of the MCA, arterial anastomoses and collateral arteries were imaged and measured in real time. BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. The reperfusion produced an extensive vasodilation and a sustained hyperemia. Although expression of NOSs was not altered at 3 hours of reperfusion, L-NAME (a non-specific NOS inhibitor) abolished reperfusion-induced vasodilation/hyperemia and significantly reduced EB and Na-F extravasation. L-NIO (an endothelial NOS (eNOS) inhibitor) significantly attenuated cerebral vasodilation but not BBB disruption, whereas L-NPA and 7-NI (neuronal NOS (nNOS) inhibitors) significantly reduced BBB disruption but not cerebral vasodilation. In contrast, aminoguanidine (AG) (an inducible NOS (iNOS) inhibitor) had less effect on either cerebral vasodilation or BBB disruption. On the other hand, papaverine (PV) not only increased the vasodilation/hyperemia but also significantly reduced BBB disruption. Combined treatment with L-NAME and PV preserved the vasodilation/hyperemia and significantly reduced BBB disruption. Our findings suggest that nNOS may play a major role in early BBB disruption following transient focal cerebral ischemia via a hyperemia-independent mechanism.

Development of a Physiologically-Based Pharmacokinetic Model of the Rat Central Nervous System

PubMed Central

Badhan, Raj K. Singh; Chenel, Marylore; Penny, Jeffrey I.

2014-01-01

Central nervous system (CNS) drug disposition is dictated by a drug’s physicochemical properties and its ability to permeate physiological barriers. The blood–brain barrier (BBB), blood-cerebrospinal fluid barrier and centrally located drug transporter proteins influence drug disposition within the central nervous system. Attainment of adequate brain-to-plasma and cerebrospinal fluid-to-plasma partitioning is important in determining the efficacy of centrally acting therapeutics. We have developed a physiologically-based pharmacokinetic model of the rat CNS which incorporates brain interstitial fluid (ISF), choroidal epithelial and total cerebrospinal fluid (CSF) compartments and accurately predicts CNS pharmacokinetics. The model yielded reasonable predictions of unbound brain-to-plasma partition ratio (Kpuu,brain) and CSF:plasma ratio (CSF:Plasmau) using a series of in vitro permeability and unbound fraction parameters. When using in vitro permeability data obtained from L-mdr1a cells to estimate rat in vivo permeability, the model successfully predicted, to within 4-fold, Kpuu,brain and CSF:Plasmau for 81.5% of compounds simulated. The model presented allows for simultaneous simulation and analysis of both brain biophase and CSF to accurately predict CNS pharmacokinetics from preclinical drug parameters routinely available during discovery and development pathways. PMID:24647103

PAMAM Dendrimers Cross the Blood–Brain Barrier When Administered through the Carotid Artery in C57BL/6J Mice

PubMed Central

Srinageshwar, Bhairavi; Peruzzaro, Sarah; Andrews, Melissa; Johnson, Kayla; Hietpas, Allison; Clark, Brittany; McGuire, Crystal; Petersen, Eric; Kippe, Jordyn; Stewart, Andrew; Lossia, Olivia; Al-Gharaibeh, Abeer; Antcliff, Aaron; Culver, Rebecca; Swanson, Douglas; Dunbar, Gary; Sharma, Ajit; Rossignol, Julien

2017-01-01

Drug delivery into the central nervous system (CNS) is challenging due to the blood–brain barrier (BBB) and drug delivery into the brain overcoming the BBB can be achieved using nanoparticles such as dendrimers. The conventional cationic dendrimers used are highly toxic. Therefore, the present study investigates the role of novel mixed surface dendrimers, which have potentially less toxicity and can cross the BBB when administered through the carotid artery in mice. In vitro experiments investigated the uptake of amine dendrimers (G1-NH2 and G4-NH2) and novel dendrimers (G1-90/10 and G4-90/10) by primary cortical cultures. In vivo experiments involved transplantation of G4-90/10 into mice through (1) invasive intracranial injections into the striatum; and (2) less invasive carotid injections. The animals were sacrificed 24-h and 1-week post-transplantations and their brains were analyzed. In vivo experiments proved that the G4-90/10 can cross the BBB when injected through the carotid artery and localize within neurons and glial cells. The dendrimers were found to migrate through the corpus callosum 1-week post intracranial injection. Immunohistochemistry showed that the migrating cells are the dendrimer-infected glial cells. Overall, our results suggest that poly-amidoamine (PAMAM) dendrimers may be used as a minimally invasive means to deliver biomolecules for treating neurological diseases or disorders PMID:28335421

Quantitative In Vitro and In Vivo Evaluation of Intestinal and Blood-Brain Barrier Transport Kinetics of the Plant N-Alkylamide Pellitorine.

PubMed

Veryser, Lieselotte; Bracke, Nathalie; Wynendaele, Evelien; Joshi, Tanmayee; Tatke, Pratima; Taevernier, Lien; De Spiegeleer, Bart

2016-01-01

Objective. To evaluate the gut mucosa and blood-brain barrier (BBB) pharmacokinetic permeability properties of the plant N-alkylamide pellitorine. Methods. Pure pellitorine and an Anacyclus pyrethrum extract were used to investigate the permeation of pellitorine through (1) a Caco-2 cell monolayer, (2) the rat gut after oral administration, and (3) the BBB in mice after intravenous and intracerebroventricular administration. A validated bioanalytical UPLC-MS(2) method was used to quantify pellitorine. Results. Pellitorine was able to cross the Caco-2 cell monolayer from the apical-to-basolateral and from the basolateral-to-apical side with apparent permeability coefficients between 0.6 · 10(-5) and 4.8 · 10(-5) cm/h and between 0.3 · 10(-5) and 5.8 · 10(-5) cm/h, respectively. In rats, a serum elimination rate constant of 0.3 h(-1) was obtained. Intravenous injection of pellitorine in mice resulted in a rapid and high permeation of pellitorine through the BBB with a unidirectional influx rate constant of 153 μL/(g·min). In particular, 97% of pellitorine reached the brain tissue, while only 3% remained in the brain capillaries. An efflux transfer constant of 0.05 min(-1) was obtained. Conclusion. Pellitorine shows a good gut permeation and rapidly permeates the BBB once in the blood, indicating a possible role in the treatment of central nervous system diseases.

Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis

PubMed Central

Taslimifar, Mehdi; Buoso, Stefano; Verrey, Francois; Kurtcuoglu, Vartan

2018-01-01

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor. PMID:29593549

Impairment of blood-brain barrier is an early event in R6/2 mouse model of Huntington Disease.

PubMed

Di Pardo, Alba; Amico, Enrico; Scalabrì, Francesco; Pepe, Giuseppe; Castaldo, Salvatore; Elifani, Francesca; Capocci, Luca; De Sanctis, Claudia; Comerci, Laura; Pompeo, Francesco; D'Esposito, Maurizio; Filosa, Stefania; Crispi, Stefania; Maglione, Vittorio

2017-01-24

Blood-brain barrier (BBB) breakdown, due to the concomitant disruption of the tight junctions (TJs), normally required for the maintenance of BBB function, and to the altered transport of molecules between blood and brain and vice-versa, has been suggested to significantly contribute to the development and progression of different brain disorders including Huntington's disease (HD). Although the detrimental consequence the BBB breakdown may have in the clinical settings, the timing of its alteration remains elusive for many neurodegenerative diseases. In this study we demonstrate for the first time that BBB disruption in HD is not confined to established symptoms, but occurs early in the disease progression. Despite the obvious signs of impaired BBB permeability were only detectable in concomitance with the onset of the disease, signs of deranged TJs integrity occur precociously in the disease and precede the onset of overt symptoms. To our perspective this finding may add a new dimension to the horizons of pathological mechanisms underlying this devastating disease, however much remains to be elucidated for understanding how specific BBB drug targets can be approached in the future.

Neuroprotective effect of interleukin-6 in a rat model of cerebral ischemia

PubMed Central

FENG, QILIN; WANG, YI; YANG, YINGDA

2015-01-01

Interleukin (IL)-6 is known to be a key cytokine in immune regulation in addition to serving crucial functions in various autoimmune diseases; however, the neuroprotective potential of IL-6 has not been fully investigated. The aim of the present study was to investigate the neuroprotective effects of the inflammatory cytokine IL-6 in a rat model of cerebral ischemia. Rat cerebral ischemia was induced by intraluminal middle cerebral artery occlusion. Following treatment with 500 or 50 ng IL-6, the infarct volumes and symptoms of neurological deficit were ameliorated. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining suggested that the IL-6 treatment reduced neuronal apoptosis in vivo, which was consistent with a lower percentage of annexin V- and caspase-3-positive cortical neurons. In addition, IL-6 in vitro induced the phosphorylation of signal transducer and activator of transcription (STAT) 3 and the expression of induced myeloid leukemia cell differentiation protein Mcl-1, but not the expression of B-cell lymphoma 2, suggesting the activation of the Janus kinase/STAT pathway by IL-6. IL-6 also appeared to be involved in the regulation of cytokine secretion and blood-brain barrier (BBB) integrity in cerebral ischemia. IL-6 downregulated a number of inflammatory cytokines, including tumor necrosis factor-α and IL-1β, as well as myeloperoxidase activity, indicating the accumulation of granulocytes in the ischemic brain tissue. IL-6 was also observed to support the integrity of the BBB by reducing Evans blue leakage in vivo and suppressing the expression of matrix metalloproteinase-9 in ischemic brain tissue. In conclusion, the results of the present study indicate that the neuroprotective effects of IL-6 in cerebral ischemia are the result of a range of processes, including the modulation of cell apoptosis, cytokine secretion and the integrity of the BBB. IL-6 could therefore be used as a therapeutic agent in clinical practice. PMID:26136879

Temporary disruption of the blood-brain barrier by use of ultrasound and microbubbles: safety and efficacy evaluation in rhesus macaques.

PubMed

McDannold, Nathan; Arvanitis, Costas D; Vykhodtseva, Natalia; Livingstone, Margaret S

2012-07-15

The blood-brain barrier (BBB) prevents entry of most drugs into the brain and is a major hurdle to the use of drugs for brain tumors and other central nervous system disorders. Work in small animals has shown that ultrasound combined with an intravenously circulating microbubble agent can temporarily permeabilize the BBB. Here, we evaluated whether this targeted drug delivery method can be applied safely, reliably, and in a controlled manner on rhesus macaques using a focused ultrasound system. We identified a clear safety window during which BBB disruption could be produced without evident tissue damage, and the acoustic pressure amplitude where the probability for BBB disruption was 50% and was found to be half of the value that would produce tissue damage. Acoustic emission measurements seem promising for predicting BBB disruption and damage. In addition, we conducted repeated BBB disruption to central visual field targets over several weeks in animals trained to conduct complex visual acuity tasks. All animals recovered from each session without behavioral deficits, visual deficits, or loss in visual acuity. Together, our findings show that BBB disruption can be reliably and repeatedly produced without evident histologic or functional damage in a clinically relevant animal model using a clinical device. These results therefore support clinical testing of this noninvasive-targeted drug delivery method.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

PubMed

Tran, Khiem A; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F; Göthert, Joachim R; Malik, Asrar B; Valyi-Nagy, Tibor; Zhao, You-Yang

2016-01-12

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation. © 2015 American Heart Association, Inc.

Application of optical coherence tomography for in vivo monitoring of the meningeal lymphatic vessels during opening of blood-brain barrier: mechanisms of brain clearing

NASA Astrophysics Data System (ADS)

Semyachkina-Glushkovskaya, Oxana; Abdurashitov, Arkady; Dubrovsky, Alexander; Bragin, Denis; Bragina, Olga; Shushunova, Nataliya; Maslyakova, Galina; Navolokin, Nikita; Bucharskaya, Alla; Tuchin, Valery; Kurths, Juergen; Shirokov, Alexander

2017-12-01

The meningeal lymphatic vessels were discovered 2 years ago as the drainage system involved in the mechanisms underlying the clearance of waste products from the brain. The blood-brain barrier (BBB) is a gatekeeper that strongly controls the movement of different molecules from the blood into the brain. We know the scenarios during the opening of the BBB, but there is extremely limited information on how the brain clears the substances that cross the BBB. Here, using the model of sound-induced opening of the BBB, we clearly show how the brain clears dextran after it crosses the BBB via the meningeal lymphatic vessels. We first demonstrate successful application of optical coherence tomography (OCT) for imaging of the lymphatic vessels in the meninges after opening of the BBB, which might be a new useful strategy for noninvasive analysis of lymphatic drainage in daily clinical practice. Also, we give information about the depth and size of the meningeal lymphatic vessels in mice. These new fundamental data with the applied focus on the OCT shed light on the mechanisms of brain clearance and the role of lymphatic drainage in these processes that could serve as an informative platform for a development of therapy and diagnostics of diseases associated with injuries of the BBB such as stroke, brain trauma, glioma, depression, or Alzheimer disease.

Dopamine Increases CD14+CD16+ Monocyte Transmigration across the Blood Brain Barrier: Implications for Substance Abuse and HIV Neuropathogenesis.

PubMed

Calderon, Tina M; Williams, Dionna W; Lopez, Lillie; Eugenin, Eliseo A; Cheney, Laura; Gaskill, Peter J; Veenstra, Mike; Anastos, Kathryn; Morgello, Susan; Berman, Joan W

2017-06-01

In human immunodeficiency virus-1 (HIV) infected individuals, substance abuse may accelerate the development and/or increase the severity of HIV associated neurocognitive disorders (HAND). It is proposed that CD14 + CD16 + monocytes mediate HIV entry into the central nervous system (CNS) and that uninfected and infected CD14 + CD16 + monocyte transmigration across the blood brain barrier (BBB) contributes to the establishment and propagation of CNS HIV viral reservoirs and chronic neuroinflammation, important factors in the development of HAND. The effects of substance abuse on the frequency of CD14 + CD16 + monocytes in the peripheral circulation and on the entry of these cells into the CNS during HIV neuropathogenesis are not known. PBMC from HIV infected individuals were analyzed by flow cytometry and we demonstrate that the frequency of peripheral blood CD14 + CD16 + monocytes in HIV infected substance abusers is increased when compared to those without active substance use. Since drug use elevates extracellular dopamine concentrations in the CNS, we examined the effects of dopamine on CD14 + CD16 + monocyte transmigration across our in vitro model of the human BBB. The transmigration of this monocyte subpopulation is increased by dopamine and the dopamine receptor agonist, SKF 38393, implicating D1-like dopamine receptors in the increase in transmigration elicited by this neurotransmitter. Thus, elevated extracellular CNS dopamine may be a novel common mechanism by which active substance use increases uninfected and HIV infected CD14 + CD16 + monocyte transmigration across the BBB. The influx of these cells into the CNS may increase viral seeding and neuroinflammation, contributing to the development of HIV associated neurocognitive impairments.

Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells.

PubMed

Mizutani, Taketoshi; Ishizaka, Aya; Nihei, Coh-Ichi

2016-02-05

As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Endothelial Cells Derived from the Blood-Brain Barrier and Islets of Langerhans Differ in their Response to the Effects of Bilirubin on Oxidative Stress Under Hyperglycemic Conditions.

PubMed

Kapitulnik, Jaime; Benaim, Clara; Sasson, Shlomo

2012-01-01

Unconjugated bilirubin (UCB) is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS). High glucose levels (hyperglycemia) generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB). In the current study we show that UCB (1-40 μM) induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM) levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langerhans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM) glucose levels. While UCB (0.1-40 μM) did not alter ROS production in cells exposed to normal glucose, relatively low ("physiological") UCB concentrations (0.1-5 μM) attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20-40 μM) increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions.

Endothelial Cells Derived from the Blood-Brain Barrier and Islets of Langerhans Differ in their Response to the Effects of Bilirubin on Oxidative Stress Under Hyperglycemic Conditions

PubMed Central

Kapitulnik, Jaime; Benaim, Clara; Sasson, Shlomo

2012-01-01

Unconjugated bilirubin (UCB) is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS). High glucose levels (hyperglycemia) generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB). In the current study we show that UCB (1–40 μM) induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM) levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langerhans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM) glucose levels. While UCB (0.1–40 μM) did not alter ROS production in cells exposed to normal glucose, relatively low (“physiological”) UCB concentrations (0.1–5 μM) attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20–40 μM) increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions. PMID:22811666

Protein kinase Cβ as a therapeutic target stabilizing blood–brain barrier disruption in experimental autoimmune encephalomyelitis

PubMed Central

Lanz, Tobias V.; Becker, Simon; Osswald, Matthias; Bittner, Stefan; Schuhmann, Michael K.; Opitz, Christiane A.; Gaikwad, Sadanand; Wiestler, Benedikt; Litzenburger, Ulrike M.; Sahm, Felix; Ott, Martina; Iwantscheff, Simeon; Grabitz, Carl; Mittelbronn, Michel; von Deimling, Andreas; Winkler, Frank; Meuth, Sven G.; Wick, Wolfgang; Platten, Michael

2013-01-01

Disruption of the blood–brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cβ, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cβ in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS. PMID:23959874

Toxicity of polymeric nanoparticles in vivo and in vitro

NASA Astrophysics Data System (ADS)

Voigt, Nadine; Henrich-Noack, Petra; Kockentiedt, Sarah; Hintz, Werner; Tomas, Jürgen; Sabel, Bernhard A.

2014-06-01

Polybutylcyanoacrylate nanoparticles (PBCA NPs) are candidates for a drug delivery system, which can cross the blood-brain barrier (BBB). Because little is known about their toxicity, we exposed cells to PBCA NPs in vitro and in vivo and monitored their life and death assays. PBCA NPs were fabricated with different surfactants according to the mini-emulsion technique. Viabilities of HeLa and HEK293 cells after NP incubation were quantified by analysing cellular metabolic activity (MTT-test). We then repetitively injected i.v. rhodamine-labelled PBCA NP variations into rats and monitored the survival and morphology of retrogradely labelled neurons by in vivo confocal neuroimaging (ICON) for five weeks. To test for carrier-efficacy and safety, PBCA NPs loaded with Kyotorphin were injected in rats, and a hot plate test was used to quantify analgesic effects. In vitro, we found dose-dependent cell death which was, however, only detectable at very high doses and mainly seen in the cultures incubated with NPs fabricated with the tensids SDS and Tween. However, the in vivo experiments did not show any NP-induced neuronal death, even with particles which were toxic at high dose in vitro, i.e. NPs with Tween and SDS. The increased pain threshold at the hot plate test demonstrated that PBCA NPs are able to cross the BBB and thus comprise a useful tool for drug delivery into the central nervous system (CNS). Our findings showing that different nanoparticle formulations are non-toxic have important implications for the value of NP engineering approaches in medicine.

Polyphosphate kinase 1 is required for the pathogenesis process of meningitic Escherichia coli K1 (RS218).

PubMed

Peng, Liang; Luo, Wen-Ying; Zhao, Tie; Wan, Cheng-Song; Jiang, Yong; Chi, Feng; Zhao, Wei; Cao, Hong; Huang, Sheng-He

2012-03-01

Polyphosphate kinase 1 (PPK1), encoded by the ppk1 gene, is one of the major enzymes to reversibly catalyze the synthesis of polyphosphate (poly P) from the terminal phosphate of ATP. Poly P confers resistance to stress in a number of bacterial species but its role in the virulence of meningitic bacterial pathogens is unknown. The aim of this study was to determine the role of PPK1 in the pathogenesis of Escherichia coli meningitis. An isogenic in-frame ppk1 deletion mutant (PD44) of E. coli K1 strain E44 was constructed and characterized. Human brain microvascular endothelial cells and neonatal rats were used as the in vitro and in vivo models, respectively, to evaluate bacterial adhesion/invasion and the abilities of bacteria crossing the blood-brain barrier (BBB) to cause meningitis. The survival of PD44 and E44 under osmotic and acid stress conditions were also examined. Poly P levels in E44 were clearly higher than those in PD44, especially at the stationary phase (SP). The ppk1 deletion mutant PD44 also showed poor survival rates during osmotic shock and acidic challenge, which the bacteria would face during pathogenesis. In vitro and in vivo assays revealed that PD44 was defective in bacterial adhesion and translocation across the BBB. By using the Evans blue method, we found that E44-induced permeability of the BBB in neonatal rats was significantly higher than that of the animals infected with PD44. Cytokine ELISA results showed that the TNF-α and IL-1β levels in the serum and brain tissues of the neonatal rats infected with PD44 were lower than that of the E44 group. A more obvious meningeal inflammation could be observed in the brain tissues of the rats infected with E44 when compared with that of the PD44 group by histopathological examination. Furthermore, the mRNA expression of IbeR, which is an RpoS-like regulator contributing to the SP regulation in E44, was found to be decreased in PD44 when compared with the parent strain. PD44 was also deficient in mRNA expression of the invasin IbeA, the adhesin FimH and the outer member protein A, which contributes to E44 penetration across BBB and resistance to the stimulations of low pH and high osmolarity. These results indicate that ppk1 plays an important role in stress adaption and virulence in meningitic E. coli K1 strain E44, and controls the relevant phenotypes by modulating the expression of the SP regulatory gene ibeR and the virulence genes ibeA, fimH and ompA.

Blood-brain barrier transport of an essential amino acid after cerebral ischemia reperfusion injury.

PubMed

Suzuki, Toyofumi; Miyazaki, Yumiko; Ohmuro, Aya; Watanabe, Masaki; Furuishi, Takayuki; Fukami, Toshiro; Tomono, Kazuo

2013-01-01

Under pathophysiological conditions such as -cerebral ischemia-reperfusion (IR), damage to cerebrovascular endothelial cells causes alterations in the blood-brain barrier (BBB) function that can exacerbate neuronal cell injury and death. Clarifying changes in BBB transport in the early period of IR is important for understanding BBB function during therapy after cerebral ischemia. The present study was aimed at clarifying changes during IR in the BBB transport of L-phenylalanine (Phe) as a substrate of L-type amino acid transporter 1. An IR model was produced in mice by blood recirculation following occlusion of the middle cerebral artery. Permeability of the BBB to [(3)H]Phe was measured after IR injury using the brain perfusion method. Confocal microscopy of the IR injury showed no brain penetration of fluorescent tracer, thus confirming BBB integrity during 45 min of ischemia. Tight junction opening was not observed at 30 min after reperfusion following ischemia for 45 min. At the time of IR, [(3)H]Phe uptake into the brain appeared saturated. The Michaelis constant and maximum transport velocity in the IR group was reduced by 22 % compared with those in controls. These results suggest that the intrinsic transport clearance of Phe is slightly decreased in the early phase of IR.

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Predict drug permeability to blood–brain-barrier from clinical phenotypes: drug side effects and drug indications

PubMed Central

Gao, Zhen; Chen, Yang; Cai, Xiaoshu; Xu, Rong

2017-01-01

Abstract Motivation: Blood–Brain-Barrier (BBB) is a rigorous permeability barrier for maintaining homeostasis of Central Nervous System (CNS). Determination of compound’s permeability to BBB is prerequisite in CNS drug discovery. Existing computational methods usually predict drug BBB permeability from chemical structure and they generally apply to small compounds passing BBB through passive diffusion. As abundant information on drug side effects and indications has been recorded over time through extensive clinical usage, we aim to explore BBB permeability prediction from a new angle and introduce a novel approach to predict BBB permeability from drug clinical phenotypes (drug side effects and drug indications). This method can apply to both small compounds and macro-molecules penetrating BBB through various mechanisms besides passive diffusion. Results: We composed a training dataset of 213 drugs with known brain and blood steady-state concentrations ratio and extracted their side effects and indications as features. Next, we trained SVM models with polynomial kernel and obtained accuracy of 76.0%, AUC 0.739, and F1 score (macro weighted) 0.760 with Monte Carlo cross validation. The independent test accuracy was 68.3%, AUC 0.692, F1 score 0.676. When both chemical features and clinical phenotypes were available, combining the two types of features achieved significantly better performance than chemical feature based approach (accuracy 85.5% versus 72.9%, AUC 0.854 versus 0.733, F1 score 0.854 versus 0.725; P < e−90). We also conducted de novo prediction and identified 110 drugs in SIDER database having the potential to penetrate BBB, which could serve as start point for CNS drug repositioning research. Availability and Implementation: https://github.com/bioinformatics-gao/CASE-BBB-prediction-Data Contact: rxx@case.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27993785

A dynamic in vivo-like organotypic blood-brain barrier model to probe metastatic brain tumors

NASA Astrophysics Data System (ADS)

Xu, Hui; Li, Zhongyu; Yu, Yue; Sizdahkhani, Saman; Ho, Winson S.; Yin, Fangchao; Wang, Li; Zhu, Guoli; Zhang, Min; Jiang, Lei; Zhuang, Zhengping; Qin, Jianhua

2016-11-01

The blood-brain barrier (BBB) restricts the uptake of many neuro-therapeutic molecules, presenting a formidable hurdle to drug development in brain diseases. We proposed a new and dynamic in vivo-like three-dimensional microfluidic system that replicates the key structural, functional and mechanical properties of the blood-brain barrier in vivo. Multiple factors in this system work synergistically to accentuate BBB-specific attributes-permitting the analysis of complex organ-level responses in both normal and pathological microenvironments in brain tumors. The complex BBB microenvironment is reproduced in this system via physical cell-cell interaction, vascular mechanical cues and cell migration. This model possesses the unique capability to examine brain metastasis of human lung, breast and melanoma cells and their therapeutic responses to chemotherapy. The results suggest that the interactions between cancer cells and astrocytes in BBB microenvironment might affect the ability of malignant brain tumors to traverse between brain and vascular compartments. Furthermore, quantification of spatially resolved barrier functions exists within a single assay, providing a versatile and valuable platform for pharmaceutical development, drug testing and neuroscientific research.

Impact of Blood-Brain Barrier Integrity on Tumor Growth and Therapy Response in Brain Metastases.

PubMed

Osswald, Matthias; Blaes, Jonas; Liao, Yunxiang; Solecki, Gergely; Gömmel, Miriam; Berghoff, Anna S; Salphati, Laurent; Wallin, Jeffrey J; Phillips, Heidi S; Wick, Wolfgang; Winkler, Frank

2016-12-15

The role of blood-brain barrier (BBB) integrity for brain tumor biology and therapy is a matter of debate. We developed a new experimental approach using in vivo two-photon imaging of mouse brain metastases originating from a melanoma cell line to investigate the growth kinetics of individual tumor cells in response to systemic delivery of two PI3K/mTOR inhibitors over time, and to study the impact of microregional vascular permeability. The two drugs are closely related but differ regarding a minor chemical modification that greatly increases brain penetration of one drug. Both inhibitors demonstrated a comparable inhibition of downstream targets and melanoma growth in vitro In vivo, increased BBB permeability to sodium fluorescein was associated with accelerated growth of individual brain metastases. Melanoma metastases with permeable microvessels responded similarly to equivalent doses of both inhibitors. In contrast, metastases with an intact BBB showed an exclusive response to the brain-penetrating inhibitor. The latter was true for macro- and micrometastases, and even single dormant melanoma cells. Nuclear morphology changes and single-cell regression patterns implied that both inhibitors, if extravasated, target not only perivascular melanoma cells but also those distant to blood vessels. Our study provides the first direct evidence that nonpermeable brain micro- and macrometastases can effectively be targeted by a drug designed to cross the BBB. Small-molecule inhibitors with these optimized properties are promising agents in preventing or treating brain metastases in patients. Clin Cancer Res; 22(24); 6078-87. ©2016 AACRSee related commentary by Steeg et al., p. 5953. ©2016 American Association for Cancer Research.

Optimization of Evans blue quantitation in limited rat tissue samples

PubMed Central

Wang, Hwai-Lee; Lai, Ted Weita

2014-01-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting. PMID:25300427

Optimization of Evans blue quantitation in limited rat tissue samples

NASA Astrophysics Data System (ADS)

Wang, Hwai-Lee; Lai, Ted Weita

2014-10-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

PubMed Central

Guo, Chang-Ming; Chen, Rong-Rong; Kalhoro, Dildar Hussain; Wang, Zhao-Fei; Liu, Guang-Jin; Lu, Cheng-Ping; Liu, Yong-Jie

2014-01-01

Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB. PMID:24498419

Combined computational-experimental approach to predict blood-brain barrier (BBB) permeation based on "green" salting-out thin layer chromatography supported by simple molecular descriptors.

PubMed

Ciura, Krzesimir; Belka, Mariusz; Kawczak, Piotr; Bączek, Tomasz; Markuszewski, Michał J; Nowakowska, Joanna

2017-09-05

The objective of this paper is to build QSRR/QSAR model for predicting the blood-brain barrier (BBB) permeability. The obtained models are based on salting-out thin layer chromatography (SOTLC) constants and calculated molecular descriptors. Among chromatographic methods SOTLC was chosen, since the mobile phases are free of organic solvent. As consequences, there are less toxic, and have lower environmental impact compared to classical reserved phases liquid chromatography (RPLC). During the study three stationary phase silica gel, cellulose plates and neutral aluminum oxide were examined. The model set of solutes presents a wide range of log BB values, containing compounds which cross the BBB readily and molecules poorly distributed to the brain including drugs acting on the nervous system as well as peripheral acting drugs. Additionally, the comparison of three regression models: multiple linear regression (MLR), partial least-squares (PLS) and orthogonal partial least squares (OPLS) were performed. The designed QSRR/QSAR models could be useful to predict BBB of systematically synthesized newly compounds in the drug development pipeline and are attractive alternatives of time-consuming and demanding directed methods for log BB measurement. The study also shown that among several regression techniques, significant differences can be obtained in models performance, measured by R 2 and Q 2 , hence it is strongly suggested to evaluate all available options as MLR, PLS and OPLS. Copyright © 2017 Elsevier B.V. All rights reserved.

Bundle branch block after ablation for Wolff-Parkinson-White syndrome.

PubMed

Fuenmayor A, Abdel J; Rodríguez S, Yenny A

2013-09-20

Bundle branch block (BBB) is a difficult diagnosis in the Wolff-Parkinson-White syndrome (WPW). We investigated the clinical implications of BBB that appears after performing an accessory pathway (AP) ablation. We studied 199 patients with WPW who were submitted to AP ablation. Thirty (15%) exhibited BBB after the ablation. Twenty-two patients had right BBB and 8 had left BBB. Thirteen patients had right-sided AP and 17 had left-sided AP. They were compared with 82 similar patients without BBB after the AP ablation. Among the patients with BBB, 86.66% showed delays in the middle part of the QRS in the ECG recorded before ablation vs. 18.29% of the patients without BBB (p<0.05) (sensitivity 86%, specificity 81%, positive predictive value 67% and negative predictive value 93%). Forty-four percent of the patients with BBB had BBB morphology during orthodromic tachycardia vs. 10% of the patients without BBB (p<0.05) (sensitivity 44%, specificity 89%, positive predictive value 57% and negative predictive value 82%). No relationship was found between AP location and the site of the BBB. Ejection fraction was normal before (0.61 ± 0.03) and upon completion of follow-up (0.61 ± 0.07). BBB disappeared in 95.3% of the patients. Delays in the middle portion of the QRS may predict BBB after AP ablation. BBB after performing AP ablation is frequent, transient, benign, and not related to either the ablation lesion location or progression to structural heart disease. BBB after AP ablation may be related to cardiac memory. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Ultrasound-mediated blood-brain barrier disruption for targeted drug delivery in the central nervous system

PubMed Central

Aryal, Muna; Arvanitis, Costas D.; Alexander, Phillip M.; McDannold, Nathan

2014-01-01

The physiology of the vasculature in the central nervous system (CNS), which includes the blood-brain barrier (BBB) and other factors, complicates the delivery of most drugs to the brain. Different methods have been used to bypass the BBB, but they have limitations such as being invasive, non-targeted or requiring the formulation of new drugs. Focused ultrasound (FUS), when combined with circulating microbubbles, is a noninvasive method to locally and transiently disrupt the BBB at discrete targets. This review provides insight on the current status of this unique drug delivery technique, experience in preclinical models, and potential for clinical translation. If translated to humans, this method would offer a flexible means to target therapeutics to desired points or volumes in the brain, and enable the whole arsenal of drugs in the CNS that are currently prevented by the BBB. PMID:24462453

Planar harmonic polynomials of type B

NASA Astrophysics Data System (ADS)

Dunkl, Charles F.

1999-11-01

The hyperoctahedral group acting on icons/Journals/Common/BbbR" ALT="BbbR" ALIGN="TOP"/>N is the Weyl group of type B and is associated with a two-parameter family of differential-difference operators {Ti:1icons/Journals/Common/leq" ALT="leq" ALIGN="TOP"/> iicons/Journals/Common/leq" ALT="leq" ALIGN="TOP"/> N}. These operators are analogous to partial derivative operators. This paper finds all the polynomials h on icons/Journals/Common/BbbR" ALT="BbbR" ALIGN="TOP"/>N which are harmonic, icons/Journals/Common/Delta" ALT="Delta" ALIGN="TOP"/>Bh = 0 and annihilated by Ti for i>2, where the Laplacian 0305-4470/32/46/308/img1" ALT="(sum). They are given explicitly in terms of a novel basis of polynomials, defined by generating functions. The harmonic polynomials can be used to find wavefunctions for the quantum many-body spin Calogero model.

EMP-induced BBB-disruption enhances drug delivery to glioma and increases treatment efficacy in rats.

PubMed

Li, Kangchu; Zhang, Keying; Xu, Shenglong; Wang, Xiaowu; Zhou, Yongchun; Zhou, Yan; Gao, Peng; Lin, Jiajin; Ding, Guirong; Guo, Guozhen

2018-01-01

Chemotherapy on gliomas is not satisfactorily efficient because the presence of blood-brain barriers (BBB) leads to inadequate exposure of tumor cells to administered drugs. In order to facilitate chemotherapeutics to penetrate BBB and increase the treatment efficacy of gliomas, electromagnetic pulse (EMP) was applied and the 1-(2-Chlorethyl)-cyclohexyl-nitrosourea (CCNU) lomustine concentration in tumor tissue, tumor size, tumor apoptosis, and side effects were measured in glioma-bearing rat model. The results showed that EMP exposure could enhance the delivery of CCNU to tumor tissue, facilitate tumor apoptosis, and inhibit tumor growth without obvious side effects. The data indicated that EMP-induced BBB disruption could enhance delivery of CCNU to glioblastoma multiforme and increase treatment efficacy in glioma-bearing rats. Bioelectromagnetics. 39:60-67, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Amylin Enhances Amyloid-β Peptide Brain to Blood Efflux Across the Blood-Brain Barrier

PubMed Central

Mohamed, Loqman A.; Zhu, Haihao; Mousa, Youssef M.; Wang, Erming; Qiu, Wei Qiao; Kaddoumi, Amal

2017-01-01

Findings from Alzheimer’s disease (AD) mouse models showed that amylin treatment improved AD pathology and enhanced amyloid-β (Aβ) brain to blood clearance; however, the mechanism was not investigated. Using the Tg2576 AD mouse model, a single intraperitoneal injection of amylin significantly increased Aβ serum levels, and the effect was abolished by AC253, an amylin receptor antagonist, suggesting that amylin effect could be mediated by its receptor. Subsequent mechanistic studies showed amylin enhanced Aβ transport across a cell-based model of the blood-brain barrier (BBB), an effect that was abolished when the amylin receptor was inhibited by two amylin antagonists and by siRNA knockdown of amylin receptor Ramp3. To explain this finding, amylin effect on Aβ transport proteins expressed at the BBB was evaluated. Findings indicated that cells treated with amylin induced LRP1 expression, a major receptor involved in brain Aβ efflux, in plasma membrane fraction, suggesting intracellular translocation of LRP1 from the cytoplasmic pool. Increased LRP1 in membrane fraction could explain, at least in part, the enhanced uptake and transport of Aβ across the BBB. Collectively, our findings indicated that amylin induced Aβ brain to blood clearance through amylin receptor by inducing LRP1 subcellular translocation to the plasma membrane of the BBB endothelium. PMID:28059785

Endothelial cells are critical regulators of iron transport in a model of the human blood-brain barrier.

PubMed

Chiou, Brian; Neal, Emma H; Bowman, Aaron B; Lippmann, Ethan S; Simpson, Ian A; Connor, James R

2018-01-01

Iron delivery to the brain is essential for multiple neurological processes such as myelination, neurotransmitter synthesis, and energy production. Loss of brain iron homeostasis is a significant factor in multiple neurological disorders. Understanding the mechanism by which the transport of iron across the blood-brain barrier (BBB) is regulated is crucial to address the impact of iron deficiency on brain development and excessive accumulation of iron in neurodegenerative diseases. Using induced pluripotent stem cell (iPSC)-derived brain endothelial cells (huECs) as a human BBB model, we demonstrate the ability of transferrin, hepcidin, and DMT1 to impact iron transport and release. Our model reveals a new function for H-ferritin to transport iron across the BBB by binding to the T-cell immunoglobulin and mucin receptor 1. We show that huECs secrete both transferrin and H-ferritin, which can serve as iron sources for the brain. Based on our data, brain iron status can exert control of iron transport across the endothelial cells that constitute the BBB. These data address a number of pertinent questions such as how brain iron uptake is regulated at the regional level, the source of iron delivery to the brain, and the clinical strategies for attempting to treat brain iron deficiency.

Death receptors DR6 and TROY regulate brain vascular development.

PubMed

Tam, Stephen J; Richmond, David L; Kaminker, Joshua S; Modrusan, Zora; Martin-McNulty, Baby; Cao, Tim C; Weimer, Robby M; Carano, Richard A D; van Bruggen, Nick; Watts, Ryan J

2012-02-14

Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development. Copyright © 2012 Elsevier Inc. All rights reserved.

Clematichinenoside protects blood brain barrier against ischemic stroke superimposed on systemic inflammatory challenges through up-regulating A20.

PubMed

Han, Dan; Fang, Weirong; Zhang, Rui; Wei, Jie; Kodithuwakku, Nandani Darshika; Sha, Lan; Ma, Wenhuan; Liu, Lifang; Li, Fengwen; Li, Yunman

2016-01-01

Suppression of excessive inflammation can ameliorate blood brain barrier (BBB) injury, which shows therapeutic potential for clinical treatment of brain injury induced by stroke superimposed on systemic inflammatory diseases. In this study, we investigated whether and how clematichinenoside (AR), an anti-inflammatory triterpene saponin, protects brain injury from stroke superimposed on systemic inflammation. Lipopolysaccharide (LPS) was intraperitoneally injected immediately after middle cerebral artery occlusion (MCAO) in rats. Rat microvessel endothelial cells (rBMECs) were exposed to hypoxia/reoxygenation (H/R) coexisting with LPS. The results revealed that AR suppressed the excessive inflammation, restored BBB dysfunction, alleviated brain edema, decreased neutrophil infiltration, lessened neurological dysfunction, and decreased infarct rate. Further study demonstrated that the expression of nucleus nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interlukin-1β (IL-1β) were suppressed by AR via zinc finger protein A20. Besides, AR increased in vitro BBB integrity through A20. In conclusion, AR alleviated cerebral inflammatory injury through A20-NF-κB signal pathway, offering an alternative medication for stroke associated with systemic inflammatory diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

nRGD modified lycobetaine and octreotide combination delivery system to overcome multiple barriers and enhance anti-glioma efficacy.

PubMed

Chen, Tijia; Song, Xu; Gong, Ting; Fu, Yao; Yang, Liuqing; Zhang, Zhirong; Gong, Tao

2017-08-01

For glioma as one of the most common and lethal primary brain tumors, the presence of BBB, BBTB, vasculogenic mimicry (VM) channels and tumor-associated macrophages (TAMs) are key biological barriers. Here, a novel drug delivery system which could efficiently deliver drugs to glioma by overcoming multi-barriers and increase antitumor efficacy through multi-therapeutic mechanisms was well developed. In this study, a multi-target peptide nRGD was used to transport across the BBB, mediate tumor penetration and target TAMs. Lycobetaine (LBT) was adopted to kill glioma cells and octreotide (OCT) was co-delivered to inhibit VM channels and prevent angiogenesis. LBT-OCT liposomes (LPs) showed controlled release profile in vitro, increased uptake efficiency, improved inhibitory effect against glioma cells and VM formation, and enhanced BBB-crossing capability. The median survival time of glioma-bearing mice administered with LBT-OCT LPs-nRGD was significantly longer than LBT-OCT LPs (P<0.01). Besides, nRGD achieved a stronger inhibitory effect against tumor associated macrophages (TAMs) compared to LPs-iRGD treatment groups in vivo. Thus, LPs-nRGD represented a promising versatile delivery platform for combination drug therapy in glioma treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Memory is preserved in older adults taking AT1 receptor blockers.

PubMed

Ho, Jean K; Nation, Daniel A

2017-04-26

Prior work suggests that some but not all antihypertensive treatments may benefit cognition and risk for Alzheimer's disease, independent of stroke. Angiotensin II receptor blockers (ARBs) have been highlighted as one antihypertensive drug class that may confer greatest benefit. The participants comprised 1626 nondemented adults, aged 55-91 years, recruited from Alzheimer's Disease Neuroimaging Initiative sites. Three groups were compared: ARB users (HTN-ARBs), other antihypertensive drug users (HTN-Other), and normotensives. In post hoc analyses, we also examined (1) users of ARBs and angiotensin-converting enzyme inhibitors (ACEIs), (2) users of blood-brain barrier (BBB)-crossing ARBs and users of non-BBB-crossing ARBs, and (3) users of BBB-crossing ARBs and ACEIs (BBB crossers) and users of non-BBB-crossing ARBs and ACEIs (BBB noncrossers). Groups were compared regarding cognition and magnetic resonance imaging measures of brain volume and white matter hyperintensities (WMH), using analysis of covariance and multilevel models. At baseline, the HTN-Other group performed worse than normotensives on Rey Auditory Verbal Learning Test (RAVLT) Immediate Recall (p = 0.002), Delayed Recall (p < 0.001), Recognition Memory (p = 0.001), and Trails A (p < 0.001) and B (p = 0.01). ARB users performed better than the HTN-Other group on Recognition Memory (p = 0.04) and worse than normotensives on Trails A (p = 0.04). The HTN-Other group performed worse than normotensives on Logical Memory Immediate (p = 0.02) and Delayed Recall over the 3-year follow-up (p = 0.007). Over the follow-up period, those taking BBB-crossing ARBs performed better than the HTN-Other group on AVLT Delayed Recall (p = 0.04), Logical Memory Immediate (p = 0.02), and Delayed Recall (p = 0.05). They also had fewer WMH than the HTN-Other group (p = 0.008) and those taking non-BBB-crossing ARBs (p = 0.05). There were no group differences in brain volume. Users of BBB-crossing medications (ARBs or ACEIs) showed better performance on AVLT Delayed Recall over time than all other groups, including normotensives (all p < 0.01), and had less WMH volume over time than the BBB noncrossers group (p = 0.03), although they had more WMH volume than normotensives (p = 0.01). The BBB noncrossers group performed worse than normotensives on Logical Memory Delayed Recall over time (p = 0.01), but the BBB crossers group was not significantly different (p = 0.13). Hypertensive participants demonstrated worse baseline memory and executive function, as well as greater memory decline, over the 3-year follow-up than normotensives, unless they were ARB users, who showed preserved memory compared with those taking other antihypertensive drugs. Users of BBB-crossing ARBs showed superior memory performance over time compared with other antihypertensive drug users and had less WMH volume. Users of BBB-crossing medications (ARBs or ACEIs) showed better list-learning memory performance over time than all other groups, including normotensives, and had less WMH volume over time than users of non-BBB-crossing medications. These findings demonstrate that ARBs, especially those of the BBB-crossing variety, are associated with greater memory preservation and less WMH volume than other antihypertensive medications.

Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature.

PubMed

Alvarez, Jorge I; Teale, Judy M

2007-09-12

The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption was explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) were evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity.

Alzheimer's disease-like impaired cognition in endothelial-specific megalin-null mice.

PubMed

Dietrich, Marcelo; Antequera, Desiree; Pascual, Consuelo; Castro, Nerea; Bolos, Marta; Carro, Eva

2014-01-01

Megalin has been suggested to be involved in Alzheimer's disease (AD), mediating blood-brain barrier (BBB) transport of multiple ligands, including amyloid-β peptide (Aβ), but also neuroprotective factors. Because no transgenic model is currently available to study this concept, we have obtained transgenic mice blocking megalin expression at the BBB. These endothelial megalin deficient (EMD) mice developed increased anxiety behavior and impaired learning ability and recognition memory, similar to symptoms described in AD. Degenerating neurons were also observed in the cerebral cortex of EMD mice. In view of our findings we suggest that, in mice, megalin deficiency at the BBB leads to neurodegeneration.

Detrimental role of the EP1 prostanoid receptor in blood-brain barrier damage following experimental ischemic stroke

PubMed Central

Frankowski, Jan C.; DeMars, Kelly M.; Ahmad, Abdullah S.; Hawkins, Kimberly E.; Yang, Changjun; Leclerc, Jenna L.; Doré, Sylvain; Candelario-Jalil, Eduardo

2015-01-01

Cyclooxygenase-2 (COX-2) is activated in response to ischemia and significantly contributes to the neuroinflammatory process. Accumulation of COX-2-derived prostaglandin E2 (PGE2) parallels the substantial increase in stroke-mediated blood-brain barrier (BBB) breakdown. Disruption of the BBB is a serious consequence of ischemic stroke, and is mainly mediated by matrix metalloproteinases (MMPs). This study aimed to investigate the role of PGE2 EP1 receptor in neurovascular injury in stroke. We hypothesized that pharmacological blockade or genetic deletion of EP1 protects against BBB damage and hemorrhagic transformation by decreasing the levels and activity of MMP-3 and MMP-9. We found that post-ischemic treatment with the EP1 antagonist, SC-51089, or EP1 genetic deletion results in a significant reduction in BBB disruption and reduced hemorrhagic transformation in an experimental model of transient focal cerebral ischemia. These neurovascular protective effects of EP1 inactivation are associated with a significant reduction in MMP-9/-3, less peripheral neutrophil infiltration, and a preservation of tight junction proteins (ZO-1 and occludin) composing the BBB. Our study identifies the EP1 signaling pathway as an important link between neuroinflammation and MMP-mediated BBB breakdown in ischemic stroke. Targeting the EP1 receptor could represent a novel approach to diminish the devastating consequences of stroke-induced neurovascular damage. PMID:26648273

A new descriptor via bio-mimetic chromatography and modeling for the blood brain barrier (Part II).

PubMed

Kouskoura, Maria G; Piteni, Aikaterini I; Markopoulou, Catherine K

2018-05-25

Within the context of drug design methodology for the central nervous system (CNS), a predictive model which can shorten the process of finding new candidate drugs was developed. Therefore, the retention time of 51 molecules which are clinically established to enter the blood brain barrier (BBB), were recorded on two HPLC columns. For this purpose, a lipophilic butyl (C 4 ) stationary phase was used to simulate the behavior of a drug regarding BBB permeability and a zwitterionic-HILIC to simulate blood. The results were plotted as Y variables on two Partial Least Squares (PLS) models, while 25 specific physicochemical properties (significant for lipid bilayers BBB permeation or blood) were used as X descriptors. Both models can be utilized to predict the drugability of a new molecule avoiding needless animal experiments, as well as time and material consuming syntheses. The developed models were validated (R 2  ≥ 0.90, Q 2  ≥ 0.83), and based on the results specific variables were proved to be significant for the studied phenomenon. Additionally, a new factor symbolized as MT was introduced. MT incorporated the experimental results and it was calculated by the fraction of the sum of the retention time of the drug on the two columns (t r(butyl)  + t r(HILIC) ) divided by the molecular volume (V m ) of each analyte. This new descriptor was used as an equivalent to the logarithm of BBB permeability (logBB) and may indicate the ability of a new molecule to act as a candidate drug able to enter the BBB. Comprehending the extend of contribution of several molecular attributes to the in vivo distribution of a drug may enlighten the knowledge on pharmacokinetics and clinical variation, and enable scientists to design more efficient drug molecules. Copyright © 2018 Elsevier B.V. All rights reserved.

Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells.

PubMed

McCarthy, Ryan C; Kosman, Daniel J

2014-01-01

We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.

αB-crystallin: a Novel Regulator of Breast Cancer Metastasis to the Brain

PubMed Central

Malin, Dmitry; Strekalova, Elena; Petrovic, Vladimir; Deal, Allison M.; Ahmad, Abraham Al; Adamo, Barbara; Miller, C. Ryan; Ugolkov, Andrey; Livasy, Chad; Fritchie, Karen; Hamilton, Erika; Blackwell, Kimberly; Geradts, Joseph; Ewend, Matt; Carey, Lisa; Shusta, Eric V.; Anders, Carey K.; Cryns, Vincent L.

2013-01-01

Purpose Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBC. Experimental Design αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among breast cancer patients with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMECs) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte co-culture blood-brain barrier (BBB) model were examined. Additionally, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models. Results In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among TNBC patients. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, while silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs at least in part through an α3β1 integrin-dependent mechanism. αB-crystallin overexpression promoted brain metastasis, while silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models. Conclusion αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease. PMID:24132917

αB-crystallin: a novel regulator of breast cancer metastasis to the brain.

PubMed

Malin, Dmitry; Strekalova, Elena; Petrovic, Vladimir; Deal, Allison M; Al Ahmad, Abraham; Adamo, Barbara; Miller, C Ryan; Ugolkov, Andrey; Livasy, Chad; Fritchie, Karen; Hamilton, Erika; Blackwell, Kimberly; Geradts, Joseph; Ewend, Matt; Carey, Lisa; Shusta, Eric V; Anders, Carey K; Cryns, Vincent L

2014-01-01

Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBCs. αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood-brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models. In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an α3β1 integrin-dependent mechanism. αB-crystallin overexpression promoted brain metastasis, whereas silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models. αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease.

Blood-Brain Barrier Alterations Provide Evidence of Subacute Diaschisis in an Ischemic Stroke Rat Model

PubMed Central

Garbuzova-Davis, Svitlana; Rodrigues, Maria C. O.; Hernandez-Ontiveros, Diana G.; Tajiri, Naoki; Frisina-Deyo, Aric; Boffeli, Sean M.; Abraham, Jerry V.; Pabon, Mibel; Wagner, Andrew; Ishikawa, Hiroto; Shinozuka, Kazutaka; Haller, Edward; Sanberg, Paul R.; Kaneko, Yuji; Borlongan, Cesario V.

2013-01-01

Background Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB) competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas. Methodology/Principal Findings In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO), significant BBB alterations characterized by large Evans Blue (EB) parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices. Conclusions/Significance These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke. PMID:23675488

Kv1.3 channel blocker (ImKTx88) maintains blood-brain barrier in experimental autoimmune encephalomyelitis.

PubMed

Huang, Jie; Han, Song; Sun, Qi; Zhao, Yipeng; Liu, Junchen; Yuan, Xiaolu; Mao, Wenqian; Peng, Biwen; Liu, Wanhong; Yin, Jun; He, Xiaohua

2017-01-01

Disruption of blood-brain barrier (BBB) and subsequent infiltration of auto-reactive T lymphocytes are major characteristics of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Kv1.3 channel blockers are demonstrated potential therapeutic effects on MS patients and EAE models, maybe via reducing activation of T cells. However, it remains to be explored whether Kv1.3 channel blockers maintain integrity of BBB in MS model. In this study, ImKTx88, a highly selective Kv1.3 channel blocker, was used to determine the role of Kv1.3 channel in the pathogenesis of EAE, particularly in the maintenance of BBB. ImKTx88 ameliorated pathological severity in the EAE rats, and reduced extravasation into CNS. ImKTx88 also ameliorated the severity of loss or redistribution of tight junction proteins, and inhibited over-expression of ICAM-1 and VCAM-1 in the brain from EAE rats. Furthermore ImKTx88 protection was associated with activation of Ang-1/Tie-2 axis, and might be due to decreased IL-17 production. ImKTx88 may be a novel therapeutic agent for MS treatment by stabilizing the BBB.

Nanoparticle transport across in vitro olfactory cell monolayers.

PubMed

Gartziandia, Oihane; Egusquiaguirre, Susana Patricia; Bianco, John; Pedraz, José Luis; Igartua, Manoli; Hernandez, Rosa Maria; Préat, Véronique; Beloqui, Ana

2016-02-29

Drug access to the CNS is hindered by the presence of the blood-brain barrier (BBB), and the intranasal route has risen as a non-invasive route to transport drugs directly from nose-to-brain avoiding the BBB. In addition, nanoparticles (NPs) have been described as efficient shuttles for direct nose-to-brain delivery of drugs. Nevertheless, there are few studies describing NP nose-to-brain transport. Thus, the aim of this work was (i) to develop, characterize and validate in vitro olfactory cell monolayers and (ii) to study the transport of polymeric- and lipid-based NPs across these monolayers in order to estimate NP access into the brain using cell penetrating peptide (CPPs) moieties: Tat and Penetratin (Pen). All tested poly(d,l-lactide-co-glycolide) (PLGA) and nanostructured lipid carrier (NLC) formulations were stable in transport buffer and biocompatible with the olfactory mucosa cells. Nevertheless, 0.7% of PLGA NPs was able to cross the olfactory cell monolayers, whereas 8% and 22% of NLC and chitosan-coated NLC (CS-NLC) were transported across them, respectively. Moreover, the incorporation of CPPs to NLC surface significantly increased their transport, reaching 46% of transported NPs. We conclude that CPP-CS-NLC represent a promising brain shuttle via nose-to-brain for drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

Revisiting atenolol as a low passive permeability marker.

PubMed

Chen, Xiaomei; Slättengren, Tim; de Lange, Elizabeth C M; Smith, David E; Hammarlund-Udenaes, Margareta

2017-10-31

Atenolol, a hydrophilic beta blocker, has been used as a model drug for studying passive permeability of biological membranes such as the blood-brain barrier (BBB) and the intestinal epithelium. However, the extent of S-atenolol (the active enantiomer) distribution in brain has never been evaluated, at equilibrium, to confirm that no transporters are involved in its transport at the BBB. To assess whether S-atenolol, in fact, depicts the characteristics of a low passive permeable drug at the BBB, a microdialysis study was performed in rats to monitor the unbound concentrations of S-atenolol in brain extracellular fluid (ECF) and plasma during and after intravenous infusion. A pharmacokinetic model was developed, based on the microdialysis data, to estimate the permeability clearance of S-atenolol into and out of brain. In addition, the nonspecific binding of S-atenolol in brain homogenate was evaluated using equilibrium dialysis. The steady-state ratio of unbound S-atenolol concentrations in brain ECF to that in plasma (i.e., K p,uu,brain ) was 3.5% ± 0.4%, a value much less than unity. The unbound volume of distribution in brain (V u, brain ) of S-atenolol was also calculated as 0.69 ± 0.10 mL/g brain, indicating that S-atenolol is evenly distributed within brain parenchyma. Lastly, equilibrium dialysis showed limited nonspecific binding of S-atenolol in brain homogenate with an unbound fraction (f u,brain ) of 0.88 ± 0.07. It is concluded, based on K p,uu,brain being much smaller than unity, that S-atenolol is actively effluxed at the BBB, indicating the need to re-consider S-atenolol as a model drug for passive permeability studies of BBB transport or intestinal absorption.

Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells.

PubMed

Yang, Shu; Jin, Hong; Zhao, Zhigang

2018-04-23

Objective The blood-brain barrier (BBB), regulating brain homeostasis and limiting the entry of most drugs, is characterized by intercellular tight junctions and the presence of transporters. In this study, the paracellular tightness and functional expression of efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were evaluated in mouse brain immortalized cell line bEnd3 to prove it as a useful BBB-mimicking system for biological and pharmacological research. Methods The presence of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 were validated by RT-PCR and Western blot. The tightness of bEnd3 monolayers was evaluated by measuring the permeability of hydrophilic marker Lucifer yellow. The P-gp functionality was identified by intracellular uptake assay using Rhodamine 123 (R123) as P-gp substrate and verapamil as P-gp inhibitor. The BCRP functionality was identified by flow cytometric analysis of mitoxantrone accumulation and fluorescence microscopic analysis of Hoechst 33342 accumulation using Ko-143 as BCRP inhibitor. Results The bEnd3 cells demonstrated the expression of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 at mRNA and protein levels. The permeability coefficient of Lucifer yellow was 1.3 ± 0.13 × 10 -3  cm/min, indicating the moderate paracellular tightness barrier formed by bEnd3 cells. The verapamil induced a higher cellular uptake of Rhodamine 123, and Ko-143 significantly elevated cellular accumulation of mitoxantrone and Hoechst 33342, suggesting the P-gp and BCRP functionality shown by bEnd3 cells. Conclusions The bEnd3 cell line represents a useful in vitro tool for studying BBB characteristics and drug transport mechanisms at the BBB.

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells.

PubMed

Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

2016-10-13

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis , permeabilizes the blood-brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218's effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

PubMed Central

Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

2016-01-01

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB. PMID:27754355

Examination of Blood-Brain Barrier (BBB) Integrity In A Mouse Brain Tumor Model

PubMed Central

On, Ngoc; Mitchell, Ryan; Savant, Sanjot D.; Bachmeier, Corbin. J.; Hatch, Grant M.; Miller, Donald W.

2013-01-01

The present study evaluates, both functionally and biochemically, brain tumor-induced alterations in brain capillary endothelial cells. Brain tumors were induced in Balb/c mice via intracranial injection of Lewis Lung carcinoma (3LL) cells into the right hemisphere of the mouse brain using stereotaxic apparatus. Blood-brain barrier (BBB) permeability was assessed at various stages of tumor development, using both radiolabeled tracer permeability and magnetic resonance imaging (MRI) with gadolinium diethylene-triamine-pentaacetate contrast enhancement (Gad-DTPA). The expression of the drug efflux transporter, P-glycoprotein (P-gp), in the BBB at various stages of tumor development was also evaluated by Western blot and immunohistochemistry. Median mouse survival following tumor cell injection was 17 days. The permeability of the BBB to 3H-mannitol was similar in both brain hemispheres at 7 and 10 days post-injection. By day 15, there was a 2-fold increase in 3H-mannitol permeability in the tumor bearing hemispheres compared to the non-tumor hemispheres. Examination of BBB permeability with Gad-DTPA contrast enhanced MRI indicated cerebral vascular permeability changes were confined to the tumor area. The permeability increase observed at the later stages of tumor development correlated with an increase in cerebral vascular volume suggesting angiogenesis within the tumor bearing hemisphere. Furthermore, the Gad-DPTA enhancement observed within the tumor area was significantly less than Gad-DPTA enhancement within the circumventricular organs not protected by the BBB. Expression of P-gp in both the tumor bearing and non-tumor bearing portions of the brain appeared similar at all time points examined. These studies suggest that although BBB integrity is altered within the tumor site at later stages of development, the BBB is still functional and limiting in terms of solute and drug permeability in and around the tumor. PMID:23184143

Novel Method for Analyzing Locomotor Ability after Spinal Cord Injury in Rats: Technical Note

PubMed Central

Shinozaki, Munehisa; Yasuda, Akimasa; Nori, Satoshi; Saito, Nobuhito; Toyama, Yoshiaki; Okano, Hideyuki; Nakamura, Masaya

2013-01-01

In the research for the treatment of spinal cord injury (SCI), the evaluation of motor function in model rats must be as objective, noninvasive, and ethical as possible. The maximum speed and acceleration of a mouse measured using a SCANET system were previously reported to vary significantly according to severity of SCI. In the present study, the motor performance of SCI model rats was examined with SCANET and assessed for Basso-Beattie-Bresnahan (BBB) score to determine the usefulness of the SCANET system in evaluating functional recovery after SCI. Maximum speed and acceleration within the measurement period correlated significantly with BBB scores. Furthermore, among several phased kinematic factors used in BBB scores, the capability of “plantar stepping” was associated with a drastic increase in maximum speed and acceleration after SCI. Therefore, evaluation of maximum speed and acceleration using a SCANET system is a useful method for rat models of SCI and can complement open field scoring scales. PMID:24097095

Development, Characterization, and Implementation of a System for Focused Ultrasound-Mediated Blood-Brain Barrier Opening in Mice

NASA Astrophysics Data System (ADS)

Valdez, Michael Aaron

The blood-brain barrier BBB refers to the set of specialized endothelial cells that line the vasculature in the brain and effectively control movement of molecules into and out of the brain. While necessary for proper brain function, the BBB blocks 98% of drugs from entering the brain and is the most significant barrier to developing therapies for neurodegenerative diseases. Active transport allows some specific molecules to cross the BBB, but therapeutic development using this route has had limited success. A number of techniques have been used to bypass the BBB, but are often highly invasive and ineffective. Over the last two decades, a minimally invasive technique to transiently open the BBB has been under development that utilizes transcranial focused ultrasound (FUS) in combination with intravascular microbubble contrast agents. This method is often carried out in conjunction with magnetic resonance imaging (MRI) to guide and assess BBB opening and has been referred to as MRI guided FUS (MRgFUS). Because of the utility of mouse models of neurological disease and the exploratory nature of MRgFUS, systems that allow BBB opening in mice are a useful and necessary tool to develop and evaluate this method for clinical application. In this dissertation project, a custom built, cost-effective FUS system for opening the BBB in mice was developed, with the objective of using this device to deliver therapeutics to the brain. Being a custom device, it was necessary to evaluate the ultrasound output, verify in vivo safety, and anticipate the therapeutic effect. The scope of the work herein consists of the design, construction, and evaluation of system that fulfills these requirements. The final constructed system cost was an order of magnitude less than any commercially available MRgFUS system. At this low price point, the hardware could allow the implementation of the methodology in many more research areas than previously possible. Additionally, to anticipate the therapeutic effect, molecules of pharmacologically-relevant sizes were delivered to brain with a novel, multispectral approach. Results demonstrated that the device was able to safely open the BBB, and macromolecule delivery showed that both molecule size and FUS pressure both influence the amount and distribution of molecules in the brain. Using different ultrasound pressures, the threshold for BBB opening was found to be ≥ 180 kPa (0.13 MI). The threshold for damage was found to be ≥ 420 kPa (0.30 MI), and was minor at this pressure, but extensive for higher pressure (870 kPa, 0.62 MI), in which minor damage was caused by this pressure. Performing a novel implementation of a diffusion model on the fluorescence images of 500, 70, and 3 kDa dextran resulted in calculated diffusion coefficients of 0.032 +/- 0.015, 12 +/- 6.0, and 0.13 +/- 0.094 square microns per second, respectively.

Nutraceutical agents with anti-inflammatory properties prevent dietary saturated-fat induced disturbances in blood-brain barrier function in wild-type mice.

PubMed

Takechi, Ryusuke; Pallebage-Gamarallage, Menuka M; Lam, Virginie; Giles, Corey; Mamo, John C

2013-06-19

Emerging evidence suggests that disturbances in the blood-brain barrier (BBB) may be pivotal to the pathogenesis and pathology of vascular-based neurodegenerative disorders. Studies suggest that heightened systemic and central inflammations are associated with BBB dysfunction. This study investigated the effect of the anti-inflammatory nutraceuticals garlic extract-aged (GEA), alpha lipoic acid (ALA), niacin, and nicotinamide (NA) in a murine dietary-induced model of BBB dysfunction. C57BL/6 mice were fed a diet enriched in saturated fatty acids (SFA, 40% fat of total energy) for nine months to induce systemic inflammation and BBB disturbances. Nutraceutical treatment groups included the provision of either GEA, ALA, niacin or NA in the positive control SFA-group and in low-fat fed controls. Brain parenchymal extravasation of plasma derived immunoglobulin G (IgG) and large macromolecules (apolipoprotein (apo) B lipoproteins) measured by quantitative immunofluorescent microscopy, were used as markers of disturbed BBB integrity. Parenchymal glial fibrillar acidic protein (GFAP) and cyclooxygenase-2 (COX-2) were considered in the context of surrogate markers of neurovascular inflammation and oxidative stress. Total anti-oxidant status and glutathione reductase activity were determined in plasma. Brain parenchymal abundance of IgG and apoB lipoproteins was markedly exaggerated in mice maintained on the SFA diet concomitant with significantly increased GFAP and COX-2, and reduced systemic anti-oxidative status. The nutraceutical GEA, ALA, niacin, and NA completely prevented the SFA-induced disturbances of BBB and normalized the measures of neurovascular inflammation and oxidative stress. The anti-inflammatory nutraceutical agents GEA, ALA, niacin, or NA are potent inhibitors of dietary fat-induced disturbances of BBB induced by systemic inflammations.

Surfactants, not size or zeta-potential influence blood-brain barrier passage of polymeric nanoparticles.

PubMed

Voigt, Nadine; Henrich-Noack, Petra; Kockentiedt, Sarah; Hintz, Werner; Tomas, Jürgen; Sabel, Bernhard A

2014-05-01

Nanoparticles (NP) can deliver drugs across the blood-brain barrier (BBB), but little is known which of the factors surfactant, size and zeta-potential are essential for allowing BBB passage. To this end we designed purpose-built fluorescent polybutylcyanoacrylate (PBCA) NP and imaged the NP's passage over the blood-retina barrier - which is a model of the BBB - in live animals. Rats received intravenous injections of fluorescent PBCA-NP fabricated by mini-emulsion polymerisation to obtain various NP's compositions that varied in surfactants (non-ionic, anionic, cationic), size (67-464nm) and zeta-potential. Real-time imaging of retinal blood vessels and retinal tissue was carried out with in vivo confocal neuroimaging (ICON) before, during and after NP's injection. Successful BBB passage with subsequent cellular labelling was achieved if NP were fabricated with non-ionic surfactants or cationic stabilizers but not when anionic compounds were added. NP's size and charge had no influence on BBB passage and cell labelling. This transport was not caused by an unspecific opening of the BBB because control experiments with injections of unlabelled NP and fluorescent dye (to test a "door-opener" effect) did not lead to parenchymal labelling. Thus, neither NP's size nor chemo-electric charge, but particle surface is the key factor determining BBB passage. This result has important implications for NP engineering in medicine: depending on the surfactant, NP can serve one of two opposite functions: while non-ionic tensides enhance brain up-take, addition of anionic tensides prevents it. NP can now be designed to specifically enhance drug delivery to the brain or, alternatively, to prevent brain penetration so to reduce unwanted psychoactive effects of drugs or prevent environmental nanoparticles from entering tissue of the central nervous system. Copyright © 2014 Elsevier B.V. All rights reserved.

Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction

PubMed Central

Jones, Letitia D.; Jackson, Joseph W.; Maggirwar, Sanjay B.

2016-01-01

The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND. PMID:26986758

Hypertonic saline alleviates experimentally induced cerebral oedema through suppression of vascular endothelial growth factor and its receptor VEGFR2 expression in astrocytes.

PubMed

Huang, Linqiang; Cao, Wei; Deng, Yiyu; Zhu, Gaofeng; Han, Yongli; Zeng, Hongke

2016-10-13

Cerebral oedema is closely related to the permeability of blood-brain barrier, vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) all of which are important blood-brain barrier (BBB) permeability regulatory factors. Zonula occludens 1 (ZO-1) and claudin-5 are also the key components of BBB. Hypertonic saline is widely used to alleviate cerebral oedema. This study aimed to explore the possible mechanisms underlying hypertonic saline that ameliorates cerebral oedema effectively. Middle cerebral artery occlusion (MCAO) model in Sprague-Dawley (SD) rats and of oxygen-glucose deprivation model in primary astrocytes were used in this study. The brain water content (BWC) was used to assess the effect of 10 % HS on cerebral oedema. The assessment of Evans blue (EB) extravasation was performed to evaluate the protective effect of 10 % HS on blood-brain barrier. The quantification of VEGF, VEGFR2, ZO-1 and claudin-5 was used to illustrate the mechanism of 10 % HS ameliorating cerebral oedema. BWC was analysed by wet-to-dry ratios in the ischemic hemisphere of SD rats; it was significantly decreased after 10 % HS treatment (P < 0.05). We also investigated the blood-brain barrier protective effect by 10 % HS which reduced EB extravasation effectively in the peri-ischemic brain tissue. In parallel to the above notably at 24 h following MCAO, mRNA and protein expression of VEGF and VEGFR2 in the peri-ischemic brain tissue was down-regulated after 10 % HS treatment (P < 0.05). Along with this, in vitro studies showed increased VEGF and VEGFR2 mRNA and protein expression in primary astrocytes under hypoxic condition (P < 0.05), but it was suppressed after HS treatment (P < 0.05). In addition, HS inhibited the down-regulation of ZO-1, claudin-5 effectively. The results suggest that 10 % HS could alleviate cerebral oedema possibly through reducing the ischemia induced BBB permeability as a consequence of inhibiting VEGF-VEGFR2-mediated down-regulation of ZO-1, claudin-5.

Involvement of P-glycoprotein and multidrug resistance associated protein 1 in the transport of tanshinone IIB, a primary active diterpenoid quinone from the roots of Salvia miltiorrhiza, across the blood-brain barrier.

PubMed

Zhou, Zhi-Wei; Chen, Xiao; Liang, Jun; Yu, Xi-Yong; Wen, Jing-Yuan; Zhou, Shu-Feng

2007-08-01

Tanshinone IIB (TSB) is a major constituent of Salvia miltiorrhiza, which is widely used in treatment of cardiovascular and central nervous system (CNS) diseases such as coronary heart disease and stroke. This study aimed to investigate the role of various drug transporters in the brain penetration of TSB using several in vitro and in vivo mouse and rat models. The uptake and efflux of TSB in rat primary microvascular endothelial cells (RBMVECs) were ATP-dependent and significantly altered in the presence of a P-glycoprotein (P-gp) or multidrug resistance associated protein (Mrp1/2) inhibitor. A polarized transport of TSB was found in RBMVEC monolayers with facilitated efflux from the abluminal to luminal side. Addition of a P-gp inhibitor (e.g. verapamil) in both abluminal and luminal sides attenuated the polarized transport. In an in situ rat brain perfusion model, TSB crossed the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a P-gp or Mrp1/2 inhibitor. The brain levels of TSB were only about 30% of that in the plasma and it could be increased to up to 72% of plasma levels when verapamil, quinidine, or probenecid was co-administered in rats. The entry of TSB to CNS increased by 67-97% in rats subjected to middle cerebral artery occlusion or treatment with the neurotoxin, quinolinic acid, compared to normal rats. Furthermore, The brain levels of TSB in mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold higher than those in the wild-type mice. TSB has limited brain penetration through the BBB due to the contribution of P-gp and to a lesser extent of Mrp1 in rodents. Further studies are needed to confirm whether these corresponding transporters in humans are involved in limiting the penetration of TSB across the BBB and the clinical relevance.

Investigation of the Safety of Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Natural Canine Model of Aging.

PubMed

O'Reilly, Meaghan Anne; Jones, Ryan Matthew; Barrett, Edward; Schwab, Anthony; Head, Elizabeth; Hynynen, Kullervo

2017-01-01

Rationale: Ultrasound-mediated opening of the Blood-Brain Barrier(BBB) has shown exciting potential for the treatment of Alzheimer's disease(AD). Studies in transgenic mouse models have shown that this approach can reduce plaque pathology and improve spatial memory. Before clinical translation can occur the safety of the method needs to be tested in a larger brain that allows lower frequencies be used to treat larger tissue volumes, simulating clinical situations. Here we investigate the safety of opening the BBB in half of the brain in a large aged animal model with naturally occurring amyloid deposits. Methods: Aged dogs naturally accumulate plaques and show associated cognitive declines. Low-frequency ultrasound was used to open the BBB unilaterally in aged beagles (9-11yrs, n=10) in accordance with institutionally approved protocols. Animals received either a single treatment or four weekly treatments. Magnetic resonance imaging(MRI) was used to guide the treatments and assess the tissue effects. The animals underwent neurological testing during treatment follow-up, and a follow-up MRI exam 1 week following the final treatment. Results: The permeability of the BBB was successfully increased in all animals (mean enhancement: 19±11% relative to untreated hemisphere). There was a single adverse event in the chronic treatment group that resolved within 24 hrs. Follow-up MRI showed the BBB to be intact with no evidence of tissue damage in all animals. Histological analysis showed comparable levels of microhemorrhage between the treated and control hemispheres in the prefrontal cortex (single/repeat treatment: 1.0±1.4 vs 0.4±0.5/5.2±1.8 vs. 4.0±2.0). No significant differences were observed in beta-amyloid load (single/repeat: p=0.31/p=0.98) although 3/5 animals in each group showed lower Aβ loads in the treated hemisphere. Conclusion: Whole-hemisphere opening of the BBB was well tolerated in the aged large animal brain. The treatment volumes and frequencies used are clinically relevant and indicate safety for clinical translation. Further study is warranted to determine if FUS has positive effects on naturally occurring amyloid pathology.

Investigation of the Safety of Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Natural Canine Model of Aging

PubMed Central

O'Reilly, Meaghan Anne; Jones, Ryan Matthew; Barrett, Edward; Schwab, Anthony; Head, Elizabeth; Hynynen, Kullervo

2017-01-01

Rationale: Ultrasound-mediated opening of the Blood-Brain Barrier(BBB) has shown exciting potential for the treatment of Alzheimer's disease(AD). Studies in transgenic mouse models have shown that this approach can reduce plaque pathology and improve spatial memory. Before clinical translation can occur the safety of the method needs to be tested in a larger brain that allows lower frequencies be used to treat larger tissue volumes, simulating clinical situations. Here we investigate the safety of opening the BBB in half of the brain in a large aged animal model with naturally occurring amyloid deposits. Methods: Aged dogs naturally accumulate plaques and show associated cognitive declines. Low-frequency ultrasound was used to open the BBB unilaterally in aged beagles (9-11yrs, n=10) in accordance with institutionally approved protocols. Animals received either a single treatment or four weekly treatments. Magnetic resonance imaging(MRI) was used to guide the treatments and assess the tissue effects. The animals underwent neurological testing during treatment follow-up, and a follow-up MRI exam 1 week following the final treatment. Results: The permeability of the BBB was successfully increased in all animals (mean enhancement: 19±11% relative to untreated hemisphere). There was a single adverse event in the chronic treatment group that resolved within 24 hrs. Follow-up MRI showed the BBB to be intact with no evidence of tissue damage in all animals. Histological analysis showed comparable levels of microhemorrhage between the treated and control hemispheres in the prefrontal cortex (single/repeat treatment: 1.0±1.4 vs 0.4±0.5/5.2±1.8 vs. 4.0±2.0). No significant differences were observed in beta-amyloid load (single/repeat: p=0.31/p=0.98) although 3/5 animals in each group showed lower Aβ loads in the treated hemisphere. Conclusion: Whole-hemisphere opening of the BBB was well tolerated in the aged large animal brain. The treatment volumes and frequencies used are clinically relevant and indicate safety for clinical translation. Further study is warranted to determine if FUS has positive effects on naturally occurring amyloid pathology. PMID:28912896

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

PubMed

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

A polymeric micelle magnetic resonance imaging (MRI) contrast agent reveals blood-brain barrier (BBB) permeability for macromolecules in cerebral ischemia-reperfusion injury.

PubMed

Shiraishi, Kouichi; Wang, Zuojun; Kokuryo, Daisuke; Aoki, Ichio; Yokoyama, Masayuki

2017-05-10

Blood-brain barrier (BBB) opening is a key phenomenon for understanding ischemia-reperfusion injuries that are directly linked to hemorrhagic transformation. The recombinant human tissue-type plasminogen activator (rtPA) increases the risk of symptomatic intracranial hemorrhages. Recent imaging technologies have advanced our understanding of pathological BBB disorders; however, an ongoing challenge in the pre-"rtPA treatment" stage is the task of developing a rigorous method for hemorrhage-risk assessments. Therefore, we examined a novel method for assessment of rtPA-extravasation through a hyper-permeable BBB. To examine the image diagnosis of rtPA-extravasation for a rat transient occlusion-reperfusion model, in this study we used a polymeric micelle MRI contrast-agent (Gd-micelles). Specifically, we used two MRI contrast agents at 1h after reperfusion. Gd-micelles provided very clear contrast images in 15.5±10.3% of the ischemic hemisphere at 30min after i.v. injection, whereas a classic gadolinium chelate MRI contrast agent provided no satisfactorily clear images. The obtained images indicate both the hyper-permeable BBB area for macromolecules and the distribution area of macromolecules in the ischemic hemisphere. Owing to their large molecular weight, Gd-micelles remained in the ischemic hemisphere through the hyper-permeable BBB. Our results indicate the feasibility of a novel clinical diagnosis for evaluating rtPA-related hemorrhage risks. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of immune cell infiltration into the CNS by regional neural inputs explained by the gate theory.

PubMed

Arima, Yasunobu; Kamimura, Daisuke; Sabharwal, Lavannya; Yamada, Moe; Bando, Hidenori; Ogura, Hideki; Atsumi, Toru; Murakami, Masaaki

2013-01-01

The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a "gateway" at the fifth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases.

Social stress induces neurovascular pathology promoting depression

PubMed Central

Menard, Caroline; Pfau, Madeline L.; Hodes, Georgia E.; Kana, Veronika; Wang, Victoria X.; Bouchard, Sylvain; Takahashi, Aki; Flanigan, Meghan E.; Aleyasin, Hossein; LeClair, Katherine B.; Janssen, William G.; Labonté, Benoit; Parise, Eric M.; Lorsch, Zachary S.; Golden, Sam A.; Heshmati, Mitra; Tamminga, Carol; Turecki, Gustavo; Campbell, Matthew; Fayad, Zahi; Tang, Cheuk Ying; Merad, Miriam; Russo, Scott J.

2017-01-01

Studies suggest that heightened peripheral inflammation contributes to the pathogenesis of major depressive disorder. We investigated the effect of chronic social defeat stress, a mouse model of depression, on blood-brain barrier (BBB) permeability and infiltration of peripheral immune signals. We found reduced expression of endothelial cell tight junction protein claudin-5 (cldn5) and abnormal blood vessel morphology in nucleus accumbens (NAc) of stress-susceptible but not resilient mice. CLDN5 expression was also decreased in NAc of depressed patients. Cldn5 down-regulation was sufficient to induce depression-like behaviors following subthreshold social stress while chronic antidepressant treatment rescued cldn5 loss and promoted resilience. Reduced BBB integrity in NAc of stress-susceptible or AAV-shRNA-cldn5-injected mice caused infiltration of peripheral cytokine interleukin-6 (IL-6) into brain parenchyma and subsequent expression of depression-like behaviors. These findings suggest that chronic social stress alters BBB integrity through loss of tight junction protein cldn5, promoting peripheral IL-6 passage across the BBB and depression. PMID:29184215

Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

PubMed

Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

2016-08-01

Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

Sestrin2 Induced by Hypoxia Inducible Factor 1 alpha protects the Blood-Brain Barrier via Inhibiting VEGF after Severe Hypoxic-Ischemic Injury in Neonatal Rats

PubMed Central

Shi, Xudan; Doycheva, Desislava Met; Xu, Liang; Tang, Jiping; Yan, Min; Zhang, John H

2016-01-01

Objective Hypoxic ischemic (HI) encephalopathy remains the leading cause of perinatal brain injury resulting in long term disabilities. Stabilization of blood brain barrier (BBB) after HI is an important target, therefore, in this study we aim to determine the role of sestrin2, a stress inducible protein which is elevated after various insults, on BBB stabilization after moderate and severe HI injury. Methods Rat pups underwent common carotid artery ligation followed by either 150 min (severe model) or 100 min (moderate model) of hypoxia. 1h post HI, rats were intranasally administered with recombinant human sestrin2 (rh-sestrin2) and sacrificed for infarct area, brain water content, righting reflex and geotaxis reflex. Sestrin2 was silenced using siRNA and an activator/inhibitor of hypoxia inducible factor1α (HIF1α) were used to examine their roles on BBB permeability. Results Rats subjected to severe HI exhibited larger infarct area and higher sestrin2 expression compared to rats in the moderate HI group. rh-sestrin2 attenuated brain infarct and edema, while silencing sestrin2 reversed these protective effects after severe HI. HIF1α induced sestrin2 activation in severe HI but not in moderate HI groups. A HIF1a agonist was shown to increase permeability of the BBB via vascular endothelial growth factor (VEGF) after moderate HI. However, after severe HI, HIF1α activated both VEGF and sestrin2. But HIF1α dependent sestrin2 activation was the predominant pathway after severe HI which inhibited VEGF and attenuated BBB permeability. Conclusions rh-sestrin2 attenuated BBB permeability via upregulation of endogenous sestrin2 which was induced by HIF1α after severe HI. However, HIF1α’s effects as a prodeath or prosurvival signal were influenced by the severity of HI injury. PMID:27425892

P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

PubMed Central

O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

2013-01-01

Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported substrate of human P-gp. Microdialysis-based pharmacokinetic studies demonstrated that administration of the P-gp inhibitor cyclosporin A resulted in increased brain levels of escitalopram without altering plasma escitalopram levels in the rat, thereby showing that P-gp restricts escitalopram transport across the blood–brain barrier (BBB) in vivo. The tail suspension test (TST) was carried out to elucidate the pharmacodynamic impact of P-gp inhibition on escitalopram effect in a mouse model of antidepressant activity. Pre-treatment with the P-gp inhibitor verapamil enhanced the response to escitalopram in the TST. Taken together, these data indicate that P-gp may restrict the BBB transport of escitalopram in humans, potentially resulting in subtherapeutic brain concentrations in certain patients. Moreover, by verifying that increasing escitalopram delivery to the brain by P-gp inhibition results in enhanced antidepressant-like activity, we suggest that adjunctive treatment with a P-gp inhibitor may represent a beneficial approach to augment escitalopram therapy in depression. PMID:23670590

The construction of the multifunctional targeting ursolic acids liposomes and its apoptosis effects to C6 glioma stem cells

PubMed Central

Ying, Xue; Wang, Yahua; Xu, Haolun; Li, Xia; Yan, Helu; Tang, Hui; Wen, Chen; Li, Yingchun

2017-01-01

Brain gliomas, one of the most fatal tumors to human, severely threat the health and life of human. They are capable of extremely strong invasion ability. And invasive glioma cells could rapidly penetrate into normal brain tissues and break them. We prepared a kind of functional liposomes, which could be transported acrossing the blood-brain barrier (BBB) and afterwards induce the apoptosis of glioma stem cells. In this research, we chose ursolic acids (UA) as an anti-cancer drug to inhibit the growth of C6 glioma cells, while epigallocatechin 3-gallate(EGCG) as the agent that could induce the apoptosis of C6 glioma stem cells. With the targeting ability of MAN, the liposomes could be delivered through the BBB and finally were concentrated on the brain gliomas. Cell experiments in vitro demonstrated that the functional liposomes were able to significantly enhance the anti-cancer effects of the drugs due to promoting the apoptosis and endocytosis effects of C6 glioma cells and C6 glioma stem cells at the same time. Furthermore, the evaluations through animal models showed that the drugs could obviously prolong the survival period of brain glioma-bearing mice and inhibit the tumor growth. Consequently, multifunctional targeting ursolic acids liposomes could potentially improve the therapeutic effects on C6 glioma cells and C6 glioma stem cells. PMID:28969057

Acute lipophilicity-dependent effect of intravascular simvastatin in the early phase of focal cerebral ischemia.

PubMed

Beretta, S; Pastori, C; Sala, G; Piazza, F; Ferrarese, C; Cattalini, A; de Curtis, M; Librizzi, L

2011-05-01

The acute effects of simvastatin lactone (lipophilic) and simvastatin acid (hydrophilic) on transient focal ischemia were assessed using the isolated guinea pig brain maintained in vitro by arterial perfusion. This new model of cerebral ischemia allows the assessment of the very early phase of the ischemic process, with the functional preservation of the vascular and neuronal compartments and the blood-brain barrier (bbb). The middle cerebral artery was transiently tied for 30 min followed by reperfusion for 60 min. Statins (nanomolar doses) were administered by intravascular continuous infusion starting 60 min before ischemia induction. Brain cortical activity and arterial vascular tone were continuously recorded. At the end of the experiment immunoreactivity for microtubule-associated protein 2 (MAP-2), expression of survival kinases (ERK and Akt) and total anti-oxidant capacity were assayed. Brains treated with simvastatin lactone showed i) reduced amplitude and delayed onset of ischemic depressions, ii) preservation of MAP-2 immunoreactivity, iii) activation of ERK signaling in the ischemic hemisphere and iv) increase in whole-brain anti-oxidant capacity. Treatment with the bbb-impermeable simvastatin acid was ineffective on the above-mentioned parameters. Vascular resistance recordings and Akt signaling were unchanged by any statin treatment. Our findings suggest that intravascular-delivered simvastatin exerts an acute lipophilicity-dependent protective effect in the early phase of cerebral ischemia. Copyright © 2011 Elsevier Ltd. All rights reserved.

Glioblastoma Targeted Gene Therapy Based on pEGFP/p53-Loaded Superparamagnetic Iron Oxide Nanoparticles.

PubMed

Eslaminejad, Touba; Nematollahi-Mahani, Seyed Noureddin; Ansari, Mehdi

2017-01-01

Blood-brain barrier (BBB) separates the neural tissue from circulating blood because of its high selectivity. This study focused on the in vitro application of magnetic nanoparticles to deliver Tp53 as a gene of interest to glioblastoma (U87) cells across a simulated BBB model that comprised KB cells. After magnetic and non-magnetic nanoparticles were internalized by KB cells, their location in these cells was examined by transmission electron microscopy. Transfection efficiency of DNA to U87 cells was evaluated by fluorescence microscopy, real time PCR, flowcytometry, and Western immuno-blotting. When a magnetic field was applied, a large number of magnetic nanoparticles accumulated in KB cells, appearing as black dots scattered in the cytoplasm of cells. Fluorescence microscope examination showed that transfection of the DNA to U87 target cells was highest in cells treated with magnetic nanoparticles and exposed to a magnetic field. Also it was reflected in significantly increased mRNA level while the p53 protein level was decreased. It could be concluded that a significant increase in total apoptosis was induced in cells by magnetic nanoparticles, coupled with exposure to a magnetic force (p ≤0.01) as compared with cells that were not exposed to magnetism. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Methylmercury Causes Blood-Brain Barrier Damage in Rats via Upregulation of Vascular Endothelial Growth Factor Expression

PubMed Central

Takahashi, Tetsuya; Fujimura, Masatake; Koyama, Misaki; Kanazawa, Masato; Usuki, Fusako; Nishizawa, Masatoyo; Shimohata, Takayoshi

2017-01-01

Clinical manifestations of methylmercury (MeHg) intoxication include cerebellar ataxia, concentric constriction of visual fields, and sensory and auditory disturbances. The symptoms depend on the site of MeHg damage, such as the cerebellum and occipital lobes. However, the underlying mechanism of MeHg-induced tissue vulnerability remains to be elucidated. In the present study, we used a rat model of subacute MeHg intoxication to investigate possible MeHg-induced blood-brain barrier (BBB) damage. The model was established by exposing the rats to 20-ppm MeHg for up to 4 weeks; the rats exhibited severe cerebellar pathological changes, although there were no significant differences in mercury content among the different brain regions. BBB damage in the cerebellum after MeHg exposure was confirmed based on extravasation of endogenous immunoglobulin G (IgG) and decreased expression of rat endothelial cell antigen-1. Furthermore, expression of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, increased markedly in the cerebellum and mildly in the occipital lobe following MeHg exposure. VEGF expression was detected mainly in astrocytes of the BBB. Intravenous administration of anti-VEGF neutralizing antibody mildly reduced the rate of hind-limb crossing signs observed in MeHg-exposed rats. In conclusion, we demonstrated for the first time that MeHg induces BBB damage via upregulation of VEGF expression at the BBB in vivo. Further studies are required in order to determine whether treatment targeted at VEGF can ameliorate MeHg-induced toxicity. PMID:28118383

Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood–brain barrier permeability

PubMed Central

De Bock, Marijke; Culot, Maxime; Wang, Nan; Bol, Mélissa; Decrock, Elke; De Vuyst, Elke; da Costa, Anaelle; Dauwe, Ine; Vinken, Mathieu; Simon, Alexander M; Rogiers, Vera; De Ley, Gaspard; Evans, William Howard; Bultynck, Geert; Dupont, Geneviève; Cecchelli, Romeo; Leybaert, Luc

2011-01-01

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood–brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5′ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations. PMID:21654699

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

PubMed

Schultz, Mark L; Tecedor, Luis; Lysenko, Elena; Ramachandran, Shyam; Stein, Colleen S; Davidson, Beverly L

2018-07-01

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

PubMed

Yang, Yi; Kimura-Ohba, Shihoko; Thompson, Jeffrey F; Salayandia, Victor M; Cossé, Melissa; Raz, Limor; Jalal, Fakhreya Y; Rosenberg, Gary A

2018-06-01

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Noninvasive delivery of stealth, brain-penetrating nanoparticles across the blood-brain barrier using MRI-guided focused ultrasound

PubMed Central

Miller, G. Wilson; Song, Ji; Louttit, Cameron; Klibanov, Alexander L; Shih, Ting-Yu; Swaminathan, Ganesh; Tamargo, Rafael J.; Woodworth, Graeme F.; Hanes, Justin; Price, Richard J.

2014-01-01

The blood-brain barrier (BBB) presents a significant obstacle for the treatment of many central nervous system (CNS) disorders, including invasive brain tumors, Alzheimer’s, Parkinson’s and stroke. Therapeutics must be capable of bypassing the BBB and also penetrate the brain parenchyma to achieve a desired effect within the brain. In this study, we test the unique combination of a noninvasive approach to BBB permeabilization with a therapeutically relevant polymeric nanoparticle platform capable of rapidly penetrating within the brain microenvironment. MR-guided focused ultrasound (FUS) with intravascular microbubbles (MBs) is able to locally and reversibly disrupt the BBB with submillimeter spatial accuracy. Densely poly(ethylene-co-glycol) (PEG) coated, brain-penetrating nanoparticles (BPNs) are long-circulating and diffuse 10-fold slower in normal rat brain tissue compared to diffusion in water. Following intravenous administration of model and biodegradable BPN in normal healthy rats, we demonstrate safe, pressure-dependent delivery of 60 nm BPNs to the brain parenchyma in regions where the BBB is disrupted by FUS and MBs. Delivery of BPNs with MR-guided FUS has the potential to improve efficacy of treatments for many CNS diseases, while reducing systemic side effects by providing sustained, well-dispersed drug delivery into select regions of the brain. PMID:24979210

Essentials and Perspectives of Computational Modelling Assistance for CNS-oriented Nanoparticle-based Drug Delivery Systems.

PubMed

Kisała, Joanna; Heclik, Kinga I; Pogocki, Krzysztof; Pogocki, Dariusz

2018-05-16

The blood-brain barrier (BBB) is a complex system controlling two-way substances traffic between circulatory (cardiovascular) system and central nervous system (CNS). It is almost perfectly crafted to regulate brain homeostasis and to permit selective transport of molecules that are essential for brain function. For potential drug candidates, the CNS-oriented neuropharmaceuticals as well as for those of primary targets in the periphery, the extent to which a substance in the circulation gains access to the CNS seems crucial. With the advent of nanopharmacology the problem of the BBB permeability for drug nano-carriers gains new significance. Compare to some other fields of medicinal chemistry, the computational science of nanodelivery is still prematured to offer the black-box type solutions, especially for the BBB-case. However, even its enormous complexity can be spell out the physical principles, and as such subjected to computation. Basic understanding of various physico-chemical parameters describing the brain uptake is required to take advantage of their usage for the BBB-nanodelivery. This mini-review provides a sketchy introduction into essential concepts allowing application of computational simulation to the BBB-nanodelivery design. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A review of technical aspects of T1-weighted dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in human brain tumors.

PubMed

Bergamino, M; Bonzano, L; Levrero, F; Mancardi, G L; Roccatagliata, L

2014-09-01

In the last few years, several imaging methods, such as magnetic resonance imaging (MRI) and computed tomography, have been used to investigate the degree of blood-brain barrier (BBB) permeability in patients with neurological diseases including multiple sclerosis, ischemic stroke, and brain tumors. One promising MRI method for assessing the BBB permeability of patients with neurological diseases in vivo is T1-weighted dynamic contrast-enhanced (DCE)-MRI. Here we review the technical issues involved in DCE-MRI in the study of human brain tumors. In the first part of this paper, theoretical models for the DCE-MRI analysis will be described, including the Toft-Kety models, the adiabatic approximation to the tissue homogeneity model and the two-compartment exchange model. These models can be used to estimate important kinetic parameters related to BBB permeability. In the second part of this paper, details of the data acquisition, issues related to the arterial input function, and procedures for DCE-MRI image analysis are illustrated. Copyright © 2014 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1G93A ALS rat model.

PubMed

Stamenković, Stefan; Pavićević, Aleksandra; Mojović, Miloš; Popović-Bijelić, Ana; Selaković, Vesna; Andjus, Pavle; Bačić, Goran

2017-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1 G93A ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t 1/2 =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1 G93A rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of changes in BBB permeability and for the unprecedented in vivo monitoring of the brain tissue redox status, as early markers of ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

Early Blood-Brain Barrier Disruption after Mechanical Thrombectomy in Acute Ischemic Stroke.

PubMed

Shi, Zhong-Song; Duckwiler, Gary R; Jahan, Reza; Tateshima, Satoshi; Szeder, Viktor; Saver, Jeffrey L; Kim, Doojin; Sharma, Latisha K; Vespa, Paul M; Salamon, Noriko; Villablanca, J Pablo; Viñuela, Fernando; Feng, Lei; Loh, Yince; Liebeskind, David S

2018-05-01

The impact of blood-brain barrier (BBB) disruption can be detected by intraparenchymal hyperdense lesion on the computed tomography (CT) scan after endovascular stroke therapy. The purpose of this study was to determine whether early BBB disruption predicts intracranial hemorrhage and poor outcome in patients with acute ischemic stroke treated with mechanical thrombectomy. We analyzed patients with anterior circulation stroke treated with mechanical thrombectomy and identified BBB disruption on the noncontrast CT images immediately after endovascular treatment. Follow-up CT or magnetic resonance imaging scan was performed at 24 hours to assess intracranial hemorrhage. We dichotomized patients into those with moderate BBB disruption versus those with minor BBB disruption and no BBB disruption. We evaluated the association of moderate BBB disruption after mechanical thrombectomy with intracranial hemorrhage and clinical outcomes. Moderate BBB disruption after mechanical thrombectomy was found in 56 of 210 patients (26.7%). Moderate BBB disruption was independently associated with higher rates of hemorrhagic transformation (OR 25.33; 95% CI 9.93-64.65; P < .001), parenchymal hematoma (OR 20.57; 95% CI 5.64-74.99; P < .001), and poor outcome at discharge (OR 2.35; 95% CI 1.09-5.07; P = .03). The association of BBB disruption with intracranial hemorrhage remained in patients with successful reperfusion after mechanical thrombectomy. The location of BBB disruption was not associated with intracranial hemorrhage and poor outcome. Moderate BBB disruption is common after mechanical thrombectomy in a quarter of patients with acute ischemic stroke and increases the risk of intracranial hemorrhage and poor outcome. Copyright © 2018 by the American Society of Neuroimaging.

Drug transport across the blood–brain barrier

PubMed Central

Pardridge, William M

2012-01-01

The blood–brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. This property arises from the epithelial-like tight junctions within the brain capillary endothelium. The BBB is anatomically and functionally distinct from the blood–cerebrospinal fluid barrier at the choroid plexus. Certain small molecule drugs may cross the BBB via lipid-mediated free diffusion, providing the drug has a molecular weight <400 Da and forms <8 hydrogen bonds. These chemical properties are lacking in the majority of small molecule drugs, and all large molecule drugs. Nevertheless, drugs can be reengineered for BBB transport, based on the knowledge of the endogenous transport systems within the BBB. Small molecule drugs can be synthesized that access carrier-mediated transport (CMT) systems within the BBB. Large molecule drugs can be reengineered with molecular Trojan horse delivery systems to access receptor-mediated transport (RMT) systems within the BBB. Peptide and antisense radiopharmaceuticals are made brain-penetrating with the combined use of RMT-based delivery systems and avidin–biotin technology. Knowledge on the endogenous CMT and RMT systems expressed at the BBB enable new solutions to the problem of BBB drug transport. PMID:22929442

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine–induced blood–brain barrier dysfunction

PubMed Central

Coelho-Santos, Vanessa; Leitão, Ricardo A; Cardoso, Filipa L; Palmela, Inês; Rito, Manuel; Barbosa, Marcos; Brito, Maria A; Fontes-Ribeiro, Carlos A; Silva, Ana P

2015-01-01

Methamphetamine (METH) is a psychostimulant that causes neurologic and psychiatric abnormalities. Recent studies have suggested that its neurotoxicity may also result from its ability to compromise the blood–brain barrier (BBB). Herein, we show that METH rapidly increased the vesicular transport across endothelial cells (ECs), followed by an increase of paracellular transport. Moreover, METH triggered the release of tumor necrosis factor-alpha (TNF-α), and the blockade of this cytokine or the inhibition of nuclear factor-kappa B (NF-κB) pathway prevented endothelial dysfunction. Since astrocytes have a crucial role in modulating BBB function, we further showed that conditioned medium obtained from astrocytes previously exposed to METH had a negative impact on barrier properties also via TNF-α/NF-κB pathway. Animal studies corroborated the in vitro results. Overall, we show that METH directly interferes with EC properties or indirectly via astrocytes through the release of TNF-α and subsequent activation of NF-κB pathway culminating in barrier dysfunction. PMID:25899299

Blood-brain barrier-supported neurogenesis in healthy and diseased brain.

PubMed

Pozhilenkova, Elena A; Lopatina, Olga L; Komleva, Yulia K; Salmin, Vladimir V; Salmina, Alla B

2017-05-24

Adult neurogenesis is one of the most important mechanisms contributing to brain development, learning, and memory. Alterations in neurogenesis underlie a wide spectrum of brain diseases. Neurogenesis takes place in highly specialized neurogenic niches. The concept of neurogenic niches is becoming widely accepted due to growing evidence of the important role of the microenvironment established in the close vicinity to stem cells in order to provide adequate control of cell proliferation, differentiation, and apoptosis. Neurogenic niches represent the platform for tight integration of neurogenesis and angiogenesis supported by specific properties of cerebral microvessel endothelial cells contributing to establishment of partially compromised blood-brain barrier (BBB) for the adjustment of local conditions to the current metabolic needs of stem and progenitor cells. Here, we review up-to-date data on microvascular dynamics in activity-dependent neurogenesis, specific properties of BBB in neurogenic niches, endothelial-driven mechanisms of clonogenic activity, and future perspectives for reconstructing the neurogenic niches in vitro.

Polyphenols journey through blood-brain barrier towards neuronal protection.

PubMed

Figueira, I; Garcia, G; Pimpão, R C; Terrasso, A P; Costa, I; Almeida, A F; Tavares, L; Pais, T F; Pinto, P; Ventura, M R; Filipe, A; McDougall, G J; Stewart, D; Kim, K S; Palmela, I; Brites, D; Brito, M A; Brito, C; Santos, C N

2017-09-13

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Blood–Brain Barrier Leakage during Early Epileptogenesis Is Associated with Rapid Remodeling of the Neurovascular Unit

PubMed Central

Breuer, Heike; Leiter, Ina; Märkel, Martin; Bascuñana, Pablo; Michalski, Dominik; Bengel, Frank M.; Löscher, Wolfgang; Meier, Martin; Bankstahl, Jens P.; Härtig, Wolfgang

2018-01-01

Abstract Increased permeability of the blood–brain barrier (BBB) following cerebral injury results in regional extravasation of plasma proteins and can critically contribute to the pathogenesis of epilepsy. Here, we comprehensively explore the spatiotemporal evolution of a main extravasation component, albumin, and illuminate associated responses of the neurovascular unit (NVU) contributing to early epileptogenic neuropathology. We applied translational in vivo MR imaging and complementary immunohistochemical analyses in the widely used rat pilocarpine post–status epilepticus (SE) model. The observed rapid BBB leakage affected major epileptogenesis-associated brain regions, peaked between 1 and 2 d post-SE, and rapidly declined thereafter, accompanied by cerebral edema generally following the same time course. At peak of BBB leakage, serum albumin colocalized with NVU constituents, such as vascular components, neurons, and brain immune cells. Surprisingly, astroglial markers did not colocalize with albumin, and aquaporin-4 (AQP4) was clearly reduced in areas of leaky BBB, indicating a severe disturbance of astrocyte-mediated endothelial-neuronal coupling. In addition, a distinct adaptive reorganization process of the NVU vasculature apparently takes place at sites of albumin presence, substantiated by reduced immunoreactivity of endothelial and changes in vascular basement membrane markers. Taken together, degenerative events at the level of the NVU, affecting vessels, astrocytes, and neurons, seem to outweigh reconstructive processes. Considering the rapidly occurring BBB leakage and subsequent impairment of the NVU, our data support the necessity of a prompt BBB-restoring treatment as one component of rational therapeutic intervention to prevent epileptogenesis and the development of other detrimental sequelae of SE. PMID:29854942

Magnetic Resonance Imaging Profile of Blood–Brain Barrier Injury in Patients With Acute Intracerebral Hemorrhage

PubMed Central

Aksoy, Didem; Bammer, Roland; Mlynash, Michael; Venkatasubramanian, Chitra; Eyngorn, Irina; Snider, Ryan W.; Gupta, Sandeep N.; Narayana, Rashmi; Fischbein, Nancy; Wijman, Christine A. C.

2013-01-01

Background Spontaneous intracerebral hemorrhage (ICH) is associated with blood–brain barrier (BBB) injury, which is a poorly understood factor in ICH pathogenesis, potentially contributing to edema formation and perihematomal tissue injury. We aimed to assess and quantify BBB permeability following human spontaneous ICH using dynamic contrast‐enhanced magnetic resonance imaging (DCE MRI). We also investigated whether hematoma size or location affected the amount of BBB leakage. Methods and Results Twenty‐five prospectively enrolled patients from the Diagnostic Accuracy of MRI in Spontaneous intracerebral Hemorrhage (DASH) study were examined using DCE MRI at 1 week after symptom onset. Contrast agent dynamics in the brain tissue and general tracer kinetic modeling were used to estimate the forward leakage rate (Ktrans) in regions of interest (ROI) in and surrounding the hematoma and in contralateral mirror–image locations (control ROI). In all patients BBB permeability was significantly increased in the brain tissue immediately adjacent to the hematoma, that is, the hematoma rim, compared to the contralateral mirror ROI (P<0.0001). Large hematomas (>30 mL) had higher Ktrans values than small hematomas (P<0.005). Ktrans values of lobar hemorrhages were significantly higher than the Ktrans values of deep hemorrhages (P<0.005), independent of hematoma volume. Higher Ktrans values were associated with larger edema volumes. Conclusions BBB leakage in the brain tissue immediately bordering the hematoma can be measured and quantified by DCE MRI in human ICH. BBB leakage at 1 week is greater in larger hematomas as well as in hematomas in lobar locations and is associated with larger edema volumes. PMID:23709564

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

PubMed

van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

2014-01-15

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid.

PubMed

Porru, Manuela; Zappavigna, Silvia; Salzano, Giuseppina; Luce, Amalia; Stoppacciaro, Antonella; Balestrieri, Maria Luisa; Artuso, Simona; Lusa, Sara; De Rosa, Giuseppe; Leonetti, Carlo; Caraglia, Michele

2014-11-15

Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.

Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid

PubMed Central

Porru, Manuela; Zappavigna, Silvia; Salzano, Giuseppina; Luce, Amalia; Stoppacciaro, Antonella; Balestrieri, Maria Luisa; Artuso, Simona; Lusa, Sara; De Rosa, Giuseppe; Leonetti, Carlo; Caraglia, Michele

2014-01-01

Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB. PMID:25431953

Pharmacokinetic Assessment of Cooperative Efflux of the Multitargeted Kinase Inhibitor Ponatinib Across the Blood-Brain Barrier.

PubMed

Laramy, Janice K; Kim, Minjee; Parrish, Karen E; Sarkaria, Jann N; Elmquist, William F

2018-05-01

A compartmental blood-brain barrier (BBB) model describing drug transport across the BBB was implemented to evaluate the influence of efflux transporters on the rate and extent of the multikinase inhibitor ponatinib penetration across the BBB. In vivo pharmacokinetic studies in wild-type and transporter knockout mice showed that two major BBB efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), cooperate to modulate the brain exposure of ponatinib. The total and unbound (free) brain-to-plasma ratios were approximately 15-fold higher in the triple knockout mice lacking both P-gp and Bcrp [ Mdr1a/b(-/-)Bcrp1(-/-) ] compared with the wild-type mice. The triple knockout mice had a greater than an additive increase in the brain exposure of ponatinib when compared with single knockout mice [ Bcrp1(-/-) or Mdr1a/b(-/-) ], suggesting functional compensation of transporter-mediated drug efflux. Based on the BBB model characterizing the observed brain and plasma concentration-time profiles, the brain exit rate constant and clearance out of the brain were approximately 15-fold higher in the wild-type compared with Mdr1a/b(-/-)Bcrp1(-/-) mice, resulting in a significant increase in the mean transit time (the average time spent by ponatinib in the brain in a single passage) in the absence of efflux transporters (P-gp and Bcrp). This study characterized transporter-mediated drug efflux from the brain, a process that reduces the duration and extent of ponatinib exposure in the brain and has critical implications for the use of targeted drug delivery for brain tumors. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Intact blood-brain barrier transport of small molecular drugs in animal models of amyloid beta and alpha-synuclein pathology.

PubMed

Gustafsson, Sofia; Lindström, Veronica; Ingelsson, Martin; Hammarlund-Udenaes, Margareta; Syvänen, Stina

2018-01-01

Pathophysiological impairment of the neurovascular unit, including the integrity and dynamics of the blood-brain barrier (BBB), has been denoted both a cause and consequence of neurodegenerative diseases. Pathological impact on BBB drug delivery has also been debated. The aim of the present study was to investigate BBB drug transport, by determining the unbound brain-to-plasma concentration ratio (K p,uu,brain ), in aged AβPP-transgenic mice, α-synuclein transgenic mice, and wild type mice. Mice were dosed with a cassette of five compounds, including digoxin, levofloxacin (1 mg/kg, s.c.), paliperidone, oxycodone, and diazepam (0.25 mg/kg, s.c.). Brain and blood were collected at 0.5, 1, or 3 h after dosage. Drug concentrations were measured using LC-MS/MS. The total brain-to-plasma concentration ratio was calculated and equilibrium dialysis was used to determine the fraction of unbound drug in brain and plasma for all compounds. Together, these three measures were used to determine the K p,uu,brain value. Despite Aβ or α-synuclein pathology in the current animal models, no difference was observed in the extent of drug transport across the BBB compared to wild type animals for any of the compounds investigated. Hence, the present study shows that the concept of a leaking barrier within neurodegenerative conditions has to be interpreted with caution when estimating drug transport into the brain. The capability of the highly dynamic BBB to regulate brain drug exposure still seems to be intact despite the presence of pathology. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone.

PubMed

Harazin, András; Bocsik, Alexandra; Barna, Lilla; Kincses, András; Váradi, Judit; Fenyvesi, Ferenc; Tubak, Vilmos; Deli, Maria A; Vecsernyés, Miklós

2018-01-01

The blood-brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB.

Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone

PubMed Central

Barna, Lilla; Kincses, András; Váradi, Judit; Fenyvesi, Ferenc; Tubak, Vilmos

2018-01-01

The blood–brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB. PMID:29780671

Electroacupuncture: a new approach to open the blood-brain barrier in rats recovering from middle cerebral artery occlusion.

PubMed

Zhang, Jiangsong; Lin, Xianming; Zhou, Hui; Chen, Yuanyuan; Xiao, Shuangkai; Jiao, Junyue; Zhao, Yibin; Di, Zhong

2018-06-14

To examine for an opening effect on the blood-brain barrier (BBB) in intact rats and rats with experimental ischaemia-reperfusion (I/R) during the recovery period after various electroacupuncture (EA) treatments with different time courses, and to determine whether there is a time-dependent effect. An additional objective was to determine whether this method could induce the penetration of nerve growth factor (NGF) through the BBB. A middle cerebral artery occlusion (MCAO) model was first established. We chose different stimulation time courses and observed the effects of EA treatment (100 Hz frequency; 2 mA intensity) at GV20 and GV26 on the BBB in rats recovering from MCAO 3 weeks after modelling. The rats were injected with 2% Evans blue (EB) saline. The brain water content was measured using a wet/dry weighing method. The degree of penetration of EB was detected using spectrophotometry and laser confocal microscopy. The rats were then injected with NGF, and the concentration of NGF in the brain tissues was measured using ELISA. The increase in the BBB permeability was most notable following the 8 min EA stimulation (P<0.05), which may be advantageous for the targeted delivery of drugs (such as NGF) into the brain. Additionally, this effect did not appear to cause brain oedema (P>0.05) in healthy or MCAO rats. EA treatment for a certain stimulation time at GV20 and GV26 in MCAO rats can increase BBB permeability. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Mechanical disruption of the blood-brain barrier following experimental concussion.

PubMed

Johnson, Victoria E; Weber, Maura T; Xiao, Rui; Cullen, D Kacy; Meaney, David F; Stewart, William; Smith, Douglas H

2018-05-01

Although concussion is now recognized as a major health issue, its non-lethal nature has limited characterization of the underlying pathophysiology. In particular, potential neuropathological changes have typically been inferred from non-invasive techniques or post-mortem examinations of severe traumatic brain injury (TBI). Here, we used a swine model of head rotational acceleration based on human concussion to examine blood-brain barrier (BBB) integrity after injury in association with diffuse axonal injury and glial responses. We then determined the potential clinical relevance of the swine concussion findings through comparisons with pathological changes in human severe TBI, where post-mortem examinations are possible. At 6-72 h post-injury in swine, we observed multifocal disruption of the BBB, demonstrated by extravasation of serum proteins, fibrinogen and immunoglobulin-G, in the absence of hemorrhage or other focal pathology. BBB disruption was observed in a stereotyped distribution consistent with biomechanical insult. Specifically, extravasated serum proteins were frequently observed at interfaces between regions of tissue with differing material properties, including the gray-white boundary, periventricular and subpial regions. In addition, there was substantial overlap of BBB disruption with regions of axonal pathology in the white matter. Acute perivascular cellular uptake of blood-borne proteins was observed to be prominent in astrocytes (GFAP-positive) and neurons (MAP-2-positive), but not microglia (IBA1-positive). Parallel examination of human severe TBI revealed similar patterns of serum extravasation and glial uptake of serum proteins, but to a much greater extent than in the swine model, attributed to the higher injury severity. These data suggest that BBB disruption represents a new and important pathological feature of concussion.

Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor.

PubMed

Zhou, Qing-Hui; Sumbria, Rachita; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Boado, Ruben J; Pardridge, William M

2011-11-01

Biologic tumor necrosis factor (TNF)-α inhibitors do not cross the blood-brain barrier (BBB). A BBB-penetrating TNF-α inhibitor was engineered by fusion of the extracellular domain of the type II human TNF receptor (TNFR) to the carboxyl terminus of the heavy chain of a mouse/rat chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb-TNFR fusion protein and etanercept bound human TNF-α with high affinity and K(D) values of 374 ± 77 and 280 ± 80 pM, respectively. Neuroprotection in brain in vivo after intravenous administration of the fusion protein was examined in a mouse model of Parkinson's disease. Mice were also treated with saline or a non-BBB-penetrating TNF decoy receptor, etanercept. After intracerebral injection of the nigral-striatal toxin, 6-hydroxydopamine, mice were treated every other day for 3 weeks. Treatment with the cTfRMAb-TNFR fusion protein caused an 83% decrease in apomorphine-induced rotation, a 67% decrease in amphetamine-induced rotation, a 82% increase in vibrissae-elicited forelimb placing, and a 130% increase in striatal tyrosine hydroxylase (TH) enzyme activity. In contrast, chronic treatment with etanercept, which does not cross the BBB, had no effect on neurobehavior or striatal TH enzyme activity. A bridging enzyme-linked immunosorbent assay specific for the cTfRMAb-TNFR fusion protein showed that the immune response generated in the mice was low titer. In conclusion, a biologic TNF inhibitor is neuroprotective after intravenous administration in a mouse model of neurodegeneration, providing that the TNF decoy receptor is reengineered to cross the BBB.

Permeabilization of the blood-brain barrier via mucosal engrafting: implications for drug delivery to the brain.

PubMed

Bleier, Benjamin S; Kohman, Richie E; Feldman, Rachel E; Ramanlal, Shreshtha; Han, Xue

2013-01-01

Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson's disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS.

Permeabilization of the Blood-Brain Barrier via Mucosal Engrafting: Implications for Drug Delivery to the Brain

PubMed Central

Bleier, Benjamin S.; Kohman, Richie E.; Feldman, Rachel E.; Ramanlal, Shreshtha; Han, Xue

2013-01-01

Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson’s disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS. PMID:23637885

Two-photon microscopy for real-time monitoring of focused ultrasound-mediated drug delivery to the brain in a mouse model of Alzheimer's disease

NASA Astrophysics Data System (ADS)

Burgess, Alison; Eterman, Naomi; Aubert, Isabelle; Hynynen, Kullervo

2013-02-01

There is substantial evidence that focused ultrasound (FUS) in combination with microbubble contrast agent can cause disruption of the blood-brain barrier (BBB) to aid in drug delivery to the brain. We have previously demonstrated that FUS efficiently delivers antibodies against amyloid-β peptides (Aβ) through the BBB, leading to a reduction in amyloid pathology at 4 days in a mouse model of Alzheimer's disease. In the current study, we used two-photon microscopy to characterize the effect of FUS in real time on amyloid pathology in the mouse brain. Mice were anesthetized and a cranial window was made in the skull. A custom-built ultrasound transducer was fixed to a coverslip and attached to the skull, covering the cranial window. Methoxy-X04 [2-5mg/kg] delivered intravenously 1 hr prior to the experiment clearly labelled the Aβ surrounding the vessels and the amyloid plaques in the cortex. Dextran conjugated Texas Red (70kDa) administered intravenously, confirmed BBB disruption. BBB disruption occurred in transgenic and non-transgenic animals at similar ultrasound pressures tested. However, the time required for BBB closure following FUS was longer in the Tg mice. We have conjugated Aβ antibodies to the fluorescent molecule FITC for real time monitoring of the antibody distribution in the brain. Our current experiments are aimed at optimizing the parameters to achieve maximal fluorescent intensity of the BAM10 antibody at the plaque surface. Two-photon microscopy has proven to be a valuable tool for evaluating the efficacy of FUS mediated drug delivery, including antibodies, to the Alzheimer brain.

Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.

PubMed

Phoolcharoen, Waranyoo; Prehaud, Christophe; van Dolleweerd, Craig J; Both, Leonard; da Costa, Anaelle; Lafon, Monique; Ma, Julian K-C

2017-10-01

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVG P fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVG P fusion protein was able to cross the in celluloBBB and neutralize RABV. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Dietary Docosahexaenoic Acid Supplementation Enhances Expression of Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier and Brain Docosahexaenoic Acid Levels.

PubMed

Pan, Yijun; Morris, Elonie R; Scanlon, Martin J; Marriott, Philip J; Porter, Christopher Jh; Nicolazzo, Joseph A

2018-03-27

The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively-beneficial fatty acid, across the blood-brain barrier (BBB) is governed by fatty acid-binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, upregulating FABP5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD. DHA supplementation has been shown to be beneficial in various mouse models of AD, and therefore, the aim of this study was to determine whether DHA has the potential to upregulate the BBB expression of FABP5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC/D3) cells with the maximum tolerable concentration of DHA (12.5 μM) for 72 hr resulted in a 1.4-fold increase in FABP5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6-8 week old C57BL/6 mice were fed with a control diet or a DHA-enriched diet for 21 days. Brain microvascular FABP5 protein expression was upregulated 1.7-fold in mice fed the DHA-enriched diet, and this was associated with increased brain DHA levels (1.3-fold). Despite an increase in brain DHA levels, reduced BBB transport of 14 C-DHA was observed over a 1 min perfusion, possibly as a result of competitive binding to FABP5 between dietary DHA and 14 C-DHA. The current study has demonstrated that DHA can increase BBB expression of FABP5, as well as fatty acid transporters, overall increasing brain DHA levels. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR.

PubMed

Makani, Vishruti; Jang, Yong-Gil; Christopher, Kevin; Judy, Wesley; Eckstein, Jacob; Hensley, Kenneth; Chiaia, Nicolas; Kim, Dong-Shik; Park, Joshua

2016-01-01

An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer's disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their in vivo efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aβ42 peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD.

BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR

PubMed Central

Makani, Vishruti; Jang, Yong-gil; Christopher, Kevin; Judy, Wesley; Eckstein, Jacob; Hensley, Kenneth; Chiaia, Nicolas; Kim, Dong-Shik; Park, Joshua

2016-01-01

An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer’s disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their in vivo efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aβ42 peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD. PMID:26939023

The safest parameters for FUS-induced blood-brain barrier opening without effects on the opening volume

NASA Astrophysics Data System (ADS)

Tung, Yao-Sheng; Olumolade, Yemi; Wang, Shutao; Wu, Shih-Ying; Konofagou, Elisa E.

2012-11-01

Acoustic cavitation has been identified as the main physical mechanism for the focused ultrasound (FUS) induced blood-brain barrier (BBB) opening. In this paper, the mechanism of stable cavitation (SC) and inertial cavitation (IC) responsible for BBB opening was investigated. Thirty-three (n=33) mice were intravenously injected with bubbles of 4-5 μm in diameter. The right hippocampus was then sonicated using focused 1.5-MHz ultrasound and three different studies were carried out. First, pulse lengths (PLs) of 0.1, 0.5, 2, and 5 ms at 0.18- MPa peak rarefactional pressure with 5-Hz pulse repetition frequency (PRF) and 5-minute duration were used to identify the threshold of PL using SC. Second, the effects of the duty cycle and exposure time were investigated. Third, the BBB opening size was compared between the SC and the IC. In the case of IC-induced BBB opening, a burst sequence (3-cycles PL; 5-Hz burst repetition frequency (BRF); 30 s duration) at 0.45 MPa was applied. Passive cavitation detection was performed with each sonication to detect whether a broadband response was obtained, i.e., if IC occurred, during BBB opening. The properties of BBB opening were measured through MRI. The threshold of PL for BBB opening was identified between 0.1 and 0.5 ms using SC, but the BBB can be opened in few cycles using IC. The BBB opening volume and normalized intensity increased with the PL, but reached saturation when the PL was above 2 ms. Once the PL threshold was reached, the same exposure time induced a similar BBB opening volume, but longer sonication duration induced higher MR intensity. The duty cycle was found not to play an important role on the BBB opening. Comparable BBB opening volume (20-25 mm3) could be reached between long PL (7500 cycles, i.e., 5 ms) at 0.18 MPa and 3 cycles at 0.45 MPa. 3-kDa fluorescently tagged dextran may be able to diffuse to the parenchyma after IC-induced BBB opening at 0.45 MPa but not after SC-induced BBB opening at 0.18 MPa.

Cyclosporine A alleviated matrix metalloproteinase 9 associated blood-brain barrier disruption after subarachnoid hemorrhage in mice.

PubMed

Pan, Pengyu; Zhang, Xuan; Li, Qiang; Zhao, Hengli; Qu, Jie; Zhang, John H; Liu, Xin; Feng, Hua; Chen, Yujie

2017-05-10

The aim of this study was to investigate whether Cyclosporine A (CsA) attenuates early brain injury by alleviating matrix metalloproteinase 9 (MMP-9) associated blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in C57B6J mice. Dosages of 5mg/kg, 10mg/kg and 15mg/kg CsA were evaluated for effects on neurological score, brain water content, Evans blue extravasation and fluorescence, P-p65, MMP-9 and BBB components' alterations after SAH. We found that CsA 15mg/kg is effective in attenuating BBB disruption, lowering edema, and improving neurological outcomes. In addition, Collagen IV, ZO-1, Occludin and Claudin 5 expressions in ipsilateral/left hemisphere were downregulated after SAH, but increased after CsA treatment. Our results suggest that CsA exert a neuroprotective role in SAH pathophysiology, possibly by alleviating MMP-9 associated BBB disruption. Copyright © 2017 Elsevier B.V. All rights reserved.

Prediction of Central Nervous System Side Effects Through Drug Permeability to Blood-Brain Barrier and Recommendation Algorithm.

PubMed

Fan, Jun; Yang, Jing; Jiang, Zhenran

2018-04-01

Drug side effects are one of the public health concerns. Using powerful machine-learning methods to predict potential side effects before the drugs reach the clinical stages is of great importance to reduce time consumption and protect the security of patients. Recently, researchers have proved that the central nervous system (CNS) side effects of a drug are closely related to its permeability to the blood-brain barrier (BBB). Inspired by this, we proposed an extended neighborhood-based recommendation method to predict CNS side effects using drug permeability to the BBB and other known features of drug. To the best of our knowledge, this is the first attempt to predict CNS side effects considering drug permeability to the BBB. Computational experiments demonstrated that drug permeability to the BBB is an important factor in CNS side effects prediction. Moreover, we built an ensemble recommendation model and obtained higher AUC score (area under the receiver operating characteristic curve) and AUPR score (area under the precision-recall curve) on the data set of CNS side effects by integrating various features of drug.

Magnetic targeting of nanoparticles across the intact blood–brain barrier

PubMed Central

Kong, Seong Deok; Lee, Jisook; Ramachandran, Srinivasan; Eliceiri, Brian P.; Shubayev, Veronica I.; Lal, Ratnesh; Jin, Sungho

2015-01-01

Delivery of therapeutic or diagnostic agents across an intact blood–brain barrier (BBB) remains a major challenge. Here we demonstrate in a mouse model that magnetic nanoparticles (MNPs) can cross the normal BBB when subjected to an external magnetic field. Following a systemic administration, an applied external magnetic field mediates the ability of MNPs to permeate the BBB and accumulate in a perivascular zone of the brain parenchyma. Direct tracking and localization inside endothelial cells and in the perivascular extracellular matrix in vivo was established using fluorescent MNPs. These MNPs were inert and associated with low toxicity, using a non-invasive reporter for astrogliosis, biochemical and histological studies. Atomic force microscopy demonstrated that MNPs were internalized by endothelial cells, suggesting that trans-cellular trafficking may be a mechanism for the MNP crossing of the BBB observed. The silica-coated magnetic nanocapsules (SiMNCs) allow on-demand drug release via remote radio frequency (RF) magnetic field. Together, these results establish an effective strategy for regulating the biodistribution of MNPs in the brain through the application of an external magnetic field. PMID:23063548

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood–brain barrier primary triple coculture model

PubMed Central

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Background Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. Conclusion These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes. PMID:26491287

Glial Expression of Disease-associated Poly-glutamine Proteins Impairs the Blood-Brain Barrier in Drosophila.

PubMed

Yeh, Po-An; Liu, Ya-Hsin; Chu, Wei-Chen; Liu, Jia-Yu; Sun, Y Henry

2018-05-02

Expansion of poly-glutamine (polyQ) stretches in several proteins has been linked to neurodegenerative diseases. The effects of polyQ-expanded proteins on neurons have been extensively studied, but their effects on glia remain unclear. We found that expression of distinct polyQ proteins exclusively in all glia or specifically in the blood-brain barrier (BBB) and blood-retina barrier (BRB) glia caused cell-autonomous impairment of BBB/BRB integrity, suggesting that BBB/BRB glia are most vulnerable to polyQ-expanded proteins. Furthermore, we also found that BBB/BRB leakage in Drosophila is reflected in reversed waveform polarity based on electroretinography (ERG), making ERG a sensitive method to detect BBB/BRB leakage. The polyQ-expanded protein Atxn3-84Q forms aggregates, induces BBB/BRB leakage, restricts Drosophila lifespan, and reduces the level of Repo (a pan-glial transcriptional factor required for glial differentiation). Expression of Repo in BBB/BRB glia can rescue BBB/BRB leakage, suggesting that the reduced expression of Repo is important for the effect of polyQ on BBB/BRB impairment. Coexpression of the chaperon HSP40 and HSP70 effectively rescues the effects of Atxn3-84Q, indicating that polyQ protein aggregation in glia is deleterious. Intriguingly, coexpression of wildtype Atxn3-27Q can also rescue BBB/BRB impairment, suggesting that normal polyQ protein may have a protective function.

Glioma targeted delivery strategy of doxorubicin-loaded liposomes by dual-ligand modification.

PubMed

Han, Wei; Yin, Guangfu; Pu, Ximing; Chen, Xianchun; Liao, Xiaoming; Huang, Zhongbing

2017-10-01

The blood-brain barrier (BBB) is the protective parclose of brain safety, but it is also the main obstacle of the drug delivery to cerebral parenchyma, which hamper therapy for brain diseases. In this work, a glioma targeted drug delivery system was developed through loading doxorubicin into Angiopep-2 and TAT peptide dual-modified liposomes (DOX-TAT-Ang-LIP). Low-density lipoprotein receptor-related protein-1 (LRP1) was one receptor overexpressed on both BBB and glioma cytomembranes. Angiopep-2, a specific ligand of LRP1, exhibited high LRP1 binding efficiency. Additionally, TAT could penetrate through cell membranes without selectivity via an unsaturated pathway. To avoid the receptor saturation of Angiopep-2, TAT was also conjugated on the surface of liposomes, providing that the liposomes not only have effective BBB penetrating effect, but also have the glioma targeting function. The prepared DOX liposomes appeared good stability and narrow dispersity in serum with a diameter of 90 nm, and exhibited sustained DOX release behaviors. The conjunctions of Angiopep-2 and TAT were confirmed by 1 H NMR spectra. The BBB model, cellular uptake observations, antiproliferation study, and the cell ultrastructure analyses suggested that DOX-TAT-Ang-LIP could not only penetrate through BBB via transcytosis, but also concentrate in glioma, then enter into glioma cells and finally result in the necrosis of glioma cells.

Blood-Brain Barrier Breakdown Following Traumatic Brain Injury: A Possible Role in Posttraumatic Epilepsy

PubMed Central

Tomkins, Oren; Feintuch, Akiva; Benifla, Moni; Cohen, Avi; Friedman, Alon; Shelef, Ilan

2011-01-01

Recent animal experiments indicate a critical role for opening of the blood-brain barrier (BBB) in the pathogenesis of post-traumatic epilepsy (PTE). This study aimed to investigate the frequency, extent, and functional correlates of BBB disruption in epileptic patients following mild traumatic brain injury (TBI). Thirty-seven TBI patients were included in this study, 19 of whom suffered from PTE. All underwent electroencephalographic (EEG) recordings and brain magnetic resonance imaging (bMRI). bMRIs were evaluated for BBB disruption using novel quantitative techniques. Cortical dysfunction was localized using standardized low-resolution brain electromagnetic tomography (sLORETA). TBI patients displayed significant EEG slowing compared to controls with no significant differences between PTE and nonepileptic patients. BBB disruption was found in 82.4% of PTE compared to 25% of non-epileptic patients (P = .001) and could be observed even years following the trauma. The volume of cerebral cortex with BBB disruption was significantly larger in PTE patients (P = .001). Slow wave EEG activity was localized to the same region of BBB disruption in 70% of patients and correlated to the volume of BBB disrupted cortex. We finally present a patient suffering from early cortical dysfunction and BBB breakdown with a gradual and parallel resolution of both pathologies. Our findings demonstrate that BBB pathology is frequently found following mild TBI. Lasting BBB breakdown is found with increased frequency and extent in PTE patients. Based on recent animal studies and the colocalization found between the region of disrupted BBB and abnormal EEG activity, we suggest a role for a vascular lesion in the pathogenesis of PTE. PMID:21436875

Ethyl pyruvate protects against blood-brain barrier damage and improves long-term neurological outcomes in a rat model of traumatic brain injury.

PubMed

Shi, Hong; Wang, Hai-Lian; Pu, Hong-Jian; Shi, Ye-Jie; Zhang, Jia; Zhang, Wen-Ting; Wang, Guo-Hua; Hu, Xiao-Ming; Leak, Rehana K; Chen, Jun; Gao, Yan-Qin

2015-04-01

Many traumatic brain injury (TBI) survivors sustain neurological disability and cognitive impairments due to the lack of defined therapies to reduce TBI-induced long-term brain damage. Ethyl pyruvate (EP) has shown neuroprotection in several models of acute brain injury. The present study therefore investigated the potential beneficial effect of EP on long-term outcomes after TBI and the underlying mechanisms. Male adult rats were subjected to unilateral controlled cortical impact injury. EP was injected intraperitoneally 15 min after TBI and again at 12, 24, 36, 48, and 60 h after TBI. Neurological deficits, blood-brain barrier (BBB) integrity, and neuroinflammation were assessed. Ethyl pyruvate improved sensorimotor and cognitive functions and ameliorated brain tissue damage up to 28 day post-TBI. BBB breach and brain edema were attenuated by EP at 48 h after TBI. EP suppressed matrix metalloproteinase (MMP)-9 production from peripheral neutrophils and reduced the number of MMP-9-overproducing neutrophils in the spleen, and therefore mitigated MMP-9-mediated BBB breakdown. Moreover, EP exerted potent antiinflammatory effects in cultured microglia and inhibited the elevation of inflammatory mediators in the brain after TBI. Ethyl pyruvate confers long-term neuroprotection against TBI, possibly through breaking the vicious cycle among MMP-9-mediated BBB disruption, neuroinflammation, and long-lasting brain damage. © 2014 John Wiley & Sons Ltd.

Kinetics of Transferrin and Transferrin-Receptor during Iron Transport through Blood Brain Barrier

NASA Astrophysics Data System (ADS)

Khan, Aminul; Liu, Jin; Dutta, Prashanta

2017-11-01

Transferrin and its receptors play an important role during the uptake and transcytosis of iron by blood brain barrier (BBB) endothelial cells to maintain iron homeostasis in BBB endothelium and brain. In the blood side of BBB, ferric iron binds with the apo-transferrin to form holo-transferrin which enters the endothelial cell via transferrin receptor mediated endocytosis. Depending on the initial concentration of iron inside the cell endocytosed holo-transferrin can either be acidified in the endosome or exocytosed through the basolateral membrane. Acidification of holo-transferrin in the endosome releases ferrous irons which may either be stored and used by the cell or transported into brain side. Exocytosis of the holo-transferrin through basolateral membrane leads to transport of iron bound to transferrin into brain side. In this work, kinetics of internalization, recycling and exocytosis of transferrin and its receptors are modeled by laws of mass action during iron transport in BBB endothelial cell. Kinetic parameters for the model are determined by least square analysis. Our results suggest that the cell's initial iron content determines the extent of the two possible iron transport pathways, which will be presented in this talk Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM122081.

Monitoring Blood-Brain Barrier Integrity Following Amyloid-β Immunotherapy Using Gadolinium-Enhanced MRI in a PDAPP Mouse Model.

PubMed

Blockx, Ines; Einstein, Steve; Guns, Pieter-Jan; Van Audekerke, Johan; Guglielmetti, Caroline; Zago, Wagner; Roose, Dimitri; Verhoye, Marleen; Van der Linden, Annemie; Bard, Frederique

2016-09-06

Amyloid-related imaging abnormalities (ARIA) have been reported with some anti-amyloid-β (Aβ) immunotherapy trials. They are detected with magnetic resonance imaging (MRI) and thought to represent transient accumulation of fluid/edema (ARIA-E) or microhemorrhages (ARIA-H). Although the clinical significance and pathophysiology are unknown, it has been proposed that anti-Aβimmunotherapy may affect blood-brain barrier (BBB) integrity. To examine vascular integrity in aged (12-16 months) PDAPP and wild type mice (WT), we performed a series of longitudinal in vivo MRI studies. Mice were treated on a weekly basis using anti-Aβimmunotherapy (3D6) and follow up was done longitudinally from 1-12 weeks after treatment. BBB-integrity was assessed using both visual assessment of T1-weighted scans and repeated T1 mapping in combination with gadolinium (Gd-DOTA). A subset of 3D6 treated PDAPP mice displayed numerous BBB disruptions, whereas WT and saline-treated PDAPP mice showed intact BBB integrity under the conditions tested. In addition, the contrast induced decrease in T1 value was observed in the meningeal and midline area. BBB disruption events occurred early during treatment (between 1 and 5 weeks), were transient, and resolved quickly. Finally, BBB-leakages associated with microhemorrhages were confirmed by Perls'Prussian blue histopathological analysis. Our preclinical findings support the hypothesis that 3D6 leads to transient leakage from amyloid-positive vessels. The current study has provided valuable insights on the time course of vascular alterations during immunization treatment and supports further research in relation to the nature of ARIA and the utility of in vivo repeated T1 MRI as a translational tool.

A novel animal model to evaluate the ability of a drug delivery system to promote the passage through the BBB.

PubMed

Iannone, Michelangelo; Cosco, Donato; Cilurzo, Felisa; Celia, Christian; Paolino, Donatella; Mollace, Vincenzo; Rotiroti, Domenicantonio; Fresta, Massimo

2010-01-18

The purpose of this investigation was to explore the potentiality of a novel animal model to be used for the in vivo evaluation of the ability of a drug delivery system to promote the passage through the blood-brain barrier (BBB) and/or to improve the brain localization of a bioactive compound. A Tween 80-coated poly-L-lactid acid nanoparticles was used as a model of colloidal drug delivery system, able to trespass the BBB. Tacrine, administered in LiCl pre-treated rats, induces electrocorticographic seizures and delayed hippocampal damage. The toxic effects of tacrine-loaded poly-L-lactid acid nanoparticles (5mg/kg), a saline solution of tacrine (5mg/kg) and an empty colloidal nanoparticle suspension were compared following i.p. administration in LiCl-pre-treated Wistar rats. All the animals treated with tacrine-loaded nanoparticles showed an earlier outcome of CNS adverse symptoms, i.e. epileptic onset, with respect to those animals treated with the free compound (10 min vs. 22 min respectively). In addition, tacrine-loaded nanoparticles administration induced damage of neuronal cells in CA1 field of the hippocampus in all treated animals, while the saline solution of tacrine only in 60% of animals. Empty nanoparticles provided similar results to control (saline-treated) group of animals. In conclusion, the evaluation of time-to-onset of symptoms and the severity of neurodegenerative processes induced by the tacrine-lithium model of epilepsy in the rat, could be used to evaluate preliminarily the capability of a drug delivery system to trespass (or not) the BBB in vivo. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Transferrin-modified liposome promotes α-mangostin to penetrate the blood-brain barrier.

PubMed

Chen, Zhi-Lan; Huang, Man; Wang, Xia-Rong; Fu, Jun; Han, Min; Shen, You-Qing; Xia, Zheng; Gao, Jian-Qing

2016-02-01

α-Mangostin (α-M) is a polyphenolic xanthone that protects and improves the survival of cerebral cortical neurons against Aβ oligomer-induced toxicity in rats. α-M is a potential candidate as a treatment for Alzheimer's disease (AD). However, the efficacy was limited by the poor penetration of the drug through the blood-brain barrier (BBB). In this study, we modified the α-M liposome with transferrin (Tf) and investigated the intracellular distribution of liposomes in bEnd3 cells. In addition, the transport of α-M across the BBB in the Tf(α-M) liposome group was examined. In vitro studies demonstrated that the Tf(α-M) liposome could cross the BBB in the form of an integrated liposome. Results of the in vivo studies on the α-M distribution in the brain demonstrated that the Tf(α-M) liposome improved the brain delivery of α-M. These results indicated that the Tf liposome is a potential carrier of α-M against AD. The use of α-Mangostin (α-M) as a potential agent to treat Alzheimer's disease (AD) has been reported. However, its use is limited by the poor penetration through the blood brain barrier. The delivery of this agent by transferrin-modified liposomes was investigated by the authors in this study. The positive results could point to a better drug delivery system for brain targeting. Copyright © 2015 Elsevier Inc. All rights reserved.

Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin.

PubMed

Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C; Malle, Ernst; Sattler, Wolfgang

2015-02-15

Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl(-) system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood-brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120min, decaying at a rate of 5.9×10(-3)min(-1). NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC-MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin

PubMed Central

Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C.; Malle, Ernst; Sattler, Wolfgang

2015-01-01

Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

Strategies for transporting nanoparticles across the blood-brain barrier.

PubMed

Zhang, Tian-Tian; Li, Wen; Meng, Guanmin; Wang, Pei; Liao, Wenzhen

2016-02-01

The existence of blood-brain barrier (BBB) hampers the effective treatment of central nervous system (CNS) diseases. Almost all macromolecular drugs and more than 98% of small molecule drugs cannot pass the BBB. Therefore, the BBB remains a big challenge for delivery of therapeutics to the central nervous system. With the structural and mechanistic elucidation of the BBB under both physiological and pathological conditions, it is now possible to design delivery systems that could cross the BBB effectively. Because of their advantageous properties, nanoparticles have been widely deployed for brain-targeted delivery. This review paper presents the current understanding of the BBB under physiological and pathological conditions, and summarizes strategies and systems for BBB crossing with a focus on nanoparticle-based drug delivery systems. In summary, with wider applications and broader prospection the treatment of brain targeted therapy, nano-medicines have proved to be more potent, more specific and less toxic than traditional drug therapy.

Drug Delivery to the Brain in Alzheimer’s Disease: Consideration of the Blood-brain Barrier

PubMed Central

Banks, William A.

2012-01-01

The successful treatment of Alzheimer’s disease (AD) will require drugs that can negotiate the blood-brain barrier (BBB). However, the BBB is not simply a physical barrier, but a complex interface that is in intimate communication with the rest of the central nervous system (CNS) and influenced by peripheral tissues. This review examines three aspects of the BBB in AD. First, it considers how the BBB may be contributing to the onset and progression of AD. In this regard, the BBB itself is a therapeutic target in the treatment of AD. Second, it examines how the BBB restricts drugs that might otherwise be useful in the treatment of AD and examines strategies being developed to deliver drugs to the CNS for the treatment of AD. Third, it considers how drug penetration across the AD BBB may differ from the BBB of normal aging. In this case, those differences can complicate the treatment of CNS diseases such as depression, delirium, psychoses, and pain control in the AD population. PMID:22202501

Cytokine Signaling Modulates Blood-Brain Barrier Function

PubMed Central

Pan, Weihong; Stone, Kirsten P.; Hsuchou, Hung; Manda, Vamshi K.; Zhang, Yan; Kastin, Abba J.

2014-01-01

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease. PMID:21834767

Blood–brain barrier dysfunction and epilepsy: Pathophysiologic role and therapeutic approaches

PubMed Central

Marchi, Nicola; Granata, Tiziana; Ghosh, Chaitali; Janigro, Damir

2016-01-01

The blood–brain barrier (BBB) is located within a unique anatomic interface and has functional ramifications to most of the brain and blood cells. In the past, the BBB was considered a pharmacokinetic impediment to antiepileptic drug penetration into the brain; nowadays it is becoming increasingly evident that targeting of the damaged or dysfunctional BBB may represent a therapeutic approach to reduce seizure burden. Several studies have investigated the mechanisms linking the onset and sustainment of seizures to BBB dysfunction. These studies have shown that the BBB is at the crossroad of a multifactorial pathophysiologic process that involves changes in brain milieu, altered neuroglial physiology, development of brain inflammation, leukocyte–endothelial interactions, faulty angiogenesis, and hemodynamic changes leading to energy mismatch. A number of knowledge gaps, conflicting points of view, and discordance between clinical and experimental data currently characterize this field of neuroscience. As more pieces are added to this puzzle, it is apparent that each mechanism needs to be validated in an appropriate clinical context. We now offer a BBB-centric view of seizure disorders, linking several aspects of seizures and epilepsy physiopathology to BBB dysfunction. We have reviewed the therapeutic, antiseizure effect of drugs that promote BBB repair. We also present BBB neuroimaging as a tool to correlate BBB restoration to seizure mitigation. Add-on cerebrovascular drug could be of efficacy in reducing seizure burden when used in association with neuronal antiepileptic drugs. PMID:22905812

Selective Targeting of Brain Tumors with Gold Nanoparticle-Induced Radiosensitization

PubMed Central

Joh, Daniel Y.; Sun, Lova; Stangl, Melissa; Al Zaki, Ajlan; Murty, Surya; Santoiemma, Phillip P.; Davis, James J.; Baumann, Brian C.; Alonso-Basanta, Michelle; Bhang, Dongha; Kao, Gary D.; Tsourkas, Andrew; Dorsey, Jay F.

2013-01-01

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature. PMID:23638079

Cholera Toxin Subunit B Enabled Multifunctional Glioma-Targeted Drug Delivery.

PubMed

Guan, Juan; Zhang, Zui; Hu, Xuefeng; Yang, Yang; Chai, Zhilan; Liu, Xiaoqin; Liu, Jican; Gao, Bo; Lu, Weiyue; Qian, Jun; Zhan, Changyou

2017-12-01

Glioma is among the most formidable brain cancers due to location in the brain. Cholera toxin subunit B (CTB) is investigated to facilitate multifunctional glioma-targeted drug delivery by targeting the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasulature, and glioma cells. When modified on the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CTB-NPs), CTB fully retains its bioactivity after 24 h incubation in the fresh mouse plasma. The formed protein corona (PC) of CTB-NP and plain PLGA nanoparticles (NP) after incubation in plasma is analyzed using liquid chromatography tandem massspectrometry (nano-LC-MS/MS). CTB modification does not alter the protein components of the formed PC, macrophage phagocytosis, or pharmacokinetic profiles. CTB-NP can efficiently penetrate the in vitro BBB model and target glioma cells and human umbilical vascular endothelial cells. Paclitaxel is loaded in NP (NP/PTX) and CTB-NP (CTB-NP/PTX), and their antiglioma effects are assessed in nude mice bearing intracranial glioma. CTB-NP/PTX can efficiently induce apoptosis of intracranial glioma cells and ablate neovasulature in vivo, resulting in significant prolongation of survival of nude mice bearing intracranial glioma (34 d) in comparison to those treated with NP/PTX (29 d), Taxol (24 d), and saline (21 d). The present study suggests a potential multifunctional glioma-targeted drug delivery system enabled by cholera toxin subunit B. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets.

PubMed

Li, Tengfei; Vandesquille, Matthias; Koukouli, Fani; Dudeffant, Clémence; Youssef, Ihsen; Lenormand, Pascal; Ganneau, Christelle; Maskos, Uwe; Czech, Christian; Grueninger, Fiona; Duyckaerts, Charles; Dhenain, Marc; Bay, Sylvie; Delatour, Benoît; Lafaye, Pierre

2016-12-10

Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-Target Protective Effects of Gintonin in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Mediated Model of Parkinson's Disease via Lysophosphatidic Acid Receptors.

PubMed

Choi, Jong Hee; Jang, Minhee; Oh, Seikwan; Nah, Seung-Yeol; Cho, Ik-Hyun

2018-01-01

Gintonin is a ginseng-derived lysophosphatidic acid receptor (LPAR) ligand. Although previous in vitro and in vivo studies demonstrated the therapeutic role of gintonin against Alzheimer's disease, the neuroprotective effects of gintonin in Parkinson's disease (PD) are still unknown. We investigated whether gintonin (50 and 100 mg/kg/day, p.o., daily for 12 days) had neuroprotective activities against neurotoxicity in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Pre-administration of 100 mg/kg gintonin displayed significantly ameliorating effects in neurological disorders (motor and welfare) as measuring using pole, rotarod, and nest building tests, and in the survival rate. These effects were associated to the reduction of the loss of tyrosine hydroxylase-positive neurons, microglial activation, activation of inflammatory mediators (interleukin-6, tumor necrosis factor, and cyclooxygenase-2), and alteration of blood-brain barrier (BBB) integrity in the substantia nigra pars compacta and/or striatum following MPTP injection. The benefits of gintonin treatment against MPTP also included the activation of the nuclear factor erythroid 2-related factor 2 pathways and the inhibition of phosphorylation of the mitogen-activated protein kinases and nuclear factor-kappa B signaling pathways. Interestingly, these neuroprotective effects of gintonin were blocked by LPAR1/3 antagonist, Ki16425. Overall, the present study shows that gintonin attenuates MPTP-induced neurotoxicity via multiple targets. Gintonin combats neuronal death, and acts as an anti-inflammatory and an anti-oxidant agent. It maintains BBB integrity. LPA receptors play a key role in gintonin-mediated anti-PD mechanisms. Finally, gintonin is a key agent for prevention and/or treatment of PD.

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

PubMed

Thom, George; Burrell, Matthew; Haqqani, Arsalan S; Yogi, Alvaro; Lessard, Etienne; Brunette, Eric; Delaney, Christie; Baumann, Ewa; Callaghan, Deborah; Rodrigo, Natalia; Webster, Carl I; Stanimirovic, Danica B

2018-04-02

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having K D s of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Trans-cranial opening of the blood-brain barrier in targeted regions using a stereotaxic brain atlas and focused ultrasound energy.

PubMed

Bing, Chenchen; Ladouceur-Wodzak, Michelle; Wanner, Clinton R; Shelton, John M; Richardson, James A; Chopra, Rajiv

2014-01-01

The blood-brain barrier (BBB) protects the brain by preventing the entry of large molecules; this poses a major obstacle for the delivery of drugs to the brain. A novel technique using focused ultrasound (FUS) energy combined with microbubble contrast agents has been widely used for non-invasive trans-cranial BBB opening. Traditionally, FUS research is conducted with magnetic resonance imaging (MRI) guidance, which is expensive and poses physical limitations due to the magnetic field. A system that could allow researchers to test brain therapies without MR intervention could facilitate and accelerate translational research. In this study, we present a novel FUS system that uses a custom-built FUS generator mounted on a motorized stereotaxic apparatus with embedded brain atlas to locally open the BBB in rodents. The system was initially characterized using a tissue-mimicking phantom. Rodent studies were also performed to evaluate whether non-invasive, localized BBB opening could be achieved using brain atlas-based targeting. Brains were exposed to pulsed focused ultrasound energy at 1.06 MHz in rats and 3.23 MHz in mice, with the focal pressure estimated to be 0.5-0.6 MPa through the skull. BBB opening was confirmed in gross tissue sections by the presence of Evans blue leakage in the exposed region of the brain and by histological assessment. The targeting accuracy of the stereotaxic system was better than 0.5 mm in the tissue-mimicking phantom. Reproducible localized BBB opening was verified with Evans blue dye leakage in 32/33 rats and had a targeting accuracy of ±0.3 mm. The use of higher frequency exposures in mice enabled a similar precision of localized BBB opening as was observed with the low frequency in the rat model. With this dedicated small-animal motorized stereotaxic-FUS system, we achieved accurate targeting of focused ultrasound exposures in the brain for non-invasive opening of the BBB. This system can be used as an alternative to MR-guided FUS and offers researchers the ability to perform efficient studies (30 min per experiment including preparation) at a reduced cost in a conventional laboratory environment.

Limitations of the hCMEC/D3 cell line as a model for Aβ clearance by the human blood-brain barrier.

PubMed

Biemans, Elisanne A L M; Jäkel, Lieke; de Waal, Robert M W; Kuiperij, H Bea; Verbeek, Marcel M

2017-07-01

Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-β (Aβ) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Aβ clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aβ clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aβ was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aβ. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aβ clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Ultrasound-Induced Blood-Brain Barrier Opening

PubMed Central

Konofagou, Elisa E.; Tung, Yao-Sheng; Choi, James; Deffieux, Thomas; Baseri, Babak; Vlachos, Fotios

2014-01-01

Over 4 million U.S. men and women suffer from Alzheimer's disease; 1 million from Parkinson's disease; 350,000 from multiple sclerosis (MS); and 20,000 from amyotrophic lateral sclerosis (ALS). Worldwide, these four diseases account for more than 20 million patients. In addition, aging greatly increases the risk of neurodegenerative disease. Although great progress has been made in recent years toward understanding of these diseases, few effective treatments and no cures are currently available. This is mainly due to the impermeability of the blood-brain barrier (BBB) that allows only 5% of the 7000 small-molecule drugs available to treat only a tiny fraction of these diseases. On the other hand, safe and localized opening of the BBB has been proven to present a significant challenge. Of the methods used for BBB disruption shown to be effective, Focused Ultrasound (FUS), in conjunction with microbubbles, is the only technique that can induce localized BBB opening noninvasively and regionally. FUS may thus have a huge impact in trans-BBB brain drug delivery. The primary objective in this paper is to elucidate the interactions between ultrasound, microbubbles and the local microenvironment during BBB opening with FUS, which are responsible for inducing the BBB disruption. The mechanism of the BBB opening in vivo is monitored through the MRI and passive cavitation detection (PCD), and the safety of BBB disruption is assessed using H&E histology at distinct pressures, pulse lengths and microbubble diameters. It is hereby shown that the BBB can be disrupted safely and transiently under specific acoustic pressures (under 0.45 MPa) and microbubble (diameter under 8 μm) conditions. PMID:22201586

Morphofunctional aspects of the blood-brain barrier.

PubMed

Nico, Beatrice; Ribatti, Domenico

2012-01-01

The blood-brain barrier (BBB) selectively controls the homeostasis of the Central Nervous System (CNS) environment by the specific structural and biochemical features of the endothelial cells, pericytes and glial endfeet, which represent the cellular components of the mature BBB. Endothelial tight junctions (TJs) are the most important structural component of the BBB, and molecular alteration in the phosphorylation state of some TJs proteins, like ZO-1 or occludin, are crucial in determining alterations in the control of BBB vascular permeability. Astrocytes endfeet enveloping the vessels wall, are considered important in the induction and maintenance of the BBB, through secretion of soluble factors, which modulate the expression of enzymatic complexes and antigens by endothelial cells and TJs - associated proteins. Moreover, astrocytes control water flux at BBB site by expressing a specific water channel, namely aquaporin-4 (AQP4), involved in the molecular composition of the orthogonal particles arrays (OAPs) on the perivascular glial endfeet and tightly coupled with the maintenance of the BBB integrity. Disruption of the BBB is a consistent event occurring in the development of several CNS diseases, including demyelinating lesions in the course of relapsing multiple sclerosis, stroke, Duchenne muscular dystrophy (DMD), but also mechanical injures, neurological insults, septic encephalopathy, brain tumors, permanent ischemia or transient ischemia followed by reperfusion. In most cases, these pathological conditions are associated with an increase in microvascular permeability, vasogenic edema, swollen atrocyte endfeet, and BBB disruption.

Mechanism and Safety at the Threshold of the Blood-Brain Barrier Opening In Vivo

NASA Astrophysics Data System (ADS)

Konofagou, Elisa E.; Choi, James; Baseri, Babak; Selert, Kirsten; Tung, Yao-Sheng

2010-03-01

Current treatments of neurological and neurodegenerative diseases are limited due to the blood-brain barrier (BBB). In this paper, the threshold of BBB opening and its dependence on the microbubble diameter as well as the associated mechanism and safety are identified in vivo. In vivo BBB opening in mice (n = 13) was achieved by systemically injecting microbubbles (Definity and 1-2 and 4-5-μm lipid-shelled, gas-filled) and applying pulsed FUS (frequency: 1.525 MHz, peak-rarefactional pressure: 150-600 kPa) to the left hippocampus through the intact skin and skull. Systemically administered, BBB-impermeable, fluorescent-tagged dextrans at 3 kDa were injected to confirm BBB opening. H&E histology was also performed to determine any associated vascular or neuronal damage. Detection of stable and inertial cavitation was performed using a passive cavitation device (PCD) in a blood vessel phantom at the same pressure amplitudes as those used in vivo. Larger microbubbles (4-5 μm) resulted in a lower threshold of BBB opening. Cavitation studies confirmed that stable cavitation occurs at the threshold of BBB opening. The mechanism of BBB opening at the threshold was identified to be mainly related to stable cavitation. Histological studies indicated that, at the BBB opening threshold, no red-blood cell extravasation or neuronal damage was noted.

Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System.

PubMed

Lochhead, Jeffrey J; Ronaldson, Patrick T; Davis, Thomas P

2017-07-01

A functional blood-brain barrier (BBB) is necessary to maintain central nervous system (CNS) homeostasis. Many diseases affecting the CNS, however, alter the functional integrity of the BBB. It has been shown that various diseases and physiological stressors can impact the BBB's ability to selectively restrict passage of substances from the blood to the brain. Modifications of the BBB's permeability properties can potentially contribute to the pathophysiology of CNS diseases and result in altered brain delivery of therapeutic agents. Hypoxia and/or inflammation are central components of a number of diseases affecting the CNS. A number of studies indicate hypoxia or inflammatory pain increase BBB paracellular permeability, induce changes in the expression and/or localization of tight junction proteins, and affect CNS drug uptake. In this review, we look at what is currently known with regard to BBB disruption following a hypoxic or inflammatory insult in vivo. Potential mechanisms involved in altering tight junction components at the BBB are also discussed. A more detailed understanding of the mediators involved in changing BBB functional integrity in response to hypoxia or inflammatory pain could potentially lead to new treatments for CNS diseases with hypoxic or inflammatory components. Additionally, greater insight into the mechanisms involved in TJ rearrangement at the BBB may lead to novel strategies to pharmacologically increase delivery of drugs to the CNS.

Drug delivery across the blood-brain barrier using focused ultrasound

PubMed Central

Burgess, Alison; Hynynen, Kullervo H.

2015-01-01

Introduction The presence of the blood-brain barrier (BBB) is a significant impediment to the delivery of therapeutic agents to the brain for treatment of brain diseases. Focused ultrasound (FUS) has been developed as a non-invasive method for transiently increasing the permeability of the BBB to promote drug delivery to targeted regions of the brain. Areas Covered The present review briefly compares the methods used to promote drug delivery to the brain and describes the benefits and limitations of FUS technology. We summarize the experimental data which shows that FUS, combined with intravascular microbubbles, increases therapeutic agent delivery into the brain leading to significant reductions in pathology in preclinical models of disease. The potential for translation of this technology to the clinic is also discussed. Expert Opinion The introduction of MRI guidance and intravascular administration of microbubbles to FUS treatments permits the consistent, transient, and targeted opening of the BBB. The development of feedback systems and real-time monitoring techniques improve the safety of BBB opening. Successful clinical translation of FUS has the potential to revolutionize the treatment of brain disease resulting in effective, less-invasive treatments without the need for expensive drug development. PMID:24650132

Drug delivery across the blood-brain barrier using focused ultrasound.

PubMed

Burgess, Alison; Hynynen, Kullervo

2014-05-01

The presence of the blood-brain barrier (BBB) is a significant impediment to the delivery of therapeutic agents to the brain for treatment of brain diseases. Focused ultrasound (FUS) has been developed as a noninvasive method for transiently increasing the permeability of the BBB to promote drug delivery to targeted regions of the brain. The present review briefly compares the methods used to promote drug delivery to the brain and describes the benefits and limitations of FUS technology. We summarize the experimental data which shows that FUS, combined with intravascular microbubbles, increases therapeutic agent delivery into the brain leading to significant reductions in pathology in preclinical models of disease. The potential for translation of this technology to the clinic is also discussed. The introduction of magnetic resonance imaging guidance and intravascular administration of microbubbles to FUS treatments permits the consistent, transient and targeted opening of the BBB. The development of feedback systems and real-time monitoring techniques improve the safety of BBB opening. Successful clinical translation of FUS has the potential to revolutionize the treatment of brain disease resulting in effective, less-invasive treatments without the need for expensive drug development.

Silencing microRNA-143 protects the integrity of the blood-brain barrier: implications for methamphetamine abuse

PubMed Central

Bai, Ying; Zhang, Yuan; Hua, Jun; Yang, Xiangyu; Zhang, Xiaotian; Duan, Ming; Zhu, Xinjian; Huang, Wenhui; Chao, Jie; Zhou, Rongbin; Hu, Gang; Yao, Honghong

2016-01-01

MicroRNA-143 (miR-143) plays a critical role in various cellular processes; however, the role of miR-143 in the maintenance of blood-brain barrier (BBB) integrity remains poorly defined. Silencing miR-143 in a genetic animal model or via an anti-miR-143 lentivirus prevented the BBB damage induced by methamphetamine. miR-143, which targets p53 unregulated modulator of apoptosis (PUMA), increased the permeability of human brain endothelial cells and concomitantly decreased the expression of tight junction proteins (TJPs). Silencing miR-143 increased the expression of TJPs and protected the BBB integrity against the effects of methamphetamine treatment. PUMA overexpression increased the TJP expression through a mechanism that involved the NF-κB and p53 transcription factor pathways. Mechanistically, methamphetamine mediated up-regulation of miR-143 via sigma-1 receptor with sequential activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3′ kinase (PI3K)/Akt and STAT3 pathways. These results indicated that silencing miR-143 could provide a novel therapeutic strategy for BBB damage-related vascular dysfunction. PMID:27767041

The quantification of blood-brain barrier disruption using dynamic contrast-enhanced magnetic resonance imaging in aging rhesus monkeys with spontaneous type 2 diabetes mellitus.

PubMed

Xu, Ziqian; Zeng, Wen; Sun, Jiayu; Chen, Wei; Zhang, Ruzhi; Yang, Zunyuan; Yao, Zunwei; Wang, Lei; Song, Li; Chen, Yushu; Zhang, Yu; Wang, Chunhua; Gong, Li; Wu, Bing; Wang, Tinghua; Zheng, Jie; Gao, Fabao

2017-09-01

Microvascular lesions of the body are one of the most serious complications that can affect patients with type 2 diabetes mellitus. The blood-brain barrier (BBB) is a highly selective permeable barrier around the microvessels of the brain. This study investigated BBB disruption in diabetic rhesus monkeys using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Multi-slice DCE-MRI was used to quantify BBB permeability. Five diabetic monkeys and six control monkeys underwent magnetic resonance brain imaging in 3 Tesla MRI system. Regions of the frontal cortex, the temporal cortex, the basal ganglia, the thalamus, and the hippocampus in the two groups were selected as regions of interest to calculate the value of the transport coefficient K trans using the extended Tofts model. Permeability in the diabetic monkeys was significantly increased as compared with permeability in the normal control monkeys. Histopathologically, zonula occludens protein-1 decreased, immunoglobulin G leaked out of the blood, and nuclear factor E2-related factor translocated from the cytoplasm to the nuclei. It is likely that diabetes contributed to the increased BBB permeability. Copyright © 2016 Elsevier Inc. All rights reserved.

Towards Improvements for Penetrating the Blood–Brain Barrier—Recent Progress from a Material and Pharmaceutical Perspective

PubMed Central

He, Quanguo; Liu, Jun; Liang, Jing; Liu, Xiaopeng; Li, Wen; Liu, Zhi; Ding, Ziyu; Tuo, Du

2018-01-01

The blood–brain barrier (BBB) is a critical biological structure that prevents damage to the brain and maintains its bathing microenvironment. However, this barrier is also the obstacle to deliver beneficial drugs to treat CNS (central nervous system) diseases. Many efforts have been made for improvement of delivering drugs across the BBB in recent years to treat CNS diseases. In this review, the anatomical and functional structure of the BBB is comprehensively discussed. The mechanisms of BBB penetration are summarized, and the methods and effects on increasing BBB permeability are investigated in detail. It also elaborates on the physical, chemical, biological and nanocarrier aspects to improve drug delivery penetration to the brain and introduces some specific drug delivery effects on BBB permeability. PMID:29570659

Gene therapy decreases seizures in a model of Incontinentia pigmenti.

PubMed

Dogbevia, Godwin K; Töllner, Kathrin; Körbelin, Jakob; Bröer, Sonja; Ridder, Dirk A; Grasshoff, Hanna; Brandt, Claudia; Wenzel, Jan; Straub, Beate K; Trepel, Martin; Löscher, Wolfgang; Schwaninger, Markus

2017-07-01

Incontinentia pigmenti (IP) is a genetic disease leading to severe neurological symptoms, such as epileptic seizures, but no specific treatment is available. IP is caused by pathogenic variants that inactivate the Nemo gene. Replacing Nemo through gene therapy might provide therapeutic benefits. In a mouse model of IP, we administered a single intravenous dose of the adeno-associated virus (AAV) vector, AAV-BR1-CAG-NEMO, delivering the Nemo gene to the brain endothelium. Spontaneous epileptic seizures and the integrity of the blood-brain barrier (BBB) were monitored. The endothelium-targeted gene therapy improved the integrity of the BBB. In parallel, it reduced the incidence of seizures and delayed their occurrence. Neonate mice intravenously injected with the AAV-BR1-CAG-NEMO vector developed no hepatocellular carcinoma or other major adverse effects 11 months after vector injection, demonstrating that the vector has a favorable safety profile. The data show that the BBB is a target of antiepileptic treatment and, more specifically, provide evidence for the therapeutic benefit of a brain endothelial-targeted gene therapy in IP. Ann Neurol 2017;82:93-104. © 2017 American Neurological Association.

The Application of MRI for Depiction of Subtle Blood Brain Barrier Disruption in Stroke

PubMed Central

Israeli, David; Tanne, David; Daniels, Dianne; Last, David; Shneor, Ran; Guez, David; Landau, Efrat; Roth, Yiftach; Ocherashvilli, Aharon; Bakon, Mati; Hoffman, Chen; Weinberg, Amit; Volk, Talila; Mardor, Yael

2011-01-01

The development of imaging methodologies for detecting blood-brain-barrier (BBB) disruption may help predict stroke patient's propensity to develop hemorrhagic complications following reperfusion. We have developed a delayed contrast extravasation MRI-based methodology enabling real-time depiction of subtle BBB abnormalities in humans with high sensitivity to BBB disruption and high spatial resolution. The increased sensitivity to subtle BBB disruption is obtained by acquiring T1-weighted MRI at relatively long delays (~15 minutes) after contrast injection and subtracting from them images acquired immediately after contrast administration. In addition, the relatively long delays allow for acquisition of high resolution images resulting in high resolution BBB disruption maps. The sensitivity is further increased by image preprocessing with corrections for intensity variations and with whole body (rigid+elastic) registration. Since only two separate time points are required, the time between the two acquisitions can be used for acquiring routine clinical data, keeping the total imaging time to a minimum. A proof of concept study was performed in 34 patients with ischemic stroke and 2 patients with brain metastases undergoing high resolution T1-weighted MRI acquired at 3 time points after contrast injection. The MR images were pre-processed and subtracted to produce BBB disruption maps. BBB maps of patients with brain metastases and ischemic stroke presented different patterns of BBB opening. The significant advantage of the long extravasation time was demonstrated by a dynamic-contrast-enhancement study performed continuously for 18 min. The high sensitivity of our methodology enabled depiction of clear BBB disruption in 27% of the stroke patients who did not have abnormalities on conventional contrast-enhanced MRI. In 36% of the patients, who had abnormalities detectable by conventional MRI, the BBB disruption volumes were significantly larger in the maps than in conventional MRI. These results demonstrate the advantages of delayed contrast extravasation in increasing the sensitivity to subtle BBB disruption in ischemic stroke patients. The calculated disruption maps provide clear depiction of significant volumes of BBB disruption unattainable by conventional contrast-enhanced MRI. PMID:21209786

The application of MRI for depiction of subtle blood brain barrier disruption in stroke.

PubMed

Israeli, David; Tanne, David; Daniels, Dianne; Last, David; Shneor, Ran; Guez, David; Landau, Efrat; Roth, Yiftach; Ocherashvilli, Aharon; Bakon, Mati; Hoffman, Chen; Weinberg, Amit; Volk, Talila; Mardor, Yael

2010-12-26

The development of imaging methodologies for detecting blood-brain-barrier (BBB) disruption may help predict stroke patient's propensity to develop hemorrhagic complications following reperfusion. We have developed a delayed contrast extravasation MRI-based methodology enabling real-time depiction of subtle BBB abnormalities in humans with high sensitivity to BBB disruption and high spatial resolution. The increased sensitivity to subtle BBB disruption is obtained by acquiring T1-weighted MRI at relatively long delays (~15 minutes) after contrast injection and subtracting from them images acquired immediately after contrast administration. In addition, the relatively long delays allow for acquisition of high resolution images resulting in high resolution BBB disruption maps. The sensitivity is further increased by image preprocessing with corrections for intensity variations and with whole body (rigid+elastic) registration. Since only two separate time points are required, the time between the two acquisitions can be used for acquiring routine clinical data, keeping the total imaging time to a minimum. A proof of concept study was performed in 34 patients with ischemic stroke and 2 patients with brain metastases undergoing high resolution T1-weighted MRI acquired at 3 time points after contrast injection. The MR images were pre-processed and subtracted to produce BBB disruption maps. BBB maps of patients with brain metastases and ischemic stroke presented different patterns of BBB opening. The significant advantage of the long extravasation time was demonstrated by a dynamic-contrast-enhancement study performed continuously for 18 min. The high sensitivity of our methodology enabled depiction of clear BBB disruption in 27% of the stroke patients who did not have abnormalities on conventional contrast-enhanced MRI. In 36% of the patients, who had abnormalities detectable by conventional MRI, the BBB disruption volumes were significantly larger in the maps than in conventional MRI. These results demonstrate the advantages of delayed contrast extravasation in increasing the sensitivity to subtle BBB disruption in ischemic stroke patients. The calculated disruption maps provide clear depiction of significant volumes of BBB disruption unattainable by conventional contrast-enhanced MRI.

Natural products: a hope for glioblastoma patients.

PubMed

Vengoji, Raghupathy; Macha, Muzafar A; Batra, Surinder K; Shonka, Nicole A

2018-04-24

Glioblastoma (GBM) is one of the most aggressive malignant tumors with an overall dismal survival averaging one year despite multimodality therapeutic interventions including surgery, radiotherapy and concomitant and adjuvant chemotherapy. Few drugs are FDA approved for GBM, and the addition of temozolomide (TMZ) to standard therapy increases the median survival by only 2.5 months. Targeted therapy appeared promising in in vitro monolayer cultures, but disappointed in preclinical and clinical trials, partly due to the poor penetration of drugs through the blood brain barrier (BBB). Cancer stem cells (CSCs) have intrinsic resistance to initial chemoradiation therapy (CRT) and acquire further resistance via deregulation of many signaling pathways. Due to the failure of classical chemotherapies and targeted drugs, research efforts focusing on the use of less toxic agents have increased. Interestingly, multiple natural compounds have shown antitumor and apoptotic effects in TMZ resistant and p53 mutant GBM cell lines and also displayed synergistic effects with TMZ. In this review, we have summarized the current literature on natural products or product analogs used to modulate the BBB permeability, induce cell death, eradicate CSCs and sensitize GBM to CRT.

Natural products: a hope for glioblastoma patients

PubMed Central

Vengoji, Raghupathy; Macha, Muzafar A.; Batra, Surinder K.; Shonka, Nicole A.

2018-01-01

Glioblastoma (GBM) is one of the most aggressive malignant tumors with an overall dismal survival averaging one year despite multimodality therapeutic interventions including surgery, radiotherapy and concomitant and adjuvant chemotherapy. Few drugs are FDA approved for GBM, and the addition of temozolomide (TMZ) to standard therapy increases the median survival by only 2.5 months. Targeted therapy appeared promising in in vitro monolayer cultures, but disappointed in preclinical and clinical trials, partly due to the poor penetration of drugs through the blood brain barrier (BBB). Cancer stem cells (CSCs) have intrinsic resistance to initial chemoradiation therapy (CRT) and acquire further resistance via deregulation of many signaling pathways. Due to the failure of classical chemotherapies and targeted drugs, research efforts focusing on the use of less toxic agents have increased. Interestingly, multiple natural compounds have shown antitumor and apoptotic effects in TMZ resistant and p53 mutant GBM cell lines and also displayed synergistic effects with TMZ. In this review, we have summarized the current literature on natural products or product analogs used to modulate the BBB permeability, induce cell death, eradicate CSCs and sensitize GBM to CRT. PMID:29774132

Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

NASA Technical Reports Server (NTRS)

McIntire, Larry V.; Wagner, John E.; Papadaki, Maria; Whitson, Peggy A.; Eskin, Suzanne G.

1996-01-01

Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

Focused ultrasound-mediated bbb disruption is associated with an increase in activation of AKT: experimental study in rats

PubMed Central

2010-01-01

Background The Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening; Methods The BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses; Results Immunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB; Conclusions Our data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB. PMID:21078165

Peripheral ammonia and blood brain barrier structure and function after methamphetamine.

PubMed

Northrop, Nicole A; Halpin, Laura E; Yamamoto, Bryan K

2016-08-01

An effect of the widely abuse psychostimulant, methamphetamine (Meth), is blood-brain-barrier (BBB) disruption; however, the mechanism by which Meth causes BBB disruption remains unclear. Recently it has been shown that Meth produces liver damage and consequent increases in plasma ammonia. Ammonia can mediate oxidative stress and inflammation, both of which are known to cause BBB disruption. Therefore, the current studies examined the role of peripheral ammonia in Meth-induced disruption of BBB structure and function. A neurotoxic Meth regimen (10 mg/kg, ip, q 2 h, ×4) administered to rats increased plasma ammonia and active MMP-9 in the cortex 2 h after the last Meth injection, compared to saline treated rats. At 24 h after Meth treatment, decreased immunoreactivity of BBB structural proteins, occludin and claudin-5, and increased extravasation of 10,000 Da FITC-dextran were observed, as compared to saline controls. Pretreatment with lactulose (5.3 g/kg, po, q 12 h), a drug that remains in the lumen of the intestine and promotes ammonia excretion, prevented the Meth-induced increases in plasma ammonia. These results were paralleled by the prevention of decreases in BBB structural proteins, increases in extravasation of 10,000 Da FITC-dextran and increases in active MMP-9. The results indicate that Meth-induced increases in ammonia produce BBB disruption and suggest that MMP-9 activation mediates the BBB disruption. These findings identify a novel mechanism of Meth-induced BBB disruption that is mediated by plasma ammonia and are the first to identify a peripheral contribution to Meth-induced BBB disruption. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regional differences in the blood-brain-barrier of the subfornical organs of rats and ducks (Anas platyrhynchos).

PubMed

Schmid, H A

1995-01-01

Recently published electrophysiological data investigated the effect of blood borne and brain intrinsic substances on the activity of neurons in the duck subfornical organ (SFO). This study defines histologically the region in the duck SFO, where blood borne substances can possibly influence neuronal activity. Intravenous injection of Evans blue, a dye which labels brain structures devoid of a blood brain barrier (BBB), resulted in diffuse labelling of the duck SFO from the anterior commissure to the end of the organ in rostrocaudal extension. In addition, specifically labelled neurons could be observed just rostral to the diffuse Evans blue labelling and in an area dorsomedial to the large central blood vessel. The majority of the somata of these heavily stained neurons were located inside the BBB, whereas in the areas with diffuse Evans blue labelling, thus being outside the BBB, labelled cells were rarely observed. Intravenous injection of Evans blue in rats resulted similarly in diffuse labelling of the parenchyma of the medial and caudal part of the SFO, with only a few, but heavily stained cells with fusiform somata. The rostral region of the rat SFO, which is known to have a functional BBB, shows hardly any diffuse labelling, but there the majority of neurons show strong Evans blue fluorescence. It is concluded that the heavily labelled somata inside the BBB have axonal or dendritic projections to BBB-free areas, where they can take up the dye. This study gives a functional description of the extension of the SFO areas without a BBB of rats and ducks. It is concluded that blood borne agents can affect those SFO neurons which have their somata located outside the BBB as well as those located inside the BBB which have terminals projecting to BBB free regions.

A Novel Magnetic Actuation Scheme to Disaggregate Nanoparticles and Enhance Passage across the Blood–Brain Barrier

PubMed Central

Le, Tuan-Anh; Amin, Faiz Ul; Kim, Myeong Ok

2017-01-01

The blood–brain barrier (BBB) hinders drug delivery to the brain. Despite various efforts to develop preprogramed actuation schemes for magnetic drug delivery, the unmodeled aggregation phenomenon limits drug delivery performance. This paper proposes a novel scheme with an aggregation model for a feed-forward magnetic actuation design. A simulation platform for aggregated particle delivery is developed and an actuation scheme is proposed to deliver aggregated magnetic nanoparticles (MNPs) using a discontinuous asymmetrical magnetic actuation. The experimental results with a Y-shaped channel indicated the success of the proposed scheme in steering and disaggregation. The delivery performance of the developed scheme was examined using a realistic, three-dimensional (3D) vessel simulation. Furthermore, the proposed scheme enhanced the transport and uptake of MNPs across the BBB in mice. The scheme presented here facilitates the passage of particles across the BBB to the brain using an electromagnetic actuation scheme. PMID:29271927

Unsanctifying the sanctuary: challenges and opportunities with brain metastases

PubMed Central

Puhalla, Shannon; Elmquist, William; Freyer, David; Kleinberg, Lawrence; Adkins, Chris; Lockman, Paul; McGregor, John; Muldoon, Leslie; Nesbit, Gary; Peereboom, David; Smith, Quentin; Walker, Sara; Neuwelt, Edward

2015-01-01

While the use of targeted therapies, particularly radiosurgery, has broadened therapeutic options for CNS metastases, patients respond minimally and prognosis remains poor. The inability of many systemic chemotherapeutic agents to penetrate the blood-brain barrier (BBB) has limited their use and allowed brain metastases to become a burgeoning clinical challenge. Adequate preclinical models that appropriately mimic the metastatic process, the BBB, and blood-tumor barriers (BTB) are needed to better evaluate therapies that have the ability to enhance delivery through or penetrate into these barriers and to understand the mechanisms of resistance to therapy. The heterogeneity among and within different solid tumors and subtypes of solid tumors further adds to the difficulties in determining the most appropriate treatment approaches and methods of laboratory and clinical studies. This review article discusses therapies focused on prevention and treatment of CNS metastases, particularly regarding the BBB, and the challenges and opportunities these therapies present. PMID:25846288

A Novel Magnetic Actuation Scheme to Disaggregate Nanoparticles and Enhance Passage across the Blood-Brain Barrier.

PubMed

Hoshiar, Ali Kafash; Le, Tuan-Anh; Amin, Faiz Ul; Kim, Myeong Ok; Yoon, Jungwon

2017-12-22

The blood-brain barrier (BBB) hinders drug delivery to the brain. Despite various efforts to develop preprogramed actuation schemes for magnetic drug delivery, the unmodeled aggregation phenomenon limits drug delivery performance. This paper proposes a novel scheme with an aggregation model for a feed-forward magnetic actuation design. A simulation platform for aggregated particle delivery is developed and an actuation scheme is proposed to deliver aggregated magnetic nanoparticles (MNPs) using a discontinuous asymmetrical magnetic actuation. The experimental results with a Y-shaped channel indicated the success of the proposed scheme in steering and disaggregation. The delivery performance of the developed scheme was examined using a realistic, three-dimensional (3D) vessel simulation. Furthermore, the proposed scheme enhanced the transport and uptake of MNPs across the BBB in mice. The scheme presented here facilitates the passage of particles across the BBB to the brain using an electromagnetic actuation scheme.

Interleukin-1β transfer across the blood–brain barrier in the ovine fetus

PubMed Central

Sadowska, Grazyna B; Chen, Xiaodi; Zhang, Jiyong; Lim, Yow-Pin; Cummings, Erin E; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Padbury, James F; Banks, William A; Stonestreet, Barbara S

2015-01-01

Pro-inflammatory cytokines contribute to hypoxic–ischemic brain injury. Blood–brain barrier (BBB) dysfunction represents an important component of hypoxic–ischemic brain injury in the fetus. Hypoxic–ischemic injury could accentuate systemic cytokine transfer across the fetal BBB. There has been considerable conjecture suggesting that systemic cytokines could cross the BBB during the perinatal period. Nonetheless, evidence to support this contention is sparse. We hypothesized that ischemia–reperfusion increases the transfer of systemic interleukin-1β (IL-1β) across the BBB in the fetus. Ovine fetuses at 127 days of gestation were studied 4 hours after 30 minutes of bilateral carotid artery occlusion and compared with a nonischemic group. Recombinant ovine IL-1β protein was expressed from an IL-1β pGEX-2 T vector in E. coli BL-21 cells and purified. The BBB function was quantified in 12 brain regions using a blood-to-brain transfer constant with intravenous 125I-radiolabeled IL-1β (125I-IL-1β). Interleukin-1β crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of 125I-IL-1β was higher (P<0.05) across brain regions in fetuses exposed to ischemia–reperfusion than nonischemic fetuses. We conclude that systemic IL-1β crosses the intact fetal BBB, and that ischemia–reperfusion increases transfer of this cytokine across the fetal BBB. Therefore, altered BBB function after hypoxia–ischemia facilitates entry of systemic cytokines into the brain of the fetus. PMID:26082012

Interleukin-1β transfer across the blood-brain barrier in the ovine fetus.

PubMed

Sadowska, Grazyna B; Chen, Xiaodi; Zhang, Jiyong; Lim, Yow-Pin; Cummings, Erin E; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Padbury, James F; Banks, William A; Stonestreet, Barbara S

2015-09-01

Pro-inflammatory cytokines contribute to hypoxic-ischemic brain injury. Blood-brain barrier (BBB) dysfunction represents an important component of hypoxic-ischemic brain injury in the fetus. Hypoxic-ischemic injury could accentuate systemic cytokine transfer across the fetal BBB. There has been considerable conjecture suggesting that systemic cytokines could cross the BBB during the perinatal period. Nonetheless, evidence to support this contention is sparse. We hypothesized that ischemia-reperfusion increases the transfer of systemic interleukin-1β (IL-1β) across the BBB in the fetus. Ovine fetuses at 127 days of gestation were studied 4 hours after 30 minutes of bilateral carotid artery occlusion and compared with a nonischemic group. Recombinant ovine IL-1β protein was expressed from an IL-1β pGEX-2 T vector in E. coli BL-21 cells and purified. The BBB function was quantified in 12 brain regions using a blood-to-brain transfer constant with intravenous (125)I-radiolabeled IL-1β ((125)I-IL-1β). Interleukin-1β crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of (125)I-IL-1β was higher (P<0.05) across brain regions in fetuses exposed to ischemia-reperfusion than nonischemic fetuses. We conclude that systemic IL-1β crosses the intact fetal BBB, and that ischemia-reperfusion increases transfer of this cytokine across the fetal BBB. Therefore, altered BBB function after hypoxia-ischemia facilitates entry of systemic cytokines into the brain of the fetus.

Opening the Blood-Brain Barrier with MR Imaging-guided Focused Ultrasound: Preclinical Testing on a Trans-Human Skull Porcine Model.

PubMed

Huang, Yuexi; Alkins, Ryan; Schwartz, Michael L; Hynynen, Kullervo

2017-01-01

Purpose To develop and test a protocol in preparation for a clinical trial on opening the blood-brain barrier (BBB) with magnetic resonance (MR) imaging-guided focused ultrasound for the delivery of chemotherapy drugs to brain tumors. Materials and Methods The procedures were approved by the institutional animal care committee. A trans-human skull porcine model was designed for the preclinical testing. Wide craniotomies were applied in 11 pigs (weight, approximately 15 kg). A partial human skull was positioned over the animal's brain. A modified clinical MR imaging-guided focused ultrasound brain system was used with a 3.0-T MR unit. The ultrasound beam was steered during sonications over a 3 × 3 grid at 3-mm spacing. Acoustic power levels of 3-20 W were tested. Bolus injections of microbubbles at 4 μL/kg were tested for each sonication. Levels of BBB opening, hemorrhage, and cavitation signal were measured with MR imaging, histologic examination, and cavitation receivers, respectively. A cavitation safety algorithm was developed on the basis of logistic regression of the measurements and tested to minimize the risk of hemorrhage. Results BBB openings of approximately 1 cm 3 in volume were visualized with gadolinium-enhanced MR imaging after sonication at an acoustic power of approximately 5 W. Gross examination of histologic specimens helped confirm Evans blue (bound to macromolecule albumin) extravasation, and hematoxylin-eosin staining helped detect only scattered extravasation of red blood cells. In cases where cavitation signals were higher than thresholds, sonications were terminated immediately without causing hemorrhage. Conclusion With a trans-human skull porcine model, this study demonstrated BBB opening with a 230-kHz system in preparation for a clinical trial. © RSNA, 2016 Online supplemental material is available for this article.

Selective Limbic Blood–Brain Barrier Breakdown in a Feline Model of Limbic Encephalitis with LGI1 Antibodies

PubMed Central

Tröscher, Anna R.; Klang, Andrea; French, Maria; Quemada-Garrido, Lucía; Kneissl, Sibylle Maria; Bien, Christian G.; Pákozdy, Ákos; Bauer, Jan

2017-01-01

Human leucine-rich glioma-inactivated protein 1 encephalitis (LGI1) is an autoimmune limbic encephalitis in which serum and cerebrospinal fluid contain antibodies targeting LGI1, a protein of the voltage gated potassium channel (VGKC) complex. Recently, we showed that a feline model of limbic encephalitis with LGI1 antibodies, called feline complex partial seizures with orofacial involvement (FEPSO), is highly comparable to human LGI1 encephalitis. In human LGI1 encephalitis, neuropathological investigations are difficult because very little material is available. Taking advantage of this natural animal model to study pathological mechanisms will, therefore, contribute to a better understanding of its human counterpart. Here, we present a brain-wide histopathological analysis of FEPSO. We discovered that blood–brain barrier (BBB) leakage was present not only in all regions of the hippocampus but also in other limbic structures such as the subiculum, amygdale, and piriform lobe. However, in other regions, such as the cerebellum, no leakage was observed. In addition, this brain-region-specific immunoglobulin leakage was associated with the breakdown of endothelial tight junctions. Brain areas affected by BBB dysfunction also revealed immunoglobulin and complement deposition as well as neuronal cell death. These neuropathological findings were supported by magnetic resonance imaging showing signal and volume increase in the amygdala and the piriform lobe. Importantly, we could show that BBB disturbance in LGI1 encephalitis does not depend on T cell infiltrates, which were present brain-wide. This finding points toward another, so far unknown, mechanism of opening the BBB. The limbic predilection sites of immunoglobulin antibody leakage into the brain may explain why most patients with LGI1 antibodies have a limbic phenotype even though LGI1, the target protein, is ubiquitously distributed across the central nervous system. PMID:29093718

Selective Limbic Blood-Brain Barrier Breakdown in a Feline Model of Limbic Encephalitis with LGI1 Antibodies.

PubMed

Tröscher, Anna R; Klang, Andrea; French, Maria; Quemada-Garrido, Lucía; Kneissl, Sibylle Maria; Bien, Christian G; Pákozdy, Ákos; Bauer, Jan

2017-01-01

Human leucine-rich glioma-inactivated protein 1 encephalitis (LGI1) is an autoimmune limbic encephalitis in which serum and cerebrospinal fluid contain antibodies targeting LGI1, a protein of the voltage gated potassium channel (VGKC) complex. Recently, we showed that a feline model of limbic encephalitis with LGI1 antibodies, called feline complex partial seizures with orofacial involvement (FEPSO), is highly comparable to human LGI1 encephalitis. In human LGI1 encephalitis, neuropathological investigations are difficult because very little material is available. Taking advantage of this natural animal model to study pathological mechanisms will, therefore, contribute to a better understanding of its human counterpart. Here, we present a brain-wide histopathological analysis of FEPSO. We discovered that blood-brain barrier (BBB) leakage was present not only in all regions of the hippocampus but also in other limbic structures such as the subiculum, amygdale, and piriform lobe. However, in other regions, such as the cerebellum, no leakage was observed. In addition, this brain-region-specific immunoglobulin leakage was associated with the breakdown of endothelial tight junctions. Brain areas affected by BBB dysfunction also revealed immunoglobulin and complement deposition as well as neuronal cell death. These neuropathological findings were supported by magnetic resonance imaging showing signal and volume increase in the amygdala and the piriform lobe. Importantly, we could show that BBB disturbance in LGI1 encephalitis does not depend on T cell infiltrates, which were present brain-wide. This finding points toward another, so far unknown, mechanism of opening the BBB. The limbic predilection sites of immunoglobulin antibody leakage into the brain may explain why most patients with LGI1 antibodies have a limbic phenotype even though LGI1, the target protein, is ubiquitously distributed across the central nervous system.

Neuroimmunological Blood Brain Barrier Opening in Experimental Cerebral Malaria

PubMed Central

Baer, Kerstin; Mikolajczak, Sebastian A.; Kappe, Stefan H. I.; Frevert, Ute

2012-01-01

Plasmodium falciparum malaria is responsible for nearly one million annual deaths worldwide. Because of the difficulty in monitoring the pathogenesis of cerebral malaria in humans, we conducted a study in various mouse models to better understand disease progression in experimental cerebral malaria (ECM). We compared the effect on the integrity of the blood brain barrier (BBB) and the histopathology of the brain of P. berghei ANKA, a known ECM model, P. berghei NK65, generally thought not to induce ECM, P. yoelii 17XL, originally reported to induce human cerebral malaria-like histopathology, and P. yoelii YM. As expected, P. berghei ANKA infection caused neurological signs, cerebral hemorrhages, and BBB dysfunction in CBA/CaJ and Swiss Webster mice, while Balb/c and A/J mice were resistant. Surprisingly, PbNK induced ECM in CBA/CaJ mice, while all other mice were resistant. P. yoelii 17XL and P. yoelii YM caused lethal hyperparasitemia in all mouse strains; histopathological alterations, BBB dysfunction, or neurological signs were not observed. Intravital imaging revealed that infected erythrocytes containing mature parasites passed slowly through capillaries making intimate contact with the endothelium, but did not arrest. Except for relatively rare microhemorrhages, mice with ECM presented no obvious histopathological alterations that would explain the widespread disruption of the BBB. Intravital imaging did reveal, however, that postcapillary venules, but not capillaries or arterioles, from mice with ECM, but not hyperparasitemia, exhibit platelet marginalization, extravascular fibrin deposition, CD14 expression, and extensive vascular leakage. Blockage of LFA-1 mediated cellular interactions prevented leukocyte adhesion, vascular leakage, neurological signs, and death from ECM. The endothelial barrier-stabilizing mediators imatinib and FTY720 inhibited vascular leakage and neurological signs and prolonged survival to ECM. Thus, it appears that neurological signs and coma in ECM are due to regulated opening of paracellular-junctional and transcellular-vesicular fluid transport pathways at the neuroimmunological BBB. PMID:23133375

Tracer kinetic modelling for DCE-MRI quantification of subtle blood-brain barrier permeability.

PubMed

Heye, Anna K; Thrippleton, Michael J; Armitage, Paul A; Valdés Hernández, Maria Del C; Makin, Stephen D; Glatz, Andreas; Sakka, Eleni; Wardlaw, Joanna M

2016-01-15

There is evidence that subtle breakdown of the blood-brain barrier (BBB) is a pathophysiological component of several diseases, including cerebral small vessel disease and some dementias. Dynamic contrast-enhanced MRI (DCE-MRI) combined with tracer kinetic modelling is widely used for assessing permeability and perfusion in brain tumours and body tissues where contrast agents readily accumulate in the extracellular space. However, in diseases where leakage is subtle, the optimal approach for measuring BBB integrity is likely to differ since the magnitude and rate of enhancement caused by leakage are extremely low; several methods have been reported in the literature, yielding a wide range of parameters even in healthy subjects. We hypothesised that the Patlak model is a suitable approach for measuring low-level BBB permeability with low temporal resolution and high spatial resolution and brain coverage, and that normal levels of scanner instability would influence permeability measurements. DCE-MRI was performed in a cohort of mild stroke patients (n=201) with a range of cerebral small vessel disease severity. We fitted these data to a set of nested tracer kinetic models, ranking their performance according to the Akaike information criterion. To assess the influence of scanner drift, we scanned 15 healthy volunteers that underwent a "sham" DCE-MRI procedure without administration of contrast agent. Numerical simulations were performed to investigate model validity and the effect of scanner drift. The Patlak model was found to be most appropriate for fitting low-permeability data, and the simulations showed vp and K(Trans) estimates to be reasonably robust to the model assumptions. However, signal drift (measured at approximately 0.1% per minute and comparable to literature reports in other settings) led to systematic errors in calculated tracer kinetic parameters, particularly at low permeabilities. Our findings justify the growing use of the Patlak model in low-permeability states, which has the potential to provide valuable information regarding BBB integrity in a range of diseases. However, absolute values of the resulting tracer kinetic parameters should be interpreted with extreme caution, and the size and influence of signal drift should be measured where possible. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Tracer kinetic modelling for DCE-MRI quantification of subtle blood–brain barrier permeability

PubMed Central

Heye, Anna K.; Thrippleton, Michael J.; Armitage, Paul A.; Valdés Hernández, Maria del C.; Makin, Stephen D.; Glatz, Andreas; Sakka, Eleni; Wardlaw, Joanna M.

2016-01-01

There is evidence that subtle breakdown of the blood–brain barrier (BBB) is a pathophysiological component of several diseases, including cerebral small vessel disease and some dementias. Dynamic contrast-enhanced MRI (DCE-MRI) combined with tracer kinetic modelling is widely used for assessing permeability and perfusion in brain tumours and body tissues where contrast agents readily accumulate in the extracellular space. However, in diseases where leakage is subtle, the optimal approach for measuring BBB integrity is likely to differ since the magnitude and rate of enhancement caused by leakage are extremely low; several methods have been reported in the literature, yielding a wide range of parameters even in healthy subjects. We hypothesised that the Patlak model is a suitable approach for measuring low-level BBB permeability with low temporal resolution and high spatial resolution and brain coverage, and that normal levels of scanner instability would influence permeability measurements. DCE-MRI was performed in a cohort of mild stroke patients (n = 201) with a range of cerebral small vessel disease severity. We fitted these data to a set of nested tracer kinetic models, ranking their performance according to the Akaike information criterion. To assess the influence of scanner drift, we scanned 15 healthy volunteers that underwent a “sham” DCE-MRI procedure without administration of contrast agent. Numerical simulations were performed to investigate model validity and the effect of scanner drift. The Patlak model was found to be most appropriate for fitting low-permeability data, and the simulations showed vp and KTrans estimates to be reasonably robust to the model assumptions. However, signal drift (measured at approximately 0.1% per minute and comparable to literature reports in other settings) led to systematic errors in calculated tracer kinetic parameters, particularly at low permeabilities. Our findings justify the growing use of the Patlak model in low-permeability states, which has the potential to provide valuable information regarding BBB integrity in a range of diseases. However, absolute values of the resulting tracer kinetic parameters should be interpreted with extreme caution, and the size and influence of signal drift should be measured where possible. PMID:26477653

12 CFR Appendix B to Subpart A of... - Appendix B to Subpart A of Part 327

Code of Federal Regulations, 2011 CFR

2011-01-01

... Converted value Standard & Poor's: AAA 1.00 AA+ 1.05 AA 1.15 AA− 1.30 A+ 1.50 A 1.80 A− 2.20 BBB+ 2.70 BBB... worse 3.00 Fitch's: AAA 1.00 AA+ 1.05 AA 1.15 AA− 1.30 A+ 1.50 A 1.80 A− 2.20 BBB+ 2.70 BBB or worse 3...

Cerebral amyloid angiopathy, blood-brain barrier disruption and amyloid accumulation in SAMP8 mice.

PubMed

del Valle, Jaume; Duran-Vilaregut, Joaquim; Manich, Gemma; Pallàs, Mercè; Camins, Antoni; Vilaplana, Jordi; Pelegrí, Carme

2011-01-01

Cerebrovascular dysfunction and β-amyloid peptide deposition on the walls of cerebral blood vessels might be an early event in the development of Alzheimer's disease. Here we studied the time course of amyloid deposition in blood vessels and blood-brain barrier (BBB) disruption in the CA1 subzone of the hippocampus of SAMP8 mice and the association between these two variables. We also studied the association between the amyloid deposition in blood vessels and the recently described amyloid clusters in the parenchyma, as well as the association of these clusters with vessels in which the BBB is disrupted. SAMP8 mice showed greater amyloid deposition in blood vessels than age-matched ICR-CD1 control mice. Moreover, at 12 months of age the number of vessels with a disrupted BBB had increased in both strains, especially SAMP8 animals. At this age, all the vessels with amyloid deposition showed BBB disruption, but several capillaries with an altered BBB showed no amyloid on their walls. Moreover, amyloid clusters showed no spatial association with vessels with amyloid deposition, nor with vessels in which the BBB had been disrupted. Finally, we can conclude that vascular amyloid deposition seems to induce BBB alterations, but BBB disruption may also be due to other factors. Copyright © 2011 S. Karger AG, Basel.

Delivery of Biologics Across the Blood-Brain Barrier with Molecular Trojan Horse Technology.

PubMed

Pardridge, William M

2017-12-01

Biologics are potential new therapeutics for many diseases of the central nervous system. Biologics include recombinant lysosomal enzymes, neurotrophins, decoy receptors, and therapeutic antibodies. These are large molecule drugs that do not cross the blood-brain barrier (BBB). All classes of biologics have been tested, without success, in clinical trials of brain disease over the last 25 years. In none of these past clinical trials was the biologic re-engineered to enable transport across the BBB. If the biologic does not cross the BBB, the drug cannot reach the target site in brain, and success in a clinical trial is not expected. Biologics can be re-engineered for BBB transport with the use of molecular Trojan horse technology. A BBB molecular Trojan horse is a monoclonal antibody (MAb) against an endogenous BBB receptor transporter, such as the insulin receptor or transferrin receptor. The receptor-specific MAb penetrates the brain via transport on the endogenous BBB receptor. The MAb acts as a molecular Trojan horse to deliver across the BBB the biologic pharmaceutical that is genetically fused to the MAb. The lead Trojan horse is a MAb against the human insulin receptor (HIR), and HIRMAb-derived fusion proteins have entered clinical trials for the treatment of brain disease.

Peptides and the blood–brain barrier

PubMed Central

Banks, William A.

2016-01-01

The demonstration that peptides and regulatory proteins can cross the blood–brain barrier (BBB) is one of the major contributions of Dr. Abba J. Kastin. He was the first to propose that peptides could cross the BBB, the first to show that an endogenous peptide did so, and the first to describe a saturable transport system at the BBB for peptides. His work shows that in crossing the BBB, peptides and regulatory proteins act as informational molecules, informing the brain of peripheral events. Brain-to-blood passage helps to control levels of peptides with the brain and can deliver information in the brain-to-blood direction. He showed that the transporters for peptides and proteins are not static, but respond to developmental and physiological changes and are affected by disease states. As such, the BBB is adaptive to the needs of the CNS, but when that adaption goes awry, the BBB can be a cause of disease. The mechanisms by which peptides and proteins cross the BBB offer opportunities for drug delivery of these substances or their analogs to the brain in the treatment of diseases of the central nervous system. PMID:25805003

Disruption of the blood-brain barrier as the primary effect of CNS irradiation.

PubMed

Rubin, P; Gash, D M; Hansen, J T; Nelson, D F; Williams, J P

1994-04-01

The blood-brain barrier (BBB) is believed to be unique in organ microcirculation due to the 'tight junctions' which exist between endothelial cells and, some argue, the additional functional components represented by the perivascular boundary of neuroglial cells; these selectively exclude proteins and drugs from the brain parenchyma. This study was designed to examine the effects of irradiation on the BBB and determine the impact of the altered pathophysiology on the production of central nervous system (CNS) late effects such as demyelination, gliosis and necrosis. Rats, irradiated at 60 Gy, were serially sacrificed at 2, 6, 12 and 24 weeks. Magnetic resonance image analysis (MRI) was obtained prior to sacrifice with selected animals from each group. The remaining animals underwent horse-radish peroxidase (HRP) perfusion at the time of sacrifice. The serial studies showed a detectable disruption of the BBB at 2 weeks post-irradiation and this was manifested as discrete leakage; late injury seen at 24 weeks indicated diffuse vasculature leakage, severe loss of the capillary network, cortical atrophy and white matter necrosis. Reversal or repair of radiation injury was seen between 6 and 12 weeks, indicating a bimodal peak in events. Blood-brain barrier disruption is an early, readily recognizable pathophysiological event occurring after radiation injury, is detectable in vivo/in vitro by MRI and HRP studies, and appears to precede white matter necrosis. Dose response studies over a wide range of doses, utilizing both external and interstitial irradiation, are in progress along with correlative histopathologic and ultrastructural studies.

Synthesis of cell-penetrating peptides and their application in neurobiology.

PubMed

Dietz, Gunnar P H; Bähr, Mathias

2007-01-01

Short basic amino acid sequences, often called cell-penetrating peptides (CPPs), allow the delivery of proteins and other molecules into cells and across the blood-brain barrier (BBB). Although the ability of basic proteins to facilitate such trafficking is known for a long time, only the application of genetic methods and overexpression of fusion proteins in Escherichia coli has lead to a wide application of CPP in many research areas, including signal transduction, cancer, angiogenesis, apoptosis, bone development, cardioprotection, cell cycle, neurobiology, and many others. For the neuroscientist, CPPs are particularly attractive, as a number of articles in the last 5 years have reported their use for neuronal rescue in a number of models for neurodegenerative diseases in vitro and in vivo in rats, mice, or gerbils. Here, we give a detailed description of the protein purification methodology and applications in neuroscience.

Fluorescein isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability

PubMed Central

Natarajan, Reka; Northrop, Nicole

2017-01-01

The blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels. PMID:28398646

The bubble-dependent mechanism of FUS-induced blood-brain barrier opening in mice and in monkeys in vivo

NASA Astrophysics Data System (ADS)

Tung, Yao-Sheng; Marquet, Fabrice; Vlachos, Fotios; Feshitan, Jameel A.; Borden, Mark A.; Konofagou, Elisa E.

2012-10-01

The blood-brain barrier (BBB) prevents most neurological drugs from traversing from the cerebral microvasculature into the brain parenchyma. Previous studies have shown that the presence of bubbles in an acoustic field temporarily opens the BBB. The BBB opening pressure threshold was previously identified to lie between 0.30 and 0.46 MPa in the case of the smaller bubbles and between 0.15 and 0.30 MPa in the larger bubble case. However, the physical effects responsible for BBB opening remain unknown. In addition, the noninvasive in vivo cavitation detection with mono-dispersed microbubbles has not been studied as of yet. The purpose of this study is to unveil the physical mechanism of the FUS-induced BBB opening with monodispersed microbubbles. Lipid-shelled microbubbles with three different diameters (1-2, 4-5 and 6-8 μm) were manufactured in-house and size-isolated using differential centrifugation. Sixty-seven (n=67) mice were each injected intravenously with bubbles of either 1-2, 4-5 or 6-8 μm in diameter and the concentration of 107 numbers/mL. The right hippocampus of each mouse was then sonicated using focused ultrasound (1.5 MHz frequency; 100 cycles (67 μs) pulse length; 10 Hz pulse repetition frequency; 1 minute sonication duration) while the left hippocampus served as the control. A 10-MHz transducer was used as a passive cavitation detector (PCD) to determine the threshold of inertial cavitation (IC). Each mouse was sonicated at a specific acoustic peak-rarefactional pressure at 0.15, 0.30, 0.45 or 0.60 MPa in order to identify the threshold of BBB opening and IC. T1-weighted MRI was used to verify the BBB opening and spectrograms were generated in order to detect the IC onset and duration. Our results suggest that the BBB opens as a result of nonlinear (harmonic) bubble oscillation when the bubble diameter is similar to the capillary diameter and with inertial cavitation when it is not. The bubble may thus have to be in contact with the capillary wall to induce BBB opening without inertial cavitation. The BBB opening volume was found to increase with both pressure and bubble size. Good correlation between the ICD and the BBB opening volume at distinct bubble diameters was established. No damage was induced at the BBB opening threshold in all bubble cases. The BBB opening was thus shown capable of being induced safely with nonlinear bubble oscillation at the pressure threshold and its volume was highly dependent on both the pressure and bubble diameter. The preliminary results of cavitation detection during BBB opening in monkeys are also provided.

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

PubMed

Rubio-Araiz, Ana; Porcu, Francesca; Pérez-Hernández, Mercedes; García-Gutiérrez, Mª Salud; Aracil-Fernández, María Auxiliadora; Gutierrez-López, María Dolores; Guerri, Consuelo; Manzanares, Jorge; O'Shea, Esther; Colado, María Isabel

2017-07-01

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice. © 2016 Society for the Study of Addiction.

Netrin-1 Preserves Blood-Brain Barrier Integrity Through Deleted in Colorectal Cancer/Focal Adhesion Kinase/RhoA Signaling Pathway Following Subarachnoid Hemorrhage in Rats.

PubMed

Xie, Zongyi; Enkhjargal, Budbazar; Reis, Cesar; Huang, Lei; Wan, Weifeng; Tang, Jiping; Cheng, Yuan; Zhang, John H

2017-05-19

Netrin-1 (NTN-1) has been established to be a novel intrinsic regulator of blood-brain barrier (BBB) maintenance. This study was carried out to investigate the potential roles of exogenous NTN-1 in preserving BBB integrity after experimental subarachnoid hemorrhage (SAH) as well as the underlying mechanisms of its protective effects. A total of 309 male Sprague-Dawley rats were subjected to an endovascular perforation model of SAH. Recombinant NTN-1 was administered intravenously 1 hour after SAH induction. NTN-1 small interfering RNA or Deleted in Colorectal Cancer small interfering RNA was administered intracerebroventricular at 48 hours before SAH. Focal adhesion kinase inhibitor was administered by intraperitoneal injection at 1 hour prior to SAH. Neurological scores, brain water content, BBB permeability, RhoA activity, Western blot, and immunofluorescence staining were evaluated. The expression of endogenous NTN-1 and its receptor Deleted in Colorectal Cancer were increased after SAH. Administration of exogenous NTN-1 significantly reduced brain water content and BBB permeability and ameliorated neurological deficits at 24 and 72 hours after SAH. Exogenous NTN-1 treatment significantly promoted phosphorylated focal adhesion kinase activation and inhibited RhoA activity, as well as upregulated the expression of ZO-1 and Occludin. Conversely, depletion of endogenous NTN-1 aggravated BBB breakdown and neurological impairments at 24 hours after SAH. The protective effects of NTN-1 at 24 hours after SAH were also abolished by pretreatment with Deleted in Colorectal Cancer small interfering RNA and focal adhesion kinase inhibitor. NTN-1 treatment preserved BBB integrity and improved neurological functions through a Deleted in Colorectal Cancer/focal adhesion kinase/RhoA signaling pathway after SAH. Thus, NTN-1 may serve as a promising treatment to alleviate early brain injury following SAH. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

The pathological role of NLRs and AIM2 inflammasome-mediated pyroptosis in damaged blood-brain barrier after traumatic brain injury.

PubMed

Ge, Xintong; Li, Wenzhu; Huang, Shan; Yin, Zhenyu; Xu, Xin; Chen, Fanglian; Kong, Xiaodong; Wang, Haichen; Zhang, Jianning; Lei, Ping

2018-06-07

Pyroptosis is a highly specific type of inflammatory programmed cell death that different from necrosis or apoptosis. It is initiated by cellular detection of acute damage via recognizing pathogen-associated molecular patterns (PAMPs) by NOD-like receptors (NLRs) or AIM2-like receptor (AIM2). NLRs and AIM2 could trigger the formation of a multi-protein complex, known as inflammasome. It also contains apoptotic speck-containing protein (ASC) and pro-Caspase-1, and could process the signals to induce a cascade of inflammatory response. Recently, growing evidence showed that inflammasome-mediated pyroptosis is involved in the pathogenesis of traumatic brain injury (TBI). However, less attention has been paid to their particular roles in regulating blood-brain barrier (BBB) damage, the central pathological change in secondary brain damage of TBI. Thus, we designed this research to explore the impact and mechanism of NLRs and AIM2 inflammasome-mediated pyroptosis in BBB after TBI. We employed the controlled cortical impact (CCI) mice model and manipulated the severity of pyroptosis in BBB using Caspase-1 inhibitor, Ac-YVAD-cmk. We found that TBI led to NLRs and AIM2 inflammasome-mediated pyroptosis in brain microvascular endothelial cells (BMVECs) from injured cerebral cortex. Ac-YVAD-cmk treatment inhibited pyroptosis in injured BMVECs by suppressing the expression of essential inflammasome subunit - Caspase-1 and pivotal downstream pro-inflammatory cytokines (IL-1β and IL-18), as well as hindering GSDMD cleavage and ASC oligomerization. In addition, inhibiting pyroptosis could alleviate TBI-induced BBB leakage, brain edema, loss of tight junction proteins, and the inflammatory response in injured BMVECs. These effects contributed to improving the neurological outcome of CCI mice. In conclusion, NLRs and AIM2 inflammasome-mediated pyroptosis could aggravate BBB damage after TBI. Targeting and controlling pyroptosis in injured BBB would be a promising therapeutic strategy for TBI in the future. Copyright © 2018. Published by Elsevier B.V.

CD146 coordinates brain endothelial cell–pericyte communication for blood–brain barrier development

PubMed Central

Chen, Jianan; Luo, Yongting; Hui, Hui; Cai, Tanxi; Huang, Hongxin; Yang, Fuquan; Feng, Jing; Zhang, Jingjing; Yan, Xiyun

2017-01-01

The blood–brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte–EC interaction during BBB development, the molecular mechanisms coordinating the pericyte–EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB. PMID:28827364

Nanowired Drug Delivery Across the Blood-Brain Barrier in Central Nervous System Injury and Repair.

PubMed

Sharma, Aruna; Menon, Preeti; Muresanu, Dafin F; Ozkizilcik, Asya; Tian, Z Ryan; Lafuente, José V; Sharma, Hari S

2016-01-01

The blood-brain barrier (BBB) is a physiological regulator of transport of essential items from blood to brain for the maintenance of homeostasis of the central nervous system (CNS) within narrow limits. The BBB is also responsible for export of harmful or metabolic products from brain to blood to keep the CNS fluid microenvironment healthy. However, noxious insults to the brain caused by trauma, ischemia or environmental/chemical toxins alter the BBB function to small as well as large molecules e.g., proteins. When proteins enter the CNS fluid microenvironment, development of brain edema occurs due to altered osmotic balance between blood and brain. On the other hand, almost all neurodegenerative diseases and traumatic insults to the CNS and subsequent BBB dysfunction lead to edema formation and cell injury. To treat these brain disorders suitable drug therapy reaching their brain targets is needed. However, due to edema formation or only a focal disruption of the BBB e.g., around brain tumors, many drugs are unable to reach their CNS targets in sufficient quantity. This results in poor therapeutic outcome. Thus, new technology such as nanodelivery is needed for drugs to reach their CNS targets and be effective. In this review, use of nanowires as a possible novel tool to enhance drug delivery into the CNS in various disease models is discussed based on our investigations. These data show that nanowired delivery of drugs may have superior neuroprotective ability to treat several CNS diseases effectively indicating their role in future therapeutic strategies.

Anterior thalamic nuclei deep brain stimulation reduces disruption of the blood-brain barrier, albumin extravasation, inflammation and apoptosis in kainic acid-induced epileptic rats.

PubMed

Chen, Ying-Chuan; Zhu, Guan-Yu; Wang, Xiu; Shi, Lin; Du, Ting-Ting; Liu, De-Feng; Liu, Yu-Ye; Jiang, Yin; Zhang, Xin; Zhang, Jian-Guo

2017-12-01

Objective The therapeutic efficacy of anterior thalamic nuclei deep brain stimulation (ATN-DBS) against seizures has been largely accepted; however, the effects of ATN-DBS on disruption of the blood-brain barrier (BBB), albumin extravasation, inflammation and apoptosis still remain unclear. Methods Rats were distributed into four treatment groups: physiological saline (PS, N = 12), kainic acid (KA, N = 12), KA-sham-DBS (N = 12) and KA-DBS (N = 12). Seizures were monitored using video-electroencephalogram (EEG). One day after surgery, all rats were sacrificed. Then, samples were prepared for quantitative real-time PCR (qPCR), western blot, immunofluorescence (IF) staining, and transmission electron microscopy to evaluate the disruption of the BBB, albumin extravasation, inflammation, and apoptosis. Result Because of the KA injection, the disruption of the BBB, albumin extravasation, inflammation and apoptosis were more severe in the KA and the KA-sham-DBS groups compared to the PS group (all Ps < 0.05 or < 0.01). The ideal outcomes were observed in the KA-DBS group. ATN-DBS produced a 46.3% reduction in seizure frequency and alleviated the disruption of the BBB, albumin extravasation, inflammatory reaction and apoptosis in comparison to the KA-sham-DBS group (all Ps < 0.05 or < 0.01). Conclusion (1) Seizures can be reduced using ATN-DBS in the epileptogenic stage. (2) ATN-DBS can reduce the disruption of the BBB and albumin extravasation. (3) ATN-DBS has an anti-inflammatory effect in epileptic models.

Age-dependent increase of blood-brain barrier permeability and neuron-binding autoantibodies in S100B knockout mice.

PubMed

Wu, Hao; Brown, Eric V; Acharya, Nimish K; Appelt, Denah M; Marks, Alexander; Nagele, Robert G; Venkataraman, Venkat

2016-04-15

S100B is a calcium-sensor protein that impacts multiple signal transduction pathways. It is widely considered to be an important biomarker for several neuronal diseases as well as blood-brain barrier (BBB) breakdown. In this report, we demonstrate a BBB deficiency in mice that lack S100B through detection of leaked Immunoglobulin G (IgG) in the brain parenchyma. IgG leaks and IgG-binding to selected neurons were observed in S100B knockout (S100BKO) mice at 6 months of age but not at 3 months. By 9 months, IgG leaks persisted and the density of IgG-bound neurons increased significantly. These results reveal a chronic increase in BBB permeability upon aging in S100BKO mice for the first time. Moreover, coincident with the increase in IgG-bound neurons, autoantibodies targeting brain proteins were detected in the serum via western blots. These events were concurrent with compromise of neurons, increase of activated microglia and lack of astrocytic activation as evidenced by decreased expression of microtubule-associated protein type 2 (MAP2), elevated number of CD68 positive cells and unaltered expression of glial fibrillary acidic protein (GFAP) respectively. Results suggest a key role for S100B in maintaining BBB functional integrity and, further, propose the S100BKO mouse as a valuable model system to explore the link between chronic functional compromise of the BBB, generation of brain-reactive autoantibodies and neuronal dysfunctions. Copyright © 2016. Published by Elsevier B.V.

Current Strategies for Brain Drug Delivery

PubMed Central

Dong, Xiaowei

2018-01-01

The blood-brain barrier (BBB) has been a great hurdle for brain drug delivery. The BBB in healthy brain is a diffusion barrier essential for protecting normal brain function by impeding most compounds from transiting from the blood to the brain; only small molecules can cross the BBB. Under certain pathological conditions of diseases such as stroke, diabetes, seizures, multiple sclerosis, Parkinson's disease and Alzheimer disease, the BBB is disrupted. The objective of this review is to provide a broad overview on current strategies for brain drug delivery and related subjects from the past five years. It is hoped that this review could inspire readers to discover possible approaches to deliver drugs into the brain. After an initial overview of the BBB structure and function in both healthy and pathological conditions, this review re-visits, according to recent publications, some questions that are controversial, such as whether nanoparticles by themselves could cross the BBB and whether drugs are specifically transferred to the brain by actively targeted nanoparticles. Current non-nanoparticle strategies are also reviewed, such as delivery of drugs through the permeable BBB under pathological conditions and using non-invasive techniques to enhance brain drug uptake. Finally, one particular area that is often neglected in brain drug delivery is the influence of aging on the BBB, which is captured in this review based on the limited studies in the literature. PMID:29556336

Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging

NASA Astrophysics Data System (ADS)

Chu, Po-Chun; Chai, Wen-Yen; Tsai, Chih-Hung; Kang, Shih-Tsung; Yeh, Chih-Kuang; Liu, Hao-Li

2016-09-01

Focused ultrasound (FUS) with microbubbles can temporally open the blood-brain barrier (BBB), and the cavitation activities of microbubbles play a key role in the BBB-opening process. Previous attempts used contrast-enhanced magnetic resonance imaging (CE-MRI) to correlate the mechanical index (MI) with the scale of BBB-opening, but MI only partially gauged acoustic activities, and CE-MRI did not fully explore correlations of pharmacodynamic/pharmacokinetic behaviors. Recently, the cavitation index (CI) has been derived to serve as an indicator of microbubble-ultrasound stable cavitation, and may also serve as a valid indicator to gauge the level of FUS-induced BBB opening. This study investigates the feasibility of gauging FUS-induced BBB opened level via the two indexes, MI and CI, through dynamic contrast-enhanced (DCE)-MRI analysis as well as passive cavitation detection (PCD) analysis. Pharmacodynamic/pharmacokinetic parameters derived from DCE-MRI were characterized to identify the scale of FUS-induced BBB opening. Our results demonstrated that DCE-MRI can successfully access pharmacodynamic/pharmacokinetic BBB-opened behavior, and was highly correlated both with MI and CI, implying the feasibility in using these two indices to gauge the scale of FUS-induced BBB opening. The proposed finding may facilitate the design toward using focused ultrasound as a safe and reliable noninvasive CNS drug delivery.

Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging.

PubMed

Chu, Po-Chun; Chai, Wen-Yen; Tsai, Chih-Hung; Kang, Shih-Tsung; Yeh, Chih-Kuang; Liu, Hao-Li

2016-09-15

Focused ultrasound (FUS) with microbubbles can temporally open the blood-brain barrier (BBB), and the cavitation activities of microbubbles play a key role in the BBB-opening process. Previous attempts used contrast-enhanced magnetic resonance imaging (CE-MRI) to correlate the mechanical index (MI) with the scale of BBB-opening, but MI only partially gauged acoustic activities, and CE-MRI did not fully explore correlations of pharmacodynamic/pharmacokinetic behaviors. Recently, the cavitation index (CI) has been derived to serve as an indicator of microbubble-ultrasound stable cavitation, and may also serve as a valid indicator to gauge the level of FUS-induced BBB opening. This study investigates the feasibility of gauging FUS-induced BBB opened level via the two indexes, MI and CI, through dynamic contrast-enhanced (DCE)-MRI analysis as well as passive cavitation detection (PCD) analysis. Pharmacodynamic/pharmacokinetic parameters derived from DCE-MRI were characterized to identify the scale of FUS-induced BBB opening. Our results demonstrated that DCE-MRI can successfully access pharmacodynamic/pharmacokinetic BBB-opened behavior, and was highly correlated both with MI and CI, implying the feasibility in using these two indices to gauge the scale of FUS-induced BBB opening. The proposed finding may facilitate the design toward using focused ultrasound as a safe and reliable noninvasive CNS drug delivery.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PubMed

Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

2015-06-01

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

Acoustic Cavitation-Based Monitoring of the Reversibility and Permeability of Ultrasound-Induced Blood-Brain Barrier Opening

PubMed Central

Sun, Tao; Samiotaki, Gesthimani; Wang, Shutao; Acosta, Camilo; Chen, Cherry C.; Konofagou, Elisa E.

2015-01-01

Cavitation events seeded by microbubbles have been previously reported to be associated with MR- or fluorescent-contrast enhancement after focused ultrasound (FUS)-induced blood-brain barrier (BBB) opening. However, it is still unknown whether bubble activity can be correlated with the reversibility (the duration of opening and the likelihood of safe reinstatement) and the permeability of opened BBB, which is critical for the clinical translation of using passive cavitation detection to monitor, predict and control the opening. In this study, the dependence of acoustic cavitation on the BBB opening duration, permeability coefficient and histological damage occurrence were thus investigated. Transcranial pulsed FUS at 1.5 MHz in the presence of systemically circulating microbubbles was applied in the mouse hippocampi (n = 60). The stable and inertial cavitation activities were monitored during sonication. Contrast-enhanced MRI was performed immediately after sonication and every 24 h up to 6 days thereafter, to assess BBB opening, brain tissue permeability and potential edema. Histological evaluations were used to assess the occurrence of neurovascular damages. It was found that stable cavitation was well correlated with: 1) the duration of the BBB opening (r2 = 0.77); 2) the permeability of the opened BBB (r2 = 0.82); 3) the likelihood of safe opening (P < 0.05, safe opening compared to cases of damage; P < 0.0001, no opening compared to safe opening). The inertial cavitation dose was correlated with the resulting BBB permeability (r2 = 0.72). Stable cavitation was found to be more reliable than inertial cavitation at assessing the BBB opening within the pressure range used in this study. This study demonstrates that the stable cavitation response during BBB opening holds promise for predicting and controlling the restoration and pharmacokinetics of FUS-opened BBB. The stable cavitation response therefore showed great promise in predicting the BBB opening duration, enabling thus control of opening according to the drug circulation time. In addition, avoiding adverse effects in the brain and assessing the pharmacokinetics of the compounds delivered can also be achieved by monitoring and controlling the stable cavitation emissions. PMID:26562661

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood-Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress.

PubMed

Zhang, Zong-Yong; Jiang, Ming; Fang, Jie; Yang, Ming-Feng; Zhang, Shuai; Yin, Yan-Xin; Li, Da-Wei; Mao, Lei-Lei; Fu, Xiao-Yan; Hou, Ya-Jun; Fu, Xiao-Ting; Fan, Cun-Dong; Sun, Bao-Liang

2017-01-01

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood-brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs' protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.

Endothelial NOS activation induces the blood-brain barrier disruption via ER stress following status epilepticus.

PubMed

Ko, Ah-Reum; Kim, Ji Yang; Hyun, Hye-Won; Kim, Ji-Eun

2015-10-05

The blood-brain barrier (BBB) maintains the unique brain microenvironment, which is separated from the systemic circulating system. Since the endoplasmic reticulum (ER) is an important cell organelle that is responsible for protein synthesis, the correct folding and sorting of proteins contributing to cell survivals, ER stress is a potential cause of cell damage in various diseases. Therefore, it would be worthy to explore the the relationship between the ER stress and BBB disruption during vasogenic edema formation induced by epileptogenic insults. In the present study, we investigated the roles of ER stress in vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced status epilepticus (SE). SE-induced eNOS activation induces BBB breakdown via up-regulation of GRP78 expression and dysfunction of SMI-71 (an endothelial BBB marker) in the piriform cortex (PC). In addition, caveolin-1 peptide (an eNOS inhibitor) effectively attenuated GRP78 expression and down-regulation of SMI-71. Taken together, our findings suggest that eNOS-mediated ER stress may participate in SE-induced vasogenic edema formation. Therefore, the modulation of ER stress may be a considerable strategy for therapy in impairments of endothelial cell function. Copyright © 2015 Elsevier B.V. All rights reserved.

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

PubMed Central

Chen, Sheng-cai; Huang, Ming; Wang, Yong; Gao, Yuan; Huang, Yan; Wang, Meng-die; Mao, Ling; Hu, Bo

2013-01-01

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage. PMID:23894369

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

PubMed

Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

2013-06-01

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

Blood-Brain Barrier Disruption After Cardiopulmonary Bypass: Diagnosis and Correlation to Cognition.

PubMed

Abrahamov, Dan; Levran, Oren; Naparstek, Sharon; Refaeli, Yael; Kaptson, Shani; Abu Salah, Mahmud; Ishai, Yaron; Sahar, Gideon

2017-07-01

Cardiopulmonary bypass (CPB) elicits a systemic inflammatory response that may impair blood-brain barrier (BBB) integrity. BBB disruption can currently be detected by dynamic contrast enhancement magnetic resonance imaging (MRI), reflected by an increase in the permeability constant (K trans ). We aimed to determine (1) whether CPB induces BBB disruption, (2) duration until BBB disruption resolution, and (3) the obtainable correlation between BBB injury (location and intensity) and neurocognitive dysfunction. Seven patients undergoing CPB with coronary artery bypass grafting (CABG) were assigned to serial cerebral designated MRI evaluations, preoperatively and on postoperative day (POD) 1 and 5. Examinations were analyzed for BBB disruption and microemboli using dynamic contrast enhancement MRI and diffusion-weighted imaging methods, respectively. Neuropsychologic tests were performed 1 day preoperatively and on POD 5. A significant local K trans increase (0.03 min -1 vs 0.07 min -1 , p = 0.033) compatible with BBB disruption was evident in 5 patients (71%) on POD 1. Resolution was observed by POD 5 (mean, 0.012 min -1 ). The location of the disruption was most prominent in the frontal lobes (400% vs 150% K trans levels upsurge, p = 0.05). MRI evidence of microembolization was demonstrated in only 1 patient (14%). The postoperative global cognitive score was reduced in all patients (98.2 ± 12 vs 95.1 ± 11, p = 0.032), predominantly in executive and attention (frontal lobe-related) functions (91.8 ± 13 vs 86.9 ± 12, p = 0.042). The intensity of the dynamic contrast enhancement MRI BBB impairment correlated with the magnitude of cognition reduction (r = 0.69, p = 0.04). BBB disruption was evident in most patients, primarily in the frontal lobes. The location and intensity of the BBB disruption, rather than the microembolic load, correlated with postoperative neurocognitive dysfunction. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Rapamycin decreased blood-brain barrier permeability in control but not in diabetic rats in early cerebral ischemia.

PubMed

Chi, Oak Z; Kiss, Geza K; Mellender, Scott J; Liu, Xia; Weiss, Harvey R

2017-07-27

Diabetes causes functional and structural changes in blood-brain barrier (BBB). The mammalian target of rapamycin (mTOR) has been associated with glucose metabolism, diabetes, and altering BBB permeability. Since there is only a narrow therapeutic window (3h) for stroke victims, it is important to investigate BBB disruption in the early stage of cerebral ischemia. We compared the degree of BBB disruption in diabetic and in control rats at two hours of reperfusion after one hour of middle cerebral artery (MCA) occlusion with or without inhibition of mTOR. Two weeks after streptozotocin ip to induce diabetes, MCA occlusion was performed. In half of the rats, an mTOR inhibitor, rapamycin was given for 2days before MCA occlusion. After one hour of MCA occlusion and two hours of the reperfusion, the transfer coefficient (K i ) of 14 C-α-aminoisobutyric acid was determined to quantify degree of BBB disruption. Ischemia-reperfusion increased the K i in the control animals. Streptozotocin increased the K i in the ischemic-reperfused (IR-C, +22%) as well as in the contralateral cortex (CC, +40%). Rapamycin decreased the K i in the IR-C (-32%) as well as in the CC (-26%) in the control rats. However, rapamycin did not affect K i in the IR-C or in the CC in the diabetic rats. Our data demonstrated a greater BBB disruption in diabetes in the ischemic as well as non-ischemic cortex even in the early stage of cerebral ischemia-reperfusion and that acute administration of rapamycin did not significantly affect BBB permeability in diabetes. From our quantitative analysis of BBB disruption, the vulnerability of BBB in diabetes has been emphasized in the early stage of cerebral ischemia-reperfusion and a less important role of the mTOR pathway is suggested in altering BBB permeability in diabetes. Copyright © 2017 Elsevier B.V. All rights reserved.

Blood-brain barrier disruption induced by diagnostic ultrasound combined with microbubbles in mice.

PubMed

Zhao, Bingxia; Chen, Yihan; Liu, Jinfeng; Zhang, Li; Wang, Jing; Yang, Yali; Lv, Qing; Xie, Mingxing

2018-01-12

To investigate the effects of the microbubble (MB) dose, mechanism index (MI) and sonication duration on blood-brain barrier (BBB) disruption induced by diagnostic ultrasound combined with MBs as well as to investigate the potential molecular mechanism. The extent of BBB disruption increased with MB dose, MI and sonication duration. A relatively larger extent of BBB disruption associated with minimal tissue damage was achieved by an appropriate MB dose and ultrasound exposure parameters with diagnostic ultrasound. Decreased expression of ZO-1, occludin and claudin-5 were correlated with disruption of the BBB, as confirmed by paracellular passage of the tracer lanthanum nitrate into the brain parenchyma after BBB disruption. These findings indicated that this technique is a promising tool for promoting brain delivery of diagnostic and therapeutic agents in the diagnosis and treatment of brain diseases. The extent of BBB disruption was qualitatively assessed by Evans blue (EB) staining and quantitatively analyzed by an EB extravasation measurement. A histological examination was performed to evaluate tissue damage. Expression of tight junction (TJ) related proteins ZO-1, occludin and claudin-5 was determined by western blotting analysis and immunohistofluorescence. Transmission electron microscopy was performed to observe ultrastructure changes of TJs after BBB disruption.

Peptides and the blood-brain barrier.

PubMed

Banks, William A

2015-10-01

The demonstration that peptides and regulatory proteins can cross the blood-brain barrier (BBB) is one of the major contributions of Dr. Abba J. Kastin. He was the first to propose that peptides could cross the BBB, the first to show that an endogenous peptide did so, and the first to describe a saturable transport system at the BBB for peptides. His work shows that in crossing the BBB, peptides and regulatory proteins act as informational molecules, informing the brain of peripheral events. Brain-to-blood passage helps to control levels of peptides with the brain and can deliver information in the brain-to-blood direction. He showed that the transporters for peptides and proteins are not static, but respond to developmental and physiological changes and are affected by disease states. As such, the BBB is adaptive to the needs of the CNS, but when that adaption goes awry, the BBB can be a cause of disease. The mechanisms by which peptides and proteins cross the BBB offer opportunities for drug delivery of these substances or their analogs to the brain in the treatment of diseases of the central nervous system. Published by Elsevier Inc.

Three-dimensional hydrogel cell culture systems for modeling neural tissue

NASA Astrophysics Data System (ADS)

Frampton, John

Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was designed for use as a tool to predict the transport and processing that occurs prior to drug uptake in the central nervous system (CNS), and to predict BBB permeability. Electrochemical techniques and immunohistochemistry were used to validate this model and provide detailed information about cellular organization and function. Electrochemical impedance spectroscopy (EIS) provided evidence that endothelial cells cultured in the presence of astrocytes formed tight junctions capable of occluding the flow of electrical current. In a second series of experiments, a microglia-astrocyte co-culture system was developed to assess the effects of glial cells on electrode impedance recorded from neural prosthetic devices in vitro. Impedance measurements were compared with confocal images to determine the effects of glial cell density and cell type on electrode performance. The results indicate that EIS data can be used to model components of the reactive cell responses in brain tissue, and that impedance measurements recorded in vitro can be compared to measurements recorded in vivo. Taken together, these results demonstrate that alginate hydrogels can be used for the creation of 3-D neural cell scaffolds, and that such cell scaffolds can be used to model a variety of three-dimensional neural tissues in vitro, that cannot be studied in 2-D cultures.

The role of the blood-brain barrier in the development and treatment of migraine and other pain disorders.

PubMed

DosSantos, Marcos F; Holanda-Afonso, Rosenilde C; Lima, Rodrigo L; DaSilva, Alexandre F; Moura-Neto, Vivaldo

2014-01-01

The function of the blood-brain barrier (BBB) related to chronic pain has been explored for its classical role in regulating the transcellular and paracellular transport, thus controlling the flow of drugs that act at the central nervous system, such as opioid analgesics (e.g., morphine) and non-steroidal anti-inflammatory drugs. Nonetheless, recent studies have raised the possibility that changes in the BBB permeability might be associated with chronic pain. For instance, changes in the relative amounts of occludin isoforms, resulting in significant increases in the BBB permeability, have been demonstrated after inflammatory hyperalgesia. Furthermore, inflammatory pain produces structural changes in the P-glycoprotein, the major efflux transporter at the BBB. One possible explanation for these findings is the action of substances typically released at the site of peripheral injuries that could lead to changes in the brain endothelial permeability, including substance P, calcitonin gene-related peptide, and interleukin-1 beta. Interestingly, inflammatory pain also results in microglial activation, which potentiates the BBB damage. In fact, astrocytes and microglia play a critical role in maintaining the BBB integrity and the activation of those cells is considered a key mechanism underlying chronic pain. Despite the recent advances in the understanding of BBB function in pain development as well as its interference in the efficacy of analgesic drugs, there remain unknowns regarding the molecular mechanisms involved in this process. In this review, we explore the connection between the BBB as well as the blood-spinal cord barrier and blood-nerve barrier, and pain, focusing on cellular and molecular mechanisms of BBB permeabilization induced by inflammatory or neuropathic pain and migraine.

The role of the blood–brain barrier in the development and treatment of migraine and other pain disorders

PubMed Central

DosSantos, Marcos F.; Holanda-Afonso, Rosenilde C.; Lima, Rodrigo L.; DaSilva, Alexandre F.; Moura-Neto, Vivaldo

2014-01-01

The function of the blood–brain barrier (BBB) related to chronic pain has been explored for its classical role in regulating the transcellular and paracellular transport, thus controlling the flow of drugs that act at the central nervous system, such as opioid analgesics (e.g., morphine) and non-steroidal anti-inflammatory drugs. Nonetheless, recent studies have raised the possibility that changes in the BBB permeability might be associated with chronic pain. For instance, changes in the relative amounts of occludin isoforms, resulting in significant increases in the BBB permeability, have been demonstrated after inflammatory hyperalgesia. Furthermore, inflammatory pain produces structural changes in the P-glycoprotein, the major efflux transporter at the BBB. One possible explanation for these findings is the action of substances typically released at the site of peripheral injuries that could lead to changes in the brain endothelial permeability, including substance P, calcitonin gene-related peptide, and interleukin-1 beta. Interestingly, inflammatory pain also results in microglial activation, which potentiates the BBB damage. In fact, astrocytes and microglia play a critical role in maintaining the BBB integrity and the activation of those cells is considered a key mechanism underlying chronic pain. Despite the recent advances in the understanding of BBB function in pain development as well as its interference in the efficacy of analgesic drugs, there remain unknowns regarding the molecular mechanisms involved in this process. In this review, we explore the connection between the BBB as well as the blood–spinal cord barrier and blood–nerve barrier, and pain, focusing on cellular and molecular mechanisms of BBB permeabilization induced by inflammatory or neuropathic pain and migraine. PMID:25339863