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Sample records for cultured rat retinal

  1. Roller culture of free-floating retinal slices: a new system of organotypic cultures of adult rat retina.

    PubMed

    Rzeczinski, Stefan; Victorov, Ilya V; Lyjin, Anatolij A; Aleksandrova, Olga P; Harms, Christoph; Kronenberg, Golo; Freyer, Dorette; Scheibe, Franziska; Priller, Josef; Endres, Matthias; Dirnagl, Ulrich

    2006-01-01

    No experimental system exists to date for the in vitro study of retinal ganglion cell populations in a three-dimensional organotypic tissue environment. Here, we describe such a novel method for roller cultivation of adult retinas. Retinas of adult (1-3 months old) rats were cut into rectangular slices of approximately 1 mm(2). Free-floating slices were cultured on a horizontal rotating roller drum (50-60 rpm) in a dry incubator at 36.5 degrees C. During the first days of cultivation, primary flat retinal slices changed their configuration and transformed into ball-shaped tissue spheres (retinal bodies). Histological and immunocytochemical studies showed that the outer wall of the retinal bodies was formed by cell and fibre layers typical of mature retina with photoreceptors located on the outside. Initially, retinal bodies contained an inner cavity which later was completely obliterated and filled with glial cells, sprouting nerve fibres, and vascular structures. This culture system was further developed into a robust model of glutamate-induced neurotoxicity. Using a novel culture method of adult rat retina, preservation of the three-dimensional organotypic retinal cytoarchitecture was achieved, including survival of neurons in the ganglion cell layer and sprouting of nerve fibres of the axotomized retinal ganglion cells. This novel culture model promises to facilitate studies of retinal physiology and pathology. Copyright (c) 2006 S. Karger AG, Basel.

  2. Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

    PubMed

    Yahara, Tohru; Tachikawa, Masanori; Akanuma, Shin-ichi; Hosoya, Ken-ichi

    2010-01-01

    We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

  3. Comparison of two methods used to culture and purify rat retinal Müller cells.

    PubMed

    Song, Wei-Tao; Zhang, Xue-Yong; Xiong, Si-Qi; Wen, Dan; Jiang, Jian; Xia, Xiao-Bo

    2013-01-01

    To study two methods for culturing and purifying Sprague-Dawley (SD) rat retinal Müller cells and determine which one is better. The passage culture method of Müller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method. After culturing retinal cells for one month through these two methods, fluorescence-activated cell sorter (FACS), RT-PCR, and immunohistochemistry technology were performed to examine the enrichment and purity of Müller glial cells, and carried out two-sample approximate t test using SSPS 13.0 to further compare the Müller cell positive rate in both methods. The statistical results showed that the purity of Müller cells was 83.2%±5.16% in group A, and the purity was 98.5%±1.08% in group B. The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant (t=-9.178, P<0.005). The results clearly exhibited a difference between the purity of Müller cells cultured by the complete pancreatic enzyme digestion method (group A) and the repeated incomplete pancreatic enzyme digestion method (group B). Compared with the complete pancreatic enzyme digestion method, this novel method was more efficient and a higher purity of Müller cells could be obtained using this approach.

  4. Nicotinic Antagonists Enhance Process Outgrowth by Rat Retinal Ganglion Cells in Culture

    NASA Astrophysics Data System (ADS)

    Lipton, Stuart A.; Frosch, Matthew P.; Phillips, Micheal D.; Tauck, David L.; Aizenman, Elias

    1988-03-01

    Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

  5. Rat retinal transcriptome

    PubMed Central

    Kozhevnikova, Oyuna S.; Korbolina, Elena E.; Ershov, Nikita I.; Kolosova, Natalia G.

    2013-01-01

    Pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, remains poorly understood due to the paucity of animal models that fully replicate the human disease. Recently, we showed that senescence-accelerated OXYS rats develop a retinopathy similar to human AMD. To identify alterations in response to normal aging and progression of AMD-like retinopathy, we compared gene expression profiles of retina from 3- and 18-mo-old OXYS and control Wistar rats by means of high-throughput RNA sequencing (RNA-Seq). We identified 160 and 146 age-regulated genes in Wistar and OXYS retinas, respectively. The majority of them are related to the immune system and extracellular matrix turnover. Only 24 age-regulated genes were common for the two strains, suggestive of different rates and mechanisms of aging. Over 600 genes showed significant differences in expression between the two strains. These genes are involved in disease-associated pathways such as immune response, inflammation, apoptosis, Ca2+ homeostasis and oxidative stress. The altered expression for selected genes was confirmed by qRT-PCR analysis. To our knowledge, this study represents the first analysis of retinal transcriptome from young and old rats with biologic replicates generated by RNA-Seq technology. We can conclude that the development of AMD-like retinopathy in OXYS rats is associated with an imbalance in immune and inflammatory responses. Aging alters the expression profile of numerous genes in the retina, and the genetic background of OXYS rats has a profound impact on the development of AMD-like retinopathy. PMID:23656783

  6. Use of an Adult Rat Retinal Explant Model for Screening of Potential Retinal Ganglion Cell Neuroprotective Therapies

    PubMed Central

    Bull, Natalie D.; Johnson, Thomas V.; Welsapar, Guncha; DeKorver, Nicholas W.; Tomarev, Stanislav I.

    2011-01-01

    Purpose. To validate an established adult organotypic retinal explant culture system for use as an efficient medium-throughput screening tool to investigate novel retinal ganglion cell (RGC) neuroprotective therapies. Methods. Optimal culture conditions for detecting RGC neuroprotection in rat retinal explants were identified. Retinal explants were treated with various recognized, or purported, neuroprotective agents and cultured for either 4 or 7 days ex vivo. The number of cells surviving in the RGC layer (RGCL) was quantified using histologic and immunohistochemical techniques, and statistical analyses were applied to detect neuroprotective effects. Results. The ability to replicate previously reported in vivo RGC neuroprotection in retinal explants was verified by demonstrating that caspase inhibition, brain-derived neurotrophic factor treatment, and stem cell transplantation all reduced RGCL cell loss in this model. Further screening of potential neuroprotective pharmacologic agents demonstrated that betaxolol, losartan, tafluprost, and simvastatin all alleviated RGCL cell loss in retinal explants, supporting previous reports. However, treatment with brimonidine did not protect RGCL neurons from death in retinal explant cultures. Explants cultured for 4 days ex vivo proved most sensitive for detecting neuroprotection. Conclusions. The current adult rat retinal explant culture model offers advantages over other models for screening potential neuroprotective drugs, including maintenance of neurons in situ, control of environmental conditions, and dissociation from other factors such as intraocular pressure. Verification that neuroprotection by previously identified RGC-protective therapies could be replicated in adult retinal explant cultures suggests that this model could be used for efficient medium-throughput screening of novel neuroprotective therapies for retinal neurodegenerative disease. PMID:21345987

  7. Quercetin Declines Apoptosis, Ameliorates Mitochondrial Function and Improves Retinal Ganglion Cell Survival and Function in In Vivo Model of Glaucoma in Rat and Retinal Ganglion Cell Culture In Vitro.

    PubMed

    Gao, Feng-Juan; Zhang, Sheng-Hai; Xu, Ping; Yang, Bo-Qi; Zhang, Rong; Cheng, Yun; Zhou, Xu-Jiao; Huang, Wan-Jing; Wang, Min; Chen, Jun-Yi; Sun, Xing-Huai; Wu, Ji-Hong

    2017-01-01

    Glaucoma is a progressive neuropathy characterized by the loss of retinal ganglion cells (RGCs). Strategies that delay or halt RGC loss have been recognized as potentially beneficial for rescuing vision in glaucoma patients. Quercetin (Qcn) is a natural and important dietary flavonoid compound, widely distributed in fruits and vegetables. Mounting evidence suggests that Qcn has numerous neuroprotective effects. However, whether Qcn exerts neuroprotective effects on RGC in glaucoma is poorly understood. In this study, we investigated the protective effect of Qcn against RGC damage in a rat chronic ocular hypertension (COHT) model invivo and hypoxia-induced primary cultured RGC damage in vitro, and we further explored the underlying neuroprotective mechanisms. We found that Qcn not only improved RGC survival and function from a very early stage of COHT invivo, it promoted the survival of hypoxia-treated primary cultured RGCs invitro via ameliorating mitochondrial function and preventing mitochondria-mediated apoptosis. Our findings suggest that Qcn has direct protective effects on RGCs that are independent of lowering the intraocular pressure (IOP). Qcn may be a promising therapeutic agent for improving RGC survival and function in glaucomatous neurodegeneration.

  8. Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration.

    PubMed

    Qu, Zepeng; Guan, Yuan; Cui, Lu; Song, Jian; Gu, Junjie; Zhao, Hanzhi; Xu, Lei; Lu, Lixia; Jin, Ying; Xu, Guo-Tong

    2015-11-09

    Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. We have successfully differentiated

  9. A novel rat model with obesity-associated retinal degeneration.

    PubMed

    Reddy, Geereddy Bhanuprakash; Vasireddy, Vidyullatha; Mandal, Md Nawajes A; Tiruvalluru, Mrudula; Wang, Xiaofei F; Jablonski, Monica M; Nappanveettil, Giridharan; Ayyagari, Radha

    2009-07-01

    A strong association between retinal degeneration and obesity has been shown in humans. However, the molecular basis of increased risk for retinal degeneration in obesity is unknown. Thus, an animal model with obesity and retinal degeneration would greatly aid the understanding of obesity-associated retinal degeneration. The retinal abnormalities in a novel rat model (WNIN-Ob) with spontaneously developed obesity are described. Histologic and immunohistochemical examination were performed on retinal sections of 2- to 12-month-old WNIN-Ob rats, and findings were compared with those of lean littermate controls. RNA from retinas of 12-month-old WNIN-Ob and lean littermate rats was used for microarray and qRT-PCR analysis. The WNIN-Ob rats developed severe obesity, with an onset at approximately 35 days. Evaluation of retinal morphology in 2- to 12-month-old WNIN-Ob and age-matched lean littermate controls revealed progressive retinal degeneration, with an onset between 4 to 6 months of age. Immunohistochemical analysis with anti-rhodopsin, anti-cone opsin, and PSD-95 antibodies further confirmed retinal degeneration, particularly rod cell loss and thinner outer plexiform layer, in the obese rat retina. Gene expression by microarray analysis and qRT-PCR established activation of stress response, tissue remodeling, impaired phototransduction, and photoreceptor degeneration in WNIN-Ob rat retina. WNIN-Ob rats develop increased stress in retinal tissue and progressive retinal degeneration after the onset of severe obesity. The WNIN-Ob rat is the first rat model to develop retinal degeneration after the onset of obesity. This novel rat model may be a valuable tool for investigating retinal degeneration associated with obesity in humans.

  10. Expression of peptide NAP in rat retinal Müller cells prevents hypoxia-induced retinal injuries and promotes retinal neurons growth.

    PubMed

    Zheng, Yuping; Zeng, Hao; She, Huaning; Liu, Hui; Sun, Naixue

    2010-07-01

    NAP (NAPVSIPQ) is a short peptide derived from activity-dependent neuroprotective protein (ADNP) sequence, whose potent and direct neuroprotective capabilities have been widely accepted. However, due to the high risk and inconvenience of intraocular injections, NAP is difficult to be clinically administered as therapeutic agent in treating retinal diseases. Currently, stable transfection of this octapeptide into cells has not been reported, partly because of its small size and lacking of 5' signal sequence. Here, we have developed a novel NT4-NAP fusion gene by attaching the 5' nonfunctional preproregion of neurotrophin 4 (NT4) to NAP cDNA. Recombinant adeno-associated virus was established to introduce NT4-NAP construct into cultured rat retinal Müller cells (RMC), resulting in sustained high level NAP production from stable transfection. Functional analyses of RMC cells transfected with NAP revealed the remarkably reduced cytotoxicity and apoptosis of the cells under hypoxia. Furthermore, coculturing of transfected RMC-NAP cells with primary rat retinal neural cells offer marked protection to the latter against hypoxia induced cellular damages. Together our data indicate that stable transfection of NAP into retinal Müller cells with constant NAP production is possible. NAP produced from cellular transfection maintained its biological neuroprotective activities. This targeted gene expression may provide an effective treatment for retinal diseases in the near future.

  11. A New CRB1 Rat Mutation Links Müller Glial Cells to Retinal Telangiectasia

    PubMed Central

    Zhao, Min; Andrieu-Soler, Charlotte; Kowalczuk, Laura; Paz Cortés, María; Berdugo, Marianne; Dernigoghossian, Marilyn; Halili, Francisco; Jeanny, Jean-Claude; Goldenberg, Brigitte; Savoldelli, Michèle; El Sanharawi, Mohamed; Naud, Marie-Christine; van Ijcken, Wilfred; Pescini-Gobert, Rosanna; Martinet, Danielle; Maass, Alejandro; Wijnholds, Jan; Crisanti, Patricia; Rivolta, Carlo

    2015-01-01

    We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in the Crb1 gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-β and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia. PMID:25878282

  12. Glucose metabolism in rat retinal pigment epithelium.

    PubMed

    Coffe, Víctor; Carbajal, Raymundo C; Salceda, Rocío

    2006-01-01

    The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO(2) production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO(2) yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO(2) production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress.

  13. Scaffolding the retina: the interstitial extracellular matrix during rat retinal development.

    PubMed

    Taylor, Linnéa; Arnér, Karin; Engelsberg, Karl; Ghosh, Fredrik

    2015-05-01

    To examine the expression of interstitial extracellular matrix components and their role during retinal development. Fibronectin (FN), collagen IV (Coll IV) and laminin 5 (Lam 5) expression in rat retinas from developmental stages E17 to adult were studied. In addition, PN5 full-thickness retinas were cultured for 7 days with dispase, which selectively cleaves FN and Coll IV, at either 0.5 U/ml or 5.0 U/ml for 3 or 24h. Eyecups and retinal cultures were examined morphologically using hematoxylin and eosin staining and immunohistochemistry. Coll IV, Lam 5 and FN were all transiently expressed in the interstitial matrix of the retinal layers during development. The retinal layers in dispase treated explants was severely disturbed in a dose and time dependent manner. FN, Lam 5 and Coll IV, are present in the interstitial extracellular matrix during rat retinal development. Enzymatic cleavage of FN and Coll IV early in the lamination process disrupts the retinal layers implicating their pivotal role in this process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Repetitive magnetic stimulation improves retinal function in a rat model of retinal dystrophy

    NASA Astrophysics Data System (ADS)

    Rotenstreich, Ygal; Tzameret, Adi; Levi, Nir; Kalish, Sapir; Sher, Ifat; Zangen, Avraham; Belkin, Michael

    2014-02-01

    Vision incapacitation and blindness associated with retinal dystrophies affect millions of people worldwide. Retinal degeneration is characterized by photoreceptor cell death and concomitant remodeling of remaining retinal cells. Repetitive Magnetic Stimulation (RMS) is a non-invasive technique that creates alternating magnetic fields by brief electric currents transmitted through an insulated coil. These magnetic field generate action potentials in neurons, and modulate the expression of neurotransmitter receptors, growth factors and transcription factors which mediate plasticity. This technology has been proven effective and safe in various psychiatric disorders. Here we determined the effect of RMS on retinal function in Royal College of Surgeons (RCS) rats, a model for retinal dystrophy. Four week-old RCS and control Spargue Dawley (SD) rats received sham or RMS treatment over the right eye (12 sessions on 4 weeks). RMS treatment at intensity of at 40% of the maximal output of a Rapid2 stimulator significantly increased the electroretinogram (ERG) b-wave responses by up to 6- or 10-fold in the left and right eye respectively, 3-5 weeks following end of treatment. RMS treatment at intensity of 25% of the maximal output did not significant effect b-wave responses following end of treatment with no adverse effect on ERG response or retinal structure of SD rats. Our findings suggest that RMS treatment induces delayed improvement of retinal functions and may induce plasticity in the retinal tissue. Furthermore, this non-invasive treatment may possibly be used in the future as a primary or adjuvant treatment for retinal dystrophy.

  15. Culturing of retinal pigment epithelium cells.

    PubMed

    Valtink, Monika; Engelmann, Katrin

    2009-01-01

    The retinal pigment epithelium (RPE) is a monolayer of cells adjacent to the photoreceptors of the retina. It plays a crucial role in maintaining photoreceptor health and survival. Degeneration or dysfunction of the RPE can lead to photoreceptor degeneration and as a consequence to visual impairment. The most common diseased state of the RPE becomes manifest in age-related macular degeneration, an increasing cause of blindness in the elderly. RPE cells are therefore of great interest to researchers working in the field of tissue engineering and cell transplantation. In fact, studies in animal models have proven that the transplantation of RPE cells can delay the course of photoreceptor degenerative diseases. Although first attempts to transplant RPE cells into the subretinal space in human individuals suffering from age-related macular degeneration were less successful, RPE cell transplantation is still favored as a future therapeutic option, and much work is done to develop and design cell transplants. Cell banking is a prerequisite to have well-differentiated and characterized cells at hand when needed for research purposes, but also for therapeutic approaches. In this chapter the authors will describe methods to isolate, culture and preserve adult human RPE cells for the purpose of RPE cell banking. Copyright 2009 S. Karger AG, Basel.

  16. Inner retinal preservation in rat models of retinal degeneration implanted with subretinal photovoltaic arrays.

    PubMed

    Light, Jacob G; Fransen, James W; Adekunle, Adewumi N; Adkins, Alice; Pangeni, Gobinda; Loudin, James; Mathieson, Keith; Palanker, Daniel V; McCall, Maureen A; Pardue, Machelle T

    2014-11-01

    Photovoltaic arrays (PVA) implanted into the subretinal space of patients with retinitis pigmentosa (RP) are designed to electrically stimulate the remaining inner retinal circuitry in response to incident light, thereby recreating a visual signal when photoreceptor function declines or is lost. Preservation of inner retinal circuitry is critical to the fidelity of this transmitted signal to ganglion cells and beyond to higher visual targets. Post-implantation loss of retinal interneurons or excessive glial scarring could diminish and/or eliminate PVA-evoked signal transmission. As such, assessing the morphology of the inner retina in RP animal models with subretinal PVAs is an important step in defining biocompatibility and predicting success of signal transmission. In this study, we used immunohistochemical methods to qualitatively and quantitatively compare inner retinal morphology after the implantation of a PVA in two RP models: the Royal College of Surgeons (RCS) or transgenic S334ter-line 3 (S334ter-3) rhodopsin mutant rat. Two PVA designs were compared. In the RCS rat, we implanted devices in the subretinal space at 4 weeks of age and histologically examined them at 8 weeks of age and found inner retinal morphology preservation with both PVA devices. In the S334ter-3 rat, we implanted devices at 6-12 weeks of age and again, inner retinal morphology was generally preserved with either PVA design 16-26 weeks post-implantation. Specifically, the length of rod bipolar cells and numbers of cholinergic amacrine cells were maintained along with their characteristic inner plexiform lamination patterns. Throughout the implanted retinas we found nonspecific glial reaction, but none showed additional glial scarring at the implant site. Our results indicate that subretinally implanted PVAs are well-tolerated in rodent RP models and that the inner retinal circuitry is preserved, consistent with our published results showing implant-evoked signal transmission.

  17. Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture

    PubMed Central

    Johnson, Thomas V.; Oglesby, Ericka N.; Steinhart, Matthew R.; Cone-Kimball, Elizabeth; Jefferys, Joan; Quigley, Harry A.

    2016-01-01

    Purpose To develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments. Methods Thy1-YFP mouse retinas were explanted and maintained in organotypic culture. Retinal ganglion cell dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing. Results Retinal ganglion cells in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra- and interobserver measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently > 0.80). Statistically significant (P < 0.001) dendritic tree degeneration was detected following 7 days in culture including: 40% to 50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF+CNTF significantly attenuated dendritic field degeneration. Conclusions Retinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning within individual RGCs. PMID:26811145

  18. Sciatic conditioned medium increases survival, proliferation and differentiation of retinal cells in culture.

    PubMed

    Torres, Patrícia Maria Mendonça; Guilarducci, Carla Valéria Vieira; Franco, Alfred Sholl; de Araujo, Elizabeth Giestal

    2002-02-01

    Many evidences clearly demonstrate that Schwann cells provide trophic support for neurons. Different cytokines, including neurotrophins (NTs), are produced and released by Schwann cells. These trophic molecules play an important role on neuronal survival either during the development or during adult life. Cytokines have also a pivotal role on neuronal regeneration after lesions occurring during pathological conditions. The aim of this work was to study the effect of sciatic conditioned medium (SCM) on rat retinal cells maintained in culture. Our results show that treatment with SCM obtained after 14 days in vitro (SCM 14 day) induced a three-fold increase in protein content of the culture after 48 h in vitro and this value remained equally high up to 72 h. This effect was totally blocked either by addition of 30 microM BAPTA-AM, an intracellular calcium chelator, 15 microM fluorodeoxyuridine, an inhibitor of cell division, or 10 microM genistein (geni) plus 1.25 microM chelerythrine chloride (CC), the two last ones inhibitors of tyrosine kinases and protein kinase C, respectively. SCM induced an increase in [(3)H]-choline uptake and [(3)H]-thymidine incorporation of retinal cells. SCM also stimulated an increase in cytoplasmic processes outgrowth of retinal cells and survival of retinal ganglion cells. Our results clearly suggest that soluble molecules released by sciatic nerve fragments are able to increase the proliferation and survival of retinal cells in culture.

  19. Vasodilator effect of nicorandil on retinal blood vessels in rats.

    PubMed

    Ogawa, Naoto; Saito, Maki; Mori, Asami; Sakamoto, Kenji; Kametaka, Sokichi; Nakahara, Tsutomu; Ishii, Kunio

    2007-07-01

    We examined the effect of nicorandil on retinal blood vessels in rats in vivo. Male Wistar rats (8 to 10 weeks old) were anaesthetised with thiobutabarbital (120 mg/kg, intraperitoneal). Fundus images were captured with a digital camera that was equipped with a special objective lens. Diameters of retinal blood vessels were measured with a personal computer. Nicorandil (1-300 microg kg(-1) min(-1), intravenous [i.v.]) increased diameters of retinal blood vessels and decreased systemic blood pressure in a dose-dependent manner. Both responses to nicorandil were attenuated by glibenclamide (20 mg/kg, i.v.), an adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channel blocker. On the other hand, indomethacin (5 mg/kg, i.v.), a cyclooxygenase inhibitor, attenuated the vasodilation of retinal blood vessels, but not depressor response, to nicorandil and sodium nitroprusside. Pinacidil (1-300 microg kg(-1) min(-1), i.v.), a K(ATP) channel opener, also dilated retinal blood vessels and decreased systemic blood pressure. The responses to pinacidil were prevented by glibenclamide, but not by indomethacin. The vasodilation of retinal arteriole, but not depressor response, to sodium nitroprusside (1-30 microg kg(-1) min(-1), i.v.), a nitric oxide donor, was attenuated by indomethacin. These results suggest that nicorandil dilates retinal blood vessels through opening of K(ATP) channels and production of prostaglandins that are probably generated by nitric oxide.

  20. Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

    PubMed Central

    Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

    2017-01-01

    Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

  1. AAV Mediated GDNF Secretion From Retinal Glia Slows Down Retinal Degeneration in a Rat Model of Retinitis Pigmentosa

    PubMed Central

    Dalkara, Deniz; Kolstad, Kathleen D; Guerin, Karen I; Hoffmann, Natalie V; Visel, Meike; Klimczak, Ryan R; Schaffer, David V; Flannery, John G

    2011-01-01

    Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions. PMID:21522134

  2. Ih without Kir in Adult Rat Retinal Ganglion Cells

    PubMed Central

    Lee, Sherwin C.; Ishida, Andrew T.

    2011-01-01

    Antisera directed against hyperpolarization-activated mixed-cation (“Ih”) and K+ (“Kir”) channels bind to some somata in the ganglion cell layer of rat and rabbit retina. Additionally, the termination of hyperpolarizing current injections can trigger spikes in some cat retinal ganglion cells, suggesting a rebound depolarization due to activation of Ih. However, patch-clamp studies have reported that rat ganglion cells lack inward rectification, or present an inwardly rectifying K+ current. We therefore tested whether hyperpolarization activates Ih in dissociated, adult rat retinal ganglion cell somata. We report here that while we found no inward rectification in some cells, and a Kir-like current in a few cells, hyperpolarization activated Ih in roughly 75% of the cells we recorded from in voltage clamp. We show that this current is blocked by Cs+ or ZD7288 and only slightly reduced by Ba2+, that the current amplitude and reversal potential are sensitive to extracellular Na+ and K+, and that we found no evidence of Kir in cells presenting Ih. In current clamp, injecting hyperpolarizing current induced a slowly relaxing membrane hyperpolarization that rebounded to a few action potentials when the hyperpolarizing current was stopped; both the membrane potential relaxation and rebound spikes were blocked by ZD7288. These results provide the first measurement of Ih in mammalian retinal ganglion cells, and indicate that the ion channels of rat retinal ganglion cells may vary in ways not expected from previous voltage and current recordings. PMID:17488978

  3. Opsin-induced experimental autoimmune retinitis in rats.

    PubMed

    Broekhuyse, R M; Winkens, H J; Kuhlmann, E D; van Vugt, A H

    1984-12-01

    Experimental autoimmune retinitis has been induced in Lewis rats by injection of opsin in mycobacterial adjuvant and Hemophilus pertussis adjuvant. Clinical, histopathological and immunological parameters of the disease are reported. Two types of opsin were prepared from purified bovine retina outer segments, one type in Triton X-100 and the other in lithium dodecyl sulfate. Both preparations were free from S-antigen. Dodecyl sulfate-denaturated-opsin displayed lower antigenicity and pathogenicity than Triton-opsin. Triton-opsin (250 micrograms) induced moderate to severe non-granulomatous uveitis (predominantly retinitis) in 70% of the Lewis rats at the end of the second week after injection. The photoreceptor cell layer was destructed within a few days. This group displayed high responses to opsin in the lymphocyte transformation test. In view of observed histological features, the possible early involvement of vasoactive factors is discussed. Low opsin doses (50 or 100 micrograms) seldomly induced severe retinitis, while the incidence of mild pathology was low. Lewis rats appeared to be more susceptible for the development of experimental autoimmune retinitis than Wistar rats.

  4. Properties of Flicker ERGs in Rat Models with Retinal Degeneration

    PubMed Central

    An, Jing; Guo, Qun; Li, Li; Zhang, Zuoming

    2012-01-01

    Purpose. To describe the characteristics of rod and cone functions in rat models for congenital stationary night blindness (CSNB) and retinal cone dysfunction (RCD). Methods. Rod and cone function were isolated by recording the rod-/cone-driven flicker and blue light flicker electroretinograms (ERGs). Results. During dark adaptation, the amplitudes of flicker ERGs in CSNB rats were lower than those in control rats; the responses of RCD rats were similar to control rats. During light adaptation, the amplitudes of flicker ERGs in CSNB rats were reduced; whereas the responses of RCD rats were not detected. Blue flicker ERGs were not observed in CSNB rats at lower frequencies. The cone driven critical flicker frequencies (CFFs) in control rats were 62 Hz. The rod driven CFF of RCD rats was 20 Hz; whereas the rod-/cone-driven CFF of CSNB rats both were about 25 Hz. Conclusions. The function of the rod system was damaged completely, the cones were the source of vision in CSNB rats. Rod system function is excellent in RCD rat. The rods of albinism rats are sensitive to frequencies less than 20 Hz; whereas the cones are sensitive to frequencies up to 62 Hz. PMID:24555124

  5. Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa.

    PubMed

    Fernández-Sánchez, Laura; Lax, Pedro; Campello, Laura; Pinilla, Isabel; Cuenca, Nicolás

    2015-01-01

    Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas. In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

  6. Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa

    PubMed Central

    Fernández-Sánchez, Laura; Lax, Pedro; Campello, Laura; Pinilla, Isabel; Cuenca, Nicolás

    2015-01-01

    Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina. PMID:26733810

  7. Structure and function of embryonic rat retinal sheet transplants.

    PubMed

    Peng, Qing; Thomas, Biju B; Aramant, Robert B; Chen, Zhenhai; Sadda, Srinivas R; Seiler, Magdalene J

    2007-09-01

    To evaluate retinal sheet transplants in S334ter-line-3 retinal degenerate rats by comparing visual responses recorded electrophysiologically with morphology based on light and electron microscopy. S334ter-line-3 retinal degenerate rats (n = 7) received retinal sheet transplants between postnatal days 28 and 31. The donor tissue was derived from transgenic embryonic day 19 (E19) rat retinae expressing human placental alkaline phosphatase (hPAP). Fresh retinal sheets were gently transplanted into the subretinal space of the left eye with the help of a custom-made implantation tool. Selected rats (n = 5) were subjected to electrophysiologic evaluation of visual responses from the superior colliculus about 84-121 days after surgery. Transplanted eyes were processed for light microscopy (LM) and electron microscopy (EM) evaluations. All the transplanted rats that were evaluated for visual responses in the brain showed responses to very low light stimulation (-3.42 to -2.8 log cd/m(2)) of the eye in a small area of the superior colliculus corresponding with the placement of the transplant in the host retina. Histologic evaluation showed that most of the transplants contained well-laminated areas with correct polarity in the subretinal space. Inside the transplant areas, rosettes of photoreceptors with inner and outer segments were found. In the laminated areas, the outer segments of photoreceptors were facing the host retinal pigment epithelium (RPE). Immunohistochemical evaluation of hPAP donor cells revealed areas with specific staining of the transplants in the subretinal space. Electron microscopic evaluation showed a glial demarcation membrane between the host and the transplant, however, processes originating from the transplant were observed inside the host retina. Sheets of E19 rat retina transplanted into the subretinal space of S334ter-line-3 rats survived without immune rejection and continued to show visual function when tested after 3 months. Well

  8. Culturing rat hippocampal neurons.

    PubMed

    Audesirk, G; Audesirk, T; Ferguson, C

    2001-01-01

    Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

  9. Betaxolol attenuates retinal ischemia/reperfusion damage in the rat.

    PubMed

    Cheon, Eun Woo; Park, Chang Hwan; Kang, Sang Soo; Cho, Gyeong Jae; Yoo, Ji Myong; Song, Joon Kyung; Choi, Wan Sung

    2003-10-27

    This study was performed to elucidate the protection afforded by post-treatment with Betoptic (0.25% betaxolol) against neuronal cell damage after ischemia/reperfusion insult in rats. Betaxolol was applied topically after the start of reperfusion and its effect was evaluated by morphometry and choline acetyltransferase immunoreactivity of retinas at 7 days after reperfusion. In non-treated eyes, the thickness of the inner plexiform layer decreased markedly after a reperfusion period of both 3 and 7 days. However, when eyes were treated with betaxolol after ischemia/reperfusion injury, both the reduction of the inner plexiform layer thickness and the retinal choline acetyltransferase immunoreactivity were significantly attenuated. These findings suggest that betaxolol is an efficient neuroprotective agent and prevents the retinal cell damage induced by ischemic injury in rats.

  10. BDNF-Treated Retinal Progenitor Sheets Transplanted to Degenerate Rats

    PubMed Central

    Seiler, Magdalene J.; Thomas, Biju B.; Chen, Zhenhai; Arai, Shinichi; Chadalavada, Sridhar; Mahoney, Melissa J.; Sadda, Srinivas R.; Aramant, Robert B.

    2011-01-01

    The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with PLGA (Poly-lactide-co-glycolide) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter-line-3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60 days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a “low light” intensity of 1 cd/m2in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF-treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF -treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects - either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs - still needs to be elucidated. PMID:17983616

  11. Chlorogenic acid decreases retinal vascular hyperpermeability in diabetic rat model.

    PubMed

    Shin, Joo Young; Sohn, Joonhong; Park, Kyu Hyung

    2013-04-01

    To evaluate the effect of chlorogenic acid (CGA), a polyphenol abundant in coffee, on retinal vascular leakage in the rat model of diabetic retinopathy, Sprague-Dawley rats were divided into four groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with 10 and 20 mg/kg chlorogenic acid intraperitoneally daily for 14 days, respectively. Blood-retinal barrier (BRB) breakdown was evaluated using FITC-dextran. Vascular endothelial growth factor (VEGF) distribution and expression level was evaluated with immunohistochemistry and Western blot analysis. Expression of tight junction proteins, occludin and claudin-5, and zonula occludens protein, ZO-1 was also evaluated with immunohistochemistry and Western blot analysis. BRB breakdown and increased vascular leakage was found in diabetic rats, with increased VEGF expression and down-regulation of occludin, claudin-5, and ZO-1. CGA treatment effectively preserved the expression of occludin, and decreased VEGF levels, leading to less BRB breakdown and less vascular leakage. CGA may have a preventive role in BRB breakdown in diabetic retinopathy by preserving tight junction protein levels and low VEGF levels.

  12. Proteoglycan synthesis in flat cell-free cultures of chick embryo retinal neurons and photoreceptors.

    PubMed

    Needham, L K; Adler, R; Hewitt, A T

    1988-04-01

    Extracellular matrix and cell surface proteoglycans are thought to play important roles in neural development and regeneration. Central nervous system proteoglycans have been isolated and characterized from rat and sheep brain and from chick neural retina. An experimental advantage offered by the latter tissue is that it is avascular and can be isolated free of connective tissue and pigment epithelium. Therefore, proteoglycans synthesized by this tissue are derived exclusively from neural cells. However, it has not yet been determined whether neurons and photoreceptors contribute to proteoglycan synthesis or whether these molecules are largely glial in origin. In the present study we have addressed this question using cultures of chick neural retinal cells free of flat, glial-like cells. Proteoglycans synthesized by cultures of retinal neurons, photoreceptors, and undifferentiated, process-free round cells from 8-day embryonic chick neural retina were metabolically labeled in vitro using [35S]sulfate and [3H]glucosamine as precursors. Radiolabeled proteoglycans accumulated in the medium, and could also be extracted from the cell layer by sequential treatments with Triton X-100 and with guanidine HCl. The proteoglycans were isolated by ion-exchange chromatography, and characterized by gel filtration chromatography and by susceptibility to degradation by enzymatic and chemical treatments. Overall, heparan sulfate proteoglycans were the predominant type of proteoglycan synthesized in vitro by the cultured neural retinal cells at this developmental stage. The medium and the Triton extract contained different proportions of both chondroitin sulfate and heparan sulfate proteoglycans, while heparan sulfate was the only proteoglycan recovered from the guanidine extract. These studies demonstrate that heparan sulfate and chondroitin sulfate proteoglycans are actively synthesized by cultures of neural retinal cells free of flat, glial-like cells.

  13. Inner retinal metabolic rate of oxygen by oxygen tension and blood flow imaging in rat.

    PubMed

    Wanek, Justin; Teng, Pang-Yu; Albers, John; Blair, Norman P; Shahidi, Mahnaz

    2011-09-01

    The metabolic function of inner retinal cells relies on the availability of nutrients and oxygen that are supplied by the retinal circulation. Assessment of retinal tissue vitality and function requires knowledge of both the rate of oxygen delivery and consumption. The purpose of the current study is to report a novel technique for assessment of the inner retinal metabolic rate of oxygen (MO(2)) by combined measurements of retinal blood flow and vascular oxygen tension (PO(2)) in rat. The application of this technology has the potential to broaden knowledge of retinal oxygen dynamics and advance understanding of disease pathophysiology.

  14. Expression of Aquaporin-6 in Rat Retinal Ganglion Cells.

    PubMed

    Jang, Sun Young; Lee, Eung Suk; Ohn, Young-Hoon; Park, Tae Kwann

    2016-08-01

    Several aquaporins (AQPs) have been identified to be present in the eyes, and it has been suggested that they are involved in the movement of water and small solutes. AQP6, which has low water permeability and transports mainly anions, was recently discovered in the eyes. In the present study, we investigate the localization of AQP6 in the rat retina and show that AQP6 is selectively localized to the ganglion cell layer and the outer plexiform layer. Along with the gradual decrease in retinal ganglion cells after a crushing injury of optic nerve, immunofluorescence signals of AQP6 gradually decreased. Confocal microscope images confirmed AQP6 expression in retinal ganglion cells and Müller cells in vitro. Therefore, AQP6 might participate in water and anion transport in these cells.

  15. Interferon-gamma and tumour necrosis factor induce expression of major histocompatibility complex antigen on rat retinal astrocytes.

    PubMed Central

    el-Asrar, A M; Maimone, D; Morse, P H; Lascola, C; Reder, A T

    1991-01-01

    Cultured rat retinal astrocytes were tested by indirect immunofluorescence staining for their ability to express class I and II major histocompatibility complex (MHC) antigens under basal culture conditions and after three days of stimulation with two recombinant cytokines, rat interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha). Under basal culture conditions low levels of class I antigens were detected on a small percentage of cells, but there was no visible class II. IFN-gamma and TNF alpha stimulation enhanced class I expression. TNF alpha had no effect on class II expression, whereas IFN-gamma induced the expression of class II in a dose dependent manner. These findings suggest that retinal astrocytes might play a part in immunological events occurring in the retina. Images PMID:1908315

  16. Interferon-gamma and tumour necrosis factor induce expression of major histocompatibility complex antigen on rat retinal astrocytes.

    PubMed

    el-Asrar, A M; Maimone, D; Morse, P H; Lascola, C; Reder, A T

    1991-08-01

    Cultured rat retinal astrocytes were tested by indirect immunofluorescence staining for their ability to express class I and II major histocompatibility complex (MHC) antigens under basal culture conditions and after three days of stimulation with two recombinant cytokines, rat interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha). Under basal culture conditions low levels of class I antigens were detected on a small percentage of cells, but there was no visible class II. IFN-gamma and TNF alpha stimulation enhanced class I expression. TNF alpha had no effect on class II expression, whereas IFN-gamma induced the expression of class II in a dose dependent manner. These findings suggest that retinal astrocytes might play a part in immunological events occurring in the retina.

  17. Computational molecular phenotyping of retinal sheet transplants to rats with retinal degeneration

    PubMed Central

    Seiler, M.J.; Jones, B.W.; Aramant, R.B.; Yang, P.B.; Keirstead, H.S.; Marc, R.E.

    2012-01-01

    Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was the first attempt to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). - S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40d. Donor tissues were incubated with slow-releasing microspheres containing BDNF or GDNF. Up to 265 days after surgery, eyes of selected rats were vibratome sectioned through the transplant area (some slices stained for donor marker hPAP), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP, L-glutamate, L-glutamine, glutathione, glycine, taurine, GABA, and DAPI. - In large transplant areas, photoreceptor outer segments in contact with host RPE revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina. PMID:22594836

  18. Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

    PubMed

    Yuan, Jing; Yu, Jian-Xiong

    2016-05-01

    Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

  19. Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

    SciTech Connect

    Chung, Hyewon; Yoon, Young Hee; Hwang, Jung Jin; Cho, Kyung Sook; Koh, Jae Young; Kim, June-Gone

    2009-03-01

    Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.

  20. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    PubMed Central

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  1. Postconditioning with inhaled hydrogen promotes survival of retinal ganglion cells in a rat model of retinal ischemia/reperfusion injury.

    PubMed

    Wang, Ruobing; Wu, Jiangchun; Chen, Zeli; Xia, Fangzhou; Sun, Qinglei; Liu, Lin

    2016-02-01

    Retinal ischemia/reperfusion (I/R) injury plays a crucial role in the pathophysiology of various ocular diseases. Intraperitoneal injection or ocular instillation with hydrogen (H2)-rich saline was recently shown to be neuroprotective in the retina due to its anti-oxidative and anti-inflammatory effects. Our study aims to explore whether postconditioning with inhaled H2 can protect retinal ganglion cells (RGCs) in a rat model of retinal I/R injury. Retinal I/R injury was performed on the right eyes of rats and was followed by inhalation of 67% H2 mixed with 33% oxygen immediately after ischemia for 1h daily for one week. RGC density was counted using haematoxylin and eosin (HE) staining and retrograde labeling with cholera toxin beta (CTB). Visual function was assessed using flash visual evoked potentials (FVEP) and pupillary light reflex (PLR). Potential biomarkers of retinal oxidative stress and inflammatory responses were measured, including the expression of 4-Hydroxynonenalv (4-HNE), interleukin-1 beta (IL1-β) and tumor necrosis factor alpha (TNF-α). HE and CTB tracing showed that the survival rate of RGCs in the H2-treated group was significantly higher than the rate in the I/R group. Rats with H2 inhalation showed better visual function in assessments of FVEP and PLR. Moreover, H2 treatment significantly decreased the number of 4-HNE-stained cells in the ganglion cell layer and inhibited the retinal overexpression of IL1-β and TNF-α that was induced by retinal I/R injury. Our results demonstrate that postconditioning with inhaled high-dose H2 appears to confer neuroprotection against retinal I/R injury via anti-oxidative, anti-inflammatory and anti-apoptosis pathways.

  2. Vasodilator Effects of Elcatonin, a Synthetic Eel Calcitonin, on Retinal Blood Vessels in Rats.

    PubMed

    Mori, Asami; Suzawa, Hironori; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2015-01-01

    The aim of this study was to examine the effects of elcatonin, a synthetic derivative of eel calcitonin, on rat retinal blood vessels, and to determine how diabetes affects the retinal vascular responses. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring the diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate were continuously recorded. Elcatonin increased the diameter of retinal blood vessels but decreased mean blood pressure in a dose-dependent manner, whereas it had no significant effect on heart rate. A diminished retinal vasodilator response and significant pressor response to elcatonin were observed in rats injected intravenously with N(G)-nitro-L-arginine methyl ester, a nitric oxide (NO) synthase inhibitor. Intravitreal injection of indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ22536, an adenylyl cyclase inhibitor, markedly attenuated the vasodilator effects of elcatonin on retinal blood vessels. The retinal vasodilator responses to elcatonin were unaffected 2 weeks after the induction of diabetes by a combination of streptozotocin treatment and D-glucose feeding. These results suggest that elcatonin dilates rat retinal blood vessels via NO- and COX-dependent mechanisms and that the adenylyl cyclase-adenosine 3',5'-cyclic monophosphate system plays a major role in the vasodilator mechanisms. The retinal vasodilatory effects of elcatonin seem to be preserved at early stages of diabetes.

  3. Stimulation of β1- and β2-adrenoceptors dilates retinal blood vessels in rats.

    PubMed

    Mori, Asami; Sekito, Akane; Sakamoto, Kenji; Ishii, Kunio; Nakahara, Tsutomu

    2017-05-01

    Our previous studies have demonstrated that adrenaline dilates rat retinal arterioles by stimulating propranolol-sensitive β-adrenoceptors and β3-adrenoceptors, and selective stimulation of β2- or β3-adrenoceptors causes retinal vasodilator responses. In the present study, we compared the effects of β1- and β2-adrenoceptor stimulation on rat retinal arterioles in vivo. Rat ocular fundus images were captured using an original high-resolution digital fundus camera. Diameters of retinal arterioles contained in the images were measured. Systemic blood pressure and heart rate were recorded continuously. Denopamine, a β1-adrenoceptor agonist, increased the diameter of retinal arterioles and heart rate, and produced a small but statistically insignificant decrease in mean arterial pressure. CGP20712A, a β1-adrenoceptor antagonist, but not ICI118551, a β2-adrenoceptor antagonist, significantly prevented denopamine-induced retinal vasodilator and heart rate responses. Salbutamol, a β2-adrenoceptor agonist, increased the diameter of retinal arterioles and decreased mean arterial pressure without significantly changing heart rate. The effects of salbutamol were significantly prevented by ICI118551, but not by CGP20712A. These results suggest that stimulation of β1- and β2-adrenoceptors dilates retinal blood vessels and indicate that all three β-adrenoceptor subtypes (β1, β2, and β3) may be involved in the retinal vasodilator response to adrenaline in rats.

  4. Protective effect of ascorbate in retinal light damage of rats

    SciTech Connect

    Organisciak, D.T.; Wang, H.M.; Li, Z.Y.; Tso, M.O.

    1985-11-01

    Cyclic light and dark-reared rats were exposed to intense visible light for various periods and then rhodopsin-measured following recovery in darkness for up to 14 days. Animals were injected with ascorbic acid or ascorbate derivatives at various doses prior to light exposure in green Plexiglas chambers. The results show that ascorbic acid administration elevates retinal ascorbate and reduces the loss of rhodopsin and photoreceptor cell nuclei resulting from intense light. When given in comparable doses, L-ascorbic acid, sodium ascorbate, and dehydroascorbate were equally effective in preserving rhodopsin. The ascorbate protective effect in the retina is also dose dependent in both cyclic light and dark-reared rats and exhibits a requirement for the L-stereoisomer of the vitamin. Ascorbic acid is effective when administered before, but not after, light exposure, suggesting that protection from light damage in the retina occurs during the light period. In some experiments, rod outer segments were isolated from rats immediately after light exposure, lipids extracted, and fatty acid composition determined. As judged by the preservation of rod outer segment docosahexaenoic acid in rats given ascorbate, the vitamin may act in an antioxidative fashion by inhibiting oxidation of membrane lipids during intense light.

  5. Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa.

    PubMed

    Fernández-Sánchez, Laura; Lax, Pedro; Isiegas, Carolina; Ayuso, Eduard; Ruiz, José M; de la Villa, Pedro; Bosch, Fatima; de la Rosa, Enrique J; Cuenca, Nicolás

    2012-12-01

    Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi-) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

  6. Proinsulin Slows Retinal Degeneration and Vision Loss in the P23H Rat Model of Retinitis Pigmentosa

    PubMed Central

    Fernández-Sánchez, Laura; Lax, Pedro; Isiegas, Carolina; Ayuso, Eduard; Ruiz, José M.; de la Villa, Pedro; Bosch, Fatima; de la Rosa, Enrique J.

    2012-01-01

    Abstract Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa. PMID:23017108

  7. Safranal, a Saffron Constituent, Attenuates Retinal Degeneration in P23H Rats

    PubMed Central

    Esquiva, Gema; Martín-Nieto, José; Pinilla, Isabel; Cuenca, Nicolás

    2012-01-01

    Saffron, an extract from Crocus sativus, has been largely used in traditional medicine for its antiapoptotic and anticarcinogenic properties. In this work, we investigate the effects of safranal, a component of saffron stigmas, in attenuating retinal degeneration in the P23H rat model of autosomal dominant retinitis pigmentosa. We demonstrate that administration of safranal to homozygous P23H line-3 rats preserves both photoreceptor morphology and number. Electroretinographic recordings showed higher a- and b-wave amplitudes under both photopic and scotopic conditions in safranal-treated versus non-treated animals. Furthermore, the capillary network in safranal-treated animals was preserved, unlike that found in untreated animals. Our findings indicate that dietary supplementation with safranal slows photoreceptor cell degeneration and ameliorates the loss of retinal function and vascular network disruption in P23H rats. This work also suggests that safranal could be potentially useful to retard retinal degeneration in patients with retinitis pigmentosa. PMID:22900092

  8. Safranal, a saffron constituent, attenuates retinal degeneration in P23H rats.

    PubMed

    Fernández-Sánchez, Laura; Lax, Pedro; Esquiva, Gema; Martín-Nieto, José; Pinilla, Isabel; Cuenca, Nicolás

    2012-01-01

    Saffron, an extract from Crocus sativus, has been largely used in traditional medicine for its antiapoptotic and anticarcinogenic properties. In this work, we investigate the effects of safranal, a component of saffron stigmas, in attenuating retinal degeneration in the P23H rat model of autosomal dominant retinitis pigmentosa. We demonstrate that administration of safranal to homozygous P23H line-3 rats preserves both photoreceptor morphology and number. Electroretinographic recordings showed higher a- and b-wave amplitudes under both photopic and scotopic conditions in safranal-treated versus non-treated animals. Furthermore, the capillary network in safranal-treated animals was preserved, unlike that found in untreated animals. Our findings indicate that dietary supplementation with safranal slows photoreceptor cell degeneration and ameliorates the loss of retinal function and vascular network disruption in P23H rats. This work also suggests that safranal could be potentially useful to retard retinal degeneration in patients with retinitis pigmentosa.

  9. Protective Effect of Tang Wang One Decoction on the Retinal Vessels of Diabetic Rats

    PubMed Central

    Kou, Xinyun; Yang, Shufei; Qin, Yali; Yang, Chao; Deng, Tingting; Luo, Dan

    2017-01-01

    Objective. This study aimed to determine the influence of Tang Wang One Decoction (TWOD) on the retinal vessels of diabetic rats. Methods. The hemorheology of diabetic rats was observed. Morphological studies of retinal vessels were conducted using optical microscopy and electron microscopy. Immunological histochemistry assay was used to measure the expression levels of MMP-9, occludin, and claudin-5. Results. Obvious pathological damage was observed in the retinal vessels of diabetic rats. TWOD positively affected the hemorheology and morphology of retinal vessels. The decoction also decreased the expression of MMP-9 and increased the expression of occludin and claudin-5. Conclusions. The results suggest that the retinal protective effects of TWOD might be related to downregulation of MMP-9 and upregulation of occludin and claudin-5. PMID:28367226

  10. Compound 49b Restores Retinal Thickness and Reduces Degenerate Capillaries in the Rat Retina following Ischemia/Reperfusion.

    PubMed

    Liu, Li; Jiang, Youde; Steinle, Jena J

    2016-01-01

    We have recently reported that Compound 49b, a novel β-adrenergic receptor agonist, can significantly reduce VEGF levels in retinal endothelial cells (REC) grown in diabetic-like conditions. In this study, we investigated whether Compound 49b could protect the retina under hypoxic conditions using the ischemia-reperfusion (I/R)-induced model in rats, as well REC cultured in hypoxic conditions. Some rats received 1mM topical Compound 49b for the 2 (5 rats each group) or 10 (4 rats in each group) days post-I/R. Analyses for retinal thickness and cell loss in the ganglion cell layer was done at 2 days post-I/R, while numbers of degenerate capillaries and pericyte ghosts were measured at 10 days post-I/R. Additionally, REC were cultured in normal oxygen or hypoxia (5% O2) only or treated with 50 nM Compound 49b for 12 hours. Twelve hours after Compound 49b exposure, cells were collected and analyzed for protein levels of insulin-like growth factor binding protein 3 (IGFBP-3), vascular endothelial cell growth factor (VEGF) and its receptor (KDR), angiopoietin 1 and its receptor Tie2 for Western blotting. Data indicate that exposure to I/R significantly decreased retinal thickness, with increasing numbers of degenerate capillaries and pericyte ghosts. Compound 49b treatment inhibited these retinal changes. In REC cultured in hypoxia, levels of IGFBP-3 were reduced, which were significantly increased by Compound 49b. Hypoxia significantly increased protein levels of VEGF, KDR, Angiopoiein 1, and Tie2, which were reduced following Compound 49b treatment. These data strongly suggested that Compound 49b protected the retina against I/R-induced injury. This provides additional support for a role of β-adrenergic receptor actions in the retina.

  11. Rat retinal vasomotion assessed by laser speckle imaging

    PubMed Central

    Postnov, Dmitry D.; Sosnovtseva, Olga; Jacobsen, Jens Christian B.

    2017-01-01

    Vasomotion is spontaneous or induced rhythmic changes in vascular tone or vessel diameter that lead to rhythmic changes in flow. While the vascular research community debates the physiological and pathophysiological consequence of vasomotion, there is a great need for experimental techniques that can address the role and dynamical properties of vasomotion in vivo. We apply laser speckle imaging to study spontaneous and drug induced vasomotion in retinal network of anesthetized rats. The results reveal a wide variety of dynamical patterns. Wavelet-based analysis shows that (i) spontaneous vasomotion occurs in anesthetized animals and (ii) vasomotion can be initiated by systemic administration of the thromboxane analogue U-46619 and the nitric-oxide donor S-nitroso-acetylDL-penicillamine (SNAP). Although these drugs activate different cellular pathways responsible for vasomotion, our approach can track the dynamical changes they cause. PMID:28339503

  12. Severity of middle cerebral artery occlusion determines retinal deficits in rats

    PubMed Central

    Allen, Rachael S.; Sayeed, Iqbal; Cale, Heather A.; Morrison, Katherine C.; Boatright, Jeffrey H.; Pardue, Machelle T.; Stein, Donald G.

    2014-01-01

    Middle cerebral artery occlusion (MCAO) using the intraluminal suture technique is a common model used to study cerebral ischemia in rodents. Due to the proximity of the ophthalmic artery to the middle cerebral artery, MCAO blocks both arteries, causing both cerebral and retinal ischemia. While previous studies have shown retinal dysfunction at 48 hours post-MCAO, we investigated whether these retinal function deficits persist until 9 days and whether they correlate with central neurological deficits. Rats received 90 minutes of transient MCAO followed by electroretinography at 2 and 9 days to assess retinal function. Retinal damage was assessed with cresyl violet staining, immunohistochemistry for glial fibrillary acidic protein (GFAP) and glutamine synthetase, and TUNEL staining. Rats showed behavioral deficits as assessed with neuroscore that correlated with cerebral infarct size and retinal function at 2 days. Two days after surgery, rats with moderate MCAO (neuroscore < 5) exhibited delays in electroretinogram implicit time, while rats with severe MCAO (neuroscore ≥ 5) exhibited reductions in amplitude. Glutamine synthetase was upregulated in Müller cells 3 days after MCAO in both severe and moderate animals, however, retinal ganglion cell death was only observed in MCAO retinas from severe animals. By 9 days after MCAO, both glutamine synthetase labeling and electroretinograms had returned to normal levels in moderate animals. Early retinal function deficits correlated with behavioral deficits. However, retinal function decreases were transient and selective retinal cell loss was observed only with severe ischemia, suggesting that the retina is less susceptible to MCAO than the brain. Temporary retinal deficits caused by MCAO are likely due to ischemia-induced increases in extracellular glutamate that impair signal conduction, but resolve by 9 days after MCAO. PMID:24518488

  13. Nitric oxide dilates rat retinal blood vessels by cyclooxygenase-dependent mechanisms.

    PubMed

    Ogawa, Naoto; Mori, Asami; Hasebe, Masami; Hoshino, Maya; Saito, Maki; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2009-10-01

    It has been suggested that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent mechanisms in the ocular vasculature; however, the importance of the pathway in regulating retinal circulation in vivo remains to be elucidated. Therefore, we investigated the role of COX-dependent mechanisms in NO-induced vasodilation of retinal blood vessels in thiobutabarbital-anesthetized rats with and without neuronal blockade (tetrodotoxin treatment). Fundus images were captured with a digital camera that was equipped with a special objective lens. The retinal vascular response was assessed by measuring changes in diameter of the retinal blood vessel. The localization of COX and soluble guanylyl cyclase in rat retina was examined using immunohistochemistry. The NO donors (sodium nitroprusside and NOR3) increased the diameter of the retinal blood vessels but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a nonselective COX inhibitor, or SC-560, a selective COX-1 inhibitor, markedly attenuated the vasodilation of retinal arterioles, but not the depressor response, to the NO donors. However, both the vascular responses to NO donors were unaffected by the selective COX-2 inhibitors NS-398 and nimesulide. Indomethacin did not change the retinal vascular and depressor responses to hydralazine, 8-(4-chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (a membrane-permeable cGMP analog) and 8-(4-chlorophenylthio)-adenosine-3', 5'-cyclic monophosphate (a membrane-permeable cAMP analog). Treatment with SQ 22536, an adenylyl cyclase inhibitor, but not ODQ, a soluble guanylyl cyclase inhibitor, significantly attenuated the NOR3-induced vasodilation of retinal arterioles. The COX-1 immunoreactivity was found in retinal blood vessels. The retinal blood vessel was faintly stained for soluble guanylyl cyclase, although the apparent immunoreactivities on mesenteric and choroidal blood vessels were observed. These results suggest

  14. Establishing an experimental rat model of photodynamically-induced retinal vein occlusion using erythrosin B

    PubMed Central

    Chen, Wei; Wu, Ying; Zheng, Mi; Gu, Qing; Zheng, Zhi; Xia, Xin

    2014-01-01

    AIM To develop a reliable, reproducible rat model of retinal vein occlusion (RVO) with a novel photosensitizer (erythrosin B) and study the cellular responses in the retina. METHODS Central and branch RVOs were created in adult male rats via photochemically-induced ischemia. Retinal changes were monitored via color fundus photography and fluorescein angiography at 1 and 3h, and 1, 4, 7, 14, and 21d after irradiation. Tissue slices were evaluated histopathologically. Retinal ganglion cell survival at different times after RVO induction was quantified by nuclear density count. Retinal thickness was also observed. RESULTS For all rats in both the central and branch RVO groups, blood flow ceased immediately after laser irradiation and retinal edema was evident at one hour. The retinal detachment rate was 100% at 3h and developed into bullous retinal detachment within 24h. Retinal hemorrhages were not observed until 24h. Clearance of the occluded veins at 7d was observed by fluorescein angiography. Disease manifestation in the central RVO eyes was more severe than in the branch RVO group. A remarkable reduction in the ganglion cell count and retinal thickness was observed in the central RVO group by 21d, whereas moderate changes occurred in the branch RVO group. CONCLUSION Rat RVO created by photochemically-induced ischemia using erythrosin B is a reproducible and reliable animal model for mimicking the key features of human RVO. However, considering the 100% rate of retinal detachment, this animal model is more suitable for studying RVO with chronic retinal detachment. PMID:24790863

  15. Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

    PubMed Central

    Varano, Monica; Mallozzi, Cinzia; Gaddini, Lucia; Formisano, Giuseppe; Pricci, Flavia

    2015-01-01

    Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight. PMID:25688355

  16. Spermidine Oxidation-Mediated Degeneration of Retinal Pigment Epithelium in Rats

    PubMed Central

    Kageyama, Masaaki; Shinomiya, Katsuhiko; Fujita-Koyama, Yukie; Hirai, Shin-ichiro; Katsuta, Osamu; Nakamura, Masatsugu

    2017-01-01

    Retinal pigment epithelium (RPE) degeneration is a crucial event in dry age-related macular degeneration and gyrate atrophy. The polyamine spermidine has been shown to induce RPE cell death in vitro. The present study aimed to establish a novel in vivo model of spermidine-induced RPE degeneration and to determine whether spermidine-induced RPE cell death involves oxidative mechanisms. In this study, spermidine caused ARPE-19 cell death in a concentration-dependent manner. This effect was prevented by removal of serum from the culture medium or treatment with amine oxidase inhibitors, N-acetylcysteine (NAC), or aldehyde dehydrogenase (ALDH). Intravitreal injection of spermidine into rats significantly increased the permeability of the blood-retinal barrier and decreased the amplitudes of scotopic electroretinogram a- and b-waves. Histological analysis revealed that spermidine induced vacuolation, atrophy, and dropout of RPE cells, leading to the disruption of photoreceptor outer segments. Simultaneous intravitreal administration of NAC and ALDH with spermidine prominently inhibited the functional and morphological changes induced by spermidine. In conclusion, this study demonstrated that the intravitreal administration of spermidine induced RPE cell dysfunction and death followed by photoreceptor degeneration in rats. These effects of spermidine are thought to be mediated by oxidative stress and a toxic aldehyde generated during spermidine oxidation. PMID:28367269

  17. Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells.

    PubMed

    Cia, David; Vergnaud-Gauduchon, Juliette; Jacquemot, Nathalie; Doly, Michel

    2014-09-01

    To determine whether the green tea polyphenol epigallocatechin gallate (EGCG) could prevent H(2)O(2)-induced oxidative stress in primary rat retinal pigment epithelial cells. Primary cultures of retinal pigment epithelium (RPE) cells were established from Long-Evans newborn rats. RPE cells were pretreated with various concentrations of EGCG for 24 h before being exposed to hydrogen peroxide (H(2)O(2)) for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was quantified by flow cytometry using propidium iodide (PI). Treatment of RPE cells with EGCG alone does not affect the cell viability up to 50 µM. Exposure of RPE cells to 600 µM H(2)O(2) caused a significant decrease in cell viability; whereas pretreatment with 10, 25, and 50 µM EGCG significantly reduced this decrease in a dose-dependent manner. The proportion of PI-positive cells increased significantly in cultures treated with H(2)O(2) alone; whereas pretreatment of RPE cells with 50 µM EGCG significantly reduced H(2)O(2)-induced RPE cell death. Our study shows that EGCG pretreatment can protect primary rat RPE cells from H(2)O(2)-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H(2)O(2)-induced oxidative stress.

  18. Sodium-calcium exchanger in cultured human retinal pigment epithelium.

    PubMed

    Mangini, N J; Haugh-Scheidt, L; Valle, J E; Cragoe, E J; Ripps, H; Kennedy, B G

    1997-12-01

    Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.

  19. Behavior tests and immunohistochemical retinal response analyses in RCS rats with subretinal implantation of Okayama-University-type retinal prosthesis.

    PubMed

    Alamusi; Matsuo, Toshihiko; Hosoya, Osamu; Tsutsui, Kimiko M; Uchida, Tetsuya

    2013-09-01

    We have developed a photoelectric dye-coupled polyethylene film as a prototype of retinal prosthesis, which we named Okayama University-type retinal prosthesis. The purposes of this study are to conduct behavior tests to assess vision in Royal College of Surgeons (RCS) rats that underwent subretinal implantation of the dye-coupled film and to reveal retinal response to the dye-coupled film by immunohistochemistry. Polyethylene films were made of polyethylene powder at refined purity, and photoelectric dyes were coupled to the film surface at higher density compared with the prototype. Either dye-coupled film or dye-uncoupled plain film used as a control was implanted subretinally from a scleral incision in both eyes of an RCS rat at 6 weeks of the age. Behavior tests 2, 4, 6, and 8 weeks after implantation were conducted by observing head turning or body turning in the direction consistent with clockwise or counterclockwise rotation of a black-and-white-striped drum around a transparent cage housed with the rat. After the behavior tests at 8 weeks, rats' eyes were enucleated to confirm subretinal implantation of the films and processed for immunohistochemistry. In the behavior tests, the number of head turnings consistent with the direction of the drum rotation was significantly larger in RCS rats with dye-coupled- compared with plain-film implantation [P < 0.05, repeated-measure analysis of variance (ANOVA), n = 7]. The number of apoptotic neurons was significantly smaller in eyes with dye-coupled- compared with plain-film implantation (P < 0.05, Mann-Whitney U test, n = 6). In conclusion, subretinal implantation of photoelectric dye-coupled films restored vision in RCS rats and prevented the remaining retinal neurons from apoptosis.

  20. Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina.

    PubMed

    Zhang, Chanjuan; Wang, Zhen; Zhao, Jiayi; Li, Qin; Huang, Cuiqin; Zhu, Lihong; Lu, Daxiang

    2016-05-01

    Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p-c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.

  1. Recognition of mannose 6-phosphate ligands by dystrophic rat retinal pigment epithelium

    SciTech Connect

    Tarnowski, B.; Shepherd, V.; McLaughlin, B.

    1986-05-01

    Retinal pigment epithelium (RPE) phagocytize discarded rod outer segments (ROS) during normal eye function. In the dystrophic rat, an animal model for retinitis pigmentosa in humans, ROS phagocytosis is defective. Dystrophic RPE can phagocytize particles other than ROS, suggesting that the defect may be in the RPE phagocytic recognition. They are currently investigating the recognition markers on RPE in dystrophic rats. In studies using ligand-coated latex beads, no uptake of mannose-coated beads was found in dystrophic rat RPE. They found that dystrophic RPE could specifically phagocytize phosphomannan-coated beads. Studies were begun to examine the presence and function of a phosphomannan receptor (PMR) on dystrophic RPE. ..cap alpha..-Mannosidase, isolated from D. discoideum has been shown to be an efficient ligand for the PMR in fibroblasts and macrophages. It is also recognized by the macrophage mannose receptor. Dystrophic rat RPE and retina explants were placed in culture dishes (5-7/well). /sup 125/I-Labelled ..cap alpha..-mannosidase was added to each well in the presence or absence of 10 mM mannose 6-phosphate (M6P) or yeast mannan (lmg/ml). Explants were incubated at 37/sup 0/ for 2 hr., washed and bound /sup 125/I-mannosidase quantitated. Approximately 2-3% of total counts added were bound to the RPE via a M6P-inhibitable recognition process. The binding to RPE was not blocked by mannan. No mannan or M6P-specific binding was found in retina explants. These results support the findings of specific uptake of phosphomannan-coated beads and demonstrate the presence of a specific PMR on dystrophic RPE phagocytic membranes.

  2. Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors.

    PubMed

    Zhao, Chen; Tao, Zui; Xue, Langyue; Zeng, Yuxiao; Wang, Yi; Xu, Haiwei; Yin, Zheng Qin

    2017-03-01

    In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors.

  3. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

    PubMed Central

    Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

    2016-01-01

    Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases. PMID:27009841

  4. A new fluorescent imaging procedure in vivo for evaluation of the retinal microcirculation in rats.

    PubMed

    Kimura, H; Kiryu, J; Nishiwaki, H; Ogura, Y

    1995-03-01

    We investigated a new method for in vivo evaluation of the retinal microcirculation in rats using a cell-permeant fluorescent dye, acridine orange (AO), which stains cell nuclei and cytoplasm, and a scanning laser ophthalmoscope (SLO). AO, which binds and interacts with DNA and RNA, and thus stains cell nuclei and cytoplasm, was administered intravenously to rats. Fluorescein angiography was performed after administration of the AO, and fundus images were recorded on S-VHS videotape by means of an SLO. Argon laser was used as an exciter of the dye. The retinal vessels were stained with the dye, rendering the retinal microvasculature clearly visible. Cell nuclei and vessel walls were observed as greater fluorescence and lesser fluorescence, respectively. Leukocytes were also observed as highly fluorescent dots moving through the vessels. The results suggest that SLO visualization of AO uptake by cells may be a useful procedure for the evaluation of retinal microcirculation in vivo in rats.

  5. Effect of ATF3-deletion on apoptosis of cultured retinal ganglion cells

    PubMed Central

    Sun, Ming-Ming; Wang, Ya-Chen; Li, Yi; Guo, Xiao-Dan; Chen, Yan-Ming; Zhang, Zhong-Zhi

    2017-01-01

    AIM To investigate the effect of activating transcription factor-3 (ATF3)-deletion on apoptosis of cultured retinal ganglion cells (RGCs). METHODS Three ATF3 siRNA (ATF3-rat-651, ATF3-rat-319, ATF3-rat-520) were constructed, and were transiently transfected into RGC-5 cells. Quantitative real-time polymerase chain reaction (PCR) was used to examine ATF3 expression and the most effective ATF3 siRNA was selected for further studies. Flow cytometry was applied to investigate the effects of ATF3 deletion on RGC-5 apoptosis under elevated hydrostatic pressure. Quantitative real-time PCR and Western blot were performed to validate differentially expressed genes and proteins in ATF3-knockdown RGC-5 cells. RESULTS ATF3 specific siRNA effectively down-regulated ATF3 expression and significantly inhibited cell apoptosis in RGC-5 cells. Quantitative real-time PCR and Western blot confirmed that ATF3 knockdown remarkably decreased Jun-B and increased c-Jun at both mRNA and protein levels in RGC-5 cells. CONCLUSION ATF/cAMP-response element-binding family of transcription factors may be involved in the development of glaucoma and could be novel treatment targets for glaucoma. PMID:28546922

  6. Improved Method of Ink-Gelatin Perfusion for Visualising Rat Retinal Microvessels

    PubMed Central

    Jianbin, Tong; Liang, Huang; Jufang, Huang; Hui, Wang; Dan, Chen; Leping, Zeng; Jin, Zhou; Xuegang, Luo

    2008-01-01

    To visualize completely rat retinal microvessels, the gelatin-ink perfusion condition was systematically optimized using von Willebrand factor (vWf) immunostaining as control. Whether the vessel showed by the new perfusion condition can be used for double label with neurons or glial cells in the same retina was also tested. Our results showed that infusing rats first with 20 ml of 37°C ink plus 3% gelatin at 140% rat mean arterial pressure (MAP), and subsequently with 20 ml of 37°C ink plus 5% gelatin at 180% rat MAP allowed the ink to completely fill the rat retinal microvessels. Rat retinal microvessels labeled by the perfusion method were more in number than that by vWf immunostaining. Moreover, our data, for the first time, displayed that the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue. These data suggest that the improved gelatin-ink perfusion technique is a superior method for morphological characterization of rat retinal microvessels, compatible to the double labeling of glial cells and neurons, and it extends the practical scale of the classic method. PMID:18989466

  7. Beta-adrenoceptor-mediated vasodilation of retinal blood vessels is reduced in streptozotocin-induced diabetic rats.

    PubMed

    Nakazawa, Taisuke; Sato, Ayumi; Mori, Asami; Saito, Maki; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2008-01-01

    We investigated the effects of epinephrine and dopamine on retinal blood vessels in streptozotocin (STZ, 80 mg/kg, i.p.)-treated rats and age-matched control rats to determine whether diabetes mellitus alters the retinal vascular responses to circulating catecholamines. Experiments were performed 6-8 weeks after treatment with STZ or the vehicle. The fundus images were captured with the digital fundus camera system for small animals we developed and diameters of retinal blood vessels contained in the digital images were measured. Epinephrine increased the diameters of retinal blood vessels, but the vasodilator responses were reduced in diabetic rats. Dopamine produced a biphasic retinal vascular response with an initial vasoconstriction followed by a vasodilation. The vasoconstrictor effects of dopamine on retinal arterioles were enhanced in diabetic rats, whereas the difference between the two groups was abolished by treatment with propranolol. The vasodilator effect of isoproterenol, but not of the activator of adenylyl cyclase colforsin, on retinal blood vessels was reduced in diabetic rats. No difference in vasoconstriction of retinal blood vessels to phenylephrine between non-diabetic and diabetic rats was observed. The vasodilator responses of retinal blood vessels to 1,1-dimethyl-4-phenylpiperazinium, a ganglionic nicotinic receptor agonist, were also attenuated in diabetic rats. These results suggest that diabetes mellitus alters the retinal vascular responses to circulating catecholamines and the impairment of vasodilator responses mediated by beta-adrenoceptors contributes to the alteration.

  8. Oxygen tension and gradient measurements in the retinal microvasculature of rats.

    PubMed

    Teng, Pang-Yu; Blair, Norman P; Wanek, Justin; Shahidi, Mahnaz

    2012-03-01

    Oxygen delivery from the retinal vasculature plays a crucial role in maintaining normal retinal metabolic function. Therefore, measurements of retinal vascular oxygen tension (PO(2)) and PO(2) longitudinal gradients (gPO(2)) along retinal blood vessels may help gain fundamental knowledge of retinal physiology and pathological processes. Three-dimensional retinal vascular PO(2) maps were generated in rats by optical section phosphorescence lifetime imaging. A major retinal artery and vein pair, and a smaller blood vessel (microvessel) between them were segmented, and PO(2) along each blood vessel was measured. In each blood vessel, an average PO(2) (mPO(2)) was calculated, and gPO(2) was determined by linear regression analysis. Reproducibility of measurements was assessed by calculating intraclass correlation coefficient (ICC) of repeated measurements. The correlations of mPO(2) and gPO(2) measurements with systemic arterial oxygen tension (P(a)O(2)) and carbon dioxide tension (P(a)CO(2)) was determined. Measurements of mPO(2) and gPO(2) in retinal arteries, microvessels and veins were reproducible (ICC > 0.86; p < 0.01; N = 8), except for retinal arterial gPO(2). Retinal arterial, microvessel and venous mPO(2) were 41 ± 8, 32 ± 8 and 25 ± 7 mmHg, respectively (mean ± SD; N = 27). Retinal arterial mPO(2) was correlated with P(a)O(2) and P(a)CO(2) (R > 0.44; p < 0.03), while retinal microvessel and venous mPO(2) were only correlated with P(a)CO(2) (R > 0.68; p < 0.01). Retinal microvessel gPO(2) (-3.8 ± 1.5 mmHg/100 μm) was significantly steeper (more negative) than venous gPO(2) (0.02 ± 0.43 mmHg/100 μm) (p < 0.01; N = 27), and neither were significantly correlated with P(a)O(2) or P(a)CO(2). Quantitative measurement of mPO(2) and gPO(2) in the retinal microvasculature was demonstrated. A significant decrease in PO(2) was observed along most retinal microvessels, indicative of substantial

  9. Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

    PubMed Central

    Chen, Li; Tao, Yong; Feng, Jing; Jiang, Yan Rong

    2015-01-01

    Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases. PMID:26491547

  10. Unfolded protein response-induced dysregulation of calcium homeostasis promotes retinal degeneration in rat models of autosomal dominant retinitis pigmentosa

    PubMed Central

    Shinde, V; Kotla, P; Strang, C; Gorbatyuk, M

    2016-01-01

    The molecular mechanism of autosomal dominant retinitis pigmentosa (ADRP) in rats is closely associated with a persistently activated unfolded protein response (UPR). If unchecked, the UPR might trigger apoptosis, leading to photoreceptor death. One of the UPR-activated cellular signaling culminating in apoptotic photoreceptor cell death is linked to an increase in intracellular Ca2+. Therefore, we validated whether ADRP retinas experience a cytosolic Ca2+ overload, and whether sustained UPR in the wild-type retina could promote retinal degeneration through Ca2+-mediated calpain activation. We performed an ex vivo experiment to measure intracellular Ca2+ in ADRP retinas as well as to detect the expression levels of proteins that act as Ca2+ sensors. In separate experiments with the subretinal injection of tunicamycin (UPR inducer) and a mixture of calcium ionophore (A231278) and thapsigargin (SERCA2b inhibitor) we assessed the consequences of a sustained UPR activation and increased intracellular Ca2+ in the wild-type retina, respectively, by performing scotopic ERG, histological, and western blot analyses. Results of the study revealed that induced UPR in the retina activates calpain-mediated signaling, and increased intracellular Ca2+ is capable of promoting retinal degeneration. A significant decline in ERG amplitudes at 6 weeks post treatment was associated with photoreceptor cell loss that occurred through calpain-activated CDK5-pJNK-Csp3/7 pathway. Similar calpain activation was found in ADRP rat retinas. A twofold increase in intracellular Ca2+ and up- and downregulations of ER membrane-associated Ca2+-regulated IP3R channels and SERCA2b transporters were detected. Therefore, sustained UPR activation in the ADRP rat retinas could promote retinal degeneration through increased intracellular Ca2+ and calpain-mediated apoptosis. PMID:26844699

  11. Response of Inner Retinal Oxygen Extraction Fraction to Light Flicker Under Normoxia and Hypoxia in Rat

    PubMed Central

    Teng, Pang-yu; Wanek, Justin; Blair, Norman P.; Shahidi, Mahnaz

    2014-01-01

    Purpose. Oxygen extraction fraction (OEF), defined by the ratio of oxygen metabolism (MO2) to delivery (DO2), determines the level of compensation of MO2 by DO2. In the current study, we tested the hypothesis that inner retinal OEF remains unchanged during light flicker under systemic normoxia and hypoxia in rats due to the matching of MO2 and DO2. Methods. Retinal vascular oxygen tension (PO2) measurements were obtained in 10 rats by phosphorescence lifetime imaging. Inner retinal OEF was derived from vascular PO2 based on Fick's principle. Measurements were obtained before and during light flicker under systemic normoxia and hypoxia. The effects of light flicker and systemic oxygenation on retinal vascular PO2 and OEF were determined by ANOVA. Results. During light flicker, retinal venous PO2 decreased (P < 0.01, N = 10), while inner retinal OEF increased (P = 0.02). Under hypoxia, retinal arterial and venous PO2 decreased (P < 0.01), while OEF increased (P < 0.01). The interaction effect was not significant on OEF (P = 0.52), indicating the responses of OEF to light flicker were similar under normoxia and hypoxia. During light flicker, OEF increased from 0.46 ± 0.13 to 0.50 ± 0.11 under normoxia, while under hypoxia, OEF increased from 0.67 ± 0.16 to 0.74 ± 0.14. Conclusions. Inner retinal OEF increased during light flicker, indicating the relative change in DO2 is less than that in MO2 in rats under systemic normoxia and hypoxia. Inner retinal OEF is a potentially useful parameter for assessment of the relative changes of MO2 and DO2 under physiologic and pathologic conditions. PMID:25183761

  12. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats

    PubMed Central

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-01-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc. PMID:24639102

  13. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

    PubMed

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-09-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

  14. Role of cyclooxygenase in vasodilation of retinal blood vessels induced by bradykinin in Brown Norway rats.

    PubMed

    Kojima, Naoki; Saito, Maki; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2009-01-01

    We investigated the role of cyclooxygenase (COX) in bradykinin (BK)-induced vasodilation of retinal blood vessels in Brown Norway rats using a novel in vivo fluorescent fundus imaging technique. Under artificial ventilation, the animals were treated with tetrodotoxin, and the decreased blood pressure and heart rate were adjusted to the normal ranges by infusing pressor agents. Diameters of retinal blood vessels contained in the fluorescent fundus images were measured. Intravenous infusion of BK (1-30 microg/kg/min) increased diameters of retinal blood vessels and decreased blood pressure in a dose-dependent manner. The retinal vasodilator responses to BK were significantly attenuated by treatment with either indomethacin, a non-selective COX inhibitor, or nimesulide, a selective COX-2 inhibitor, but not with SC-560, a selective COX-1 inhibitor. These COX inhibitors failed to affect the depressor responses to BK. Under conditions of nitric oxide (NO) synthase blockade with N(G)-nitro-l-arginine methyl ester, no inhibitory effects of indomethacin on BK-induced vasodilation of retinal blood vessels were observed. These results suggest that the vasodilator effects of BK on retinal blood vessels are partly mediated through a COX-2-dependent pathway in Brown Norway rats. The prostanoid-dependent component of BK-induced retinal vasodilator response seems to be mediated by a mechanism that involves NO.

  15. Vasodilator effects of ibudilast on retinal blood vessels in anesthetized rats.

    PubMed

    Noguchi, Masahiro; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2009-11-01

    Ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-alpha]pyridine) is clinically used as a cerebral vasodilator in Japan. However, the effects of ibudilast on retinal blood vessels have not been fully examined. The aim of this study, therefore, was to examine the effects of ibudilast on retinal blood vessels in rats in vivo. Male Wistar rats (8 to 10 weeks old) were anesthetized with thiobutabarbital (120 mg/kg, intraperitoneally (i.p.)). Retinal vascular images were captured with a fundus camera system for small animals, and the diameter of retinal blood vessels was measured. Ibudilast (0.1 and 1 mg/kg, intravenously (i.v.)) elicited a sustained increase in the diameter of retinal blood vessels and heart rate without altering systemic blood pressure. The effects of ibudilast were significantly reduced by treatment with the nonselective cyclooxygenase inhibitor indomethacin (5 mg/kg, i.p.). These results suggest that ibudilast dilates retinal blood vessels through cyclooxygenase-dependent mechanisms in rats in vivo.

  16. Vasodilator effects of flunarizine on retinal blood vessels in anesthetized rats.

    PubMed

    Noguchi, Masahiro; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2009-12-01

    The aim of this study was to investigate the effects of intravenous administration of flunarizine on the diameter of retinal blood vessels and blood pressure in anesthetized rats and to compare the effects of this antagonist with those of nicardipine and nifedipine. Retinal vascular images were captured with a fundus camera system for small animals and the diameter of retinal blood vessels contained in the images was measured using image-processing softwares on a personal computer. Blood pressure was continuously measured. Flunarizine [1-30 microg/kg, intravenously (i.v.)] dose-dependently increased the diameter of retinal blood vessels without significantly changing systemic blood pressure. Nicardipine (1-30 microg/kg, i.v.) increased the retinal blood vessel diameter but decreased blood pressure in a dose-dependent manner. Nifedipine (10-100 microg/kg, i.v.) failed to dilate the retinal blood vessels, although it produced comparable depressor responses as those to nicardipine. These results suggest that flunarizine selectively acts on the retinal blood vessels rather than on the peripheral resistance vessels. Flunarizine could therefore be considered as a candidate for therapeutic drugs to treat diseases associated with disorders of retinal circulation without severe cardiovascular side-effects.

  17. The neuroprotective effect of hyperbaric oxygen treatment on laser-induced retinal damage in rats

    NASA Astrophysics Data System (ADS)

    Vishnevskia-Dai, Victoria; Belokopytov, Mark; Dubinsky, Galina; Nachum, Gal; Avni, Isaac; Belkin, Michael; Rosner, Mordechai

    2005-04-01

    Retinal damage induced by mechanical trauma, ischemia or laser photocoagulation increases considerably by secondary degeneration processes. The spread of damage may be ameliorated by neuroprotection that is aimed at reducing the extent of the secondary degeneration and promote healing processes. Hyperbaric oxygen (HBO) treatment consists of inspiration of oxygen at higher than one absolute atmospheric pressure. Improved neural function was observed in patients with acute brain trauma or ischemia treated with HBO. This study was designed to evaluate the neuroprotective effect of hyperbaric oxygen (HBO) on laser induced retinal damage in a rat model. Standard argon laser lesions were created in 25 pigmented rats divided into three groups: Ten rats were treated immediately after the irradiation with HBO three times during the first 24 hr followed by 12 consecutive daily treatments. Five rats received a shorter treatment regimen of 10 consecutive HBO treatments. The control group (10 rats) underwent the laser damage with no additional treatment. The retinal lesions were evaluated 20 days after the injury. All outcome measures were improved by the longer HBO treatment (P<0.01). The shorter HBO treatment was less effective, showing an increase only in nuclei density at the central area of lesion (P< 0.01). Hyperbaric oxygen seems to exert a neuroprotective effect on laser-induced retinal damage in a rat model. In the range of HBO exposures studied, longer exposure provides more neuroprotection. These results encourage further evaluation of the potential therapeutic use of hyperbaric oxygen in diseases and injuries of the retina.

  18. Intravenously administered phosphodiesterase 4 inhibitors dilate retinal blood vessels in rats.

    PubMed

    Miwa, Tomoyo; Mori, Asami; Nakahara, Tsutomu; Ishii, Kunio

    2009-01-05

    In the present study, we examined effects of intravenously administered inhibitors of phosphodiesterase 4 (rolipram and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro-20-1724)) and non-selective inhibitor of phosphodiesterases (theophylline) on diameter of retinal blood vessel and fundus (retinal/choroidal) blood flow in rats. Male Wistar rats (8- to 10-week-old) were treated with tetrodotoxin (50 microg/kg, i.v.) to eliminate any nerve activity and prevent the eye movement under artificial ventilation. Methoxamine was used to maintain adequate systemic circulation. Ocular fundus images were captured with an original high-resolution digital fundus camera for small animals. Diameters of retinal blood vessels contained in the digital images were measured using image-processing softwares on a personal computer. Fundus blood flow was measured using a laser Doppler flow meter. Both rolipram (0.01-10 microg/kg/min, i.v.) and Ro-20-1724 (0.01-10 microg/kg/min, i.v.) increased diameters of retinal blood vessels in a dose-dependent manner without significant effect on systemic blood pressure, heart rate and fundus blood flow. The effects of phosphodiesterase 4 inhibitors on retinal arterioles were greater than those on retinal venules. Similarly, theophylline (0.1-10 mg/kg/min, i.v.) dilated retinal blood vessels, whereas it decreased blood pressure and increased heart rate markedly. These results suggest that phosphodiesterase 4 contributes to maintenance of retinal vascular tone. Inhibitors of phosphodiesterase 4 could be considered as a candidate for therapeutic drugs to treat diseases associated with disorders of retinal circulation without severe cardiovascular side-effects.

  19. Displaced retinal ganglion cells in albino and pigmented rats

    PubMed Central

    Nadal-Nicolás, Francisco M.; Salinas-Navarro, Manuel; Jiménez-López, Manuel; Sobrado-Calvo, Paloma; Villegas-Pérez, María P.; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta

    2014-01-01

    We have studied in parallel the population of displaced retinal ganglion cells (dRGCs) and normally placed (orthotopic RGCs, oRGCs) in albino and pigmented rats. Using retrograde tracing from the optic nerve, from both superior colliculi (SC) or from the ipsilateral SC in conjunction with Brn3 and melanopsin immunodetection, we report for the first time their total number and topography as well as the number and distribution of those dRGCs and oRGCs that project ipsi- or contralaterally and/or that express any of the three Brn3 isoforms or melanopsin. The total number of RGCs (oRGCs+dRGCs) is 84,706 ± 1249 in albino and 90,440 ± 2236 in pigmented, out of which 2383 and 2428 are melanopsin positive (m-RGCs), respectively. Regarding dRGCs: i/ albino rats have a significantly lower number of dRGCs than pigmented animals (0.5% of the total number of RGCs vs. 2.5%, respectively), ii/ dRGCs project massively to the contralateral SC, iii/ the percentage of ipsilaterality is higher for dRGCs than for oRGCs, iv/ a higher proportion of ipsilateral dRGCs is observed in albino than pigmented animals, v/ dRGC topography is very specific, they predominate in the equatorial temporal retina, being densest where the oRGCs are densest, vi/ Brn3a detects all dRGCs except half of the ipsilateral ones and those that express melanopsin, vii/ the proportion of dRGCs that express Brn3b or Brn3c is slightly lower than in the oRGC population, viii/ a higher percentage of dRGCs (13% albino, 9% pigmented) than oRGCs (2.6%) express melanopsin, ix/ few m-RGCs (displaced and orthotopic) project to the ipsilateral SC, x/ the topography of m-dRGCs does not resemble the general distribution of dRGCs, xi/ The soma size in m-oRGCs ranges from 10 to 21 μm and in m-dRGCs from 8 to 15 μm, xii/ oRGCs and dRGCs have the same susceptibility to axonal injury and ocular hypertension. Although the role of mammalian dRGCs remains to be determined, our data suggest that they are not misplaced by an

  20. Neuroprotective effect of minocycline in a rat model of branch retinal vein occlusion.

    PubMed

    Sun, Chuan; Li, Xiao-Xin; He, Xiang-Jun; Zhang, Qi; Tao, Yong

    2013-08-01

    Branch retinal vein occlusion (BRVO) is the second most frequent retinal vascular disorder. Currently the first-line therapies for BRVO include anti-VEGF and dexamethasone implant treatment, however, with direct or indirect damage on retinal neurons, it has limited effect in improving patients visual acuity. Therefore, novel treatments with neuroprotective effect for BRVO retina were expected. Minocycline is a semisynthetic, broad spectrum tetracycline antibiotic with high penetration through the blood brain barrier. The neuroprotective effects of minocycline have been shown in various central nervous system (CNS) disease. Since both CNS and retina were composed of neurons and glials, it is reasonable to expect a neuroprotective effect by minocycline for BRVO retina. Therefore, the aim of the present study was to study whether minocycline has neuroprotective effect in branch retinal vein occlusion (BRVO) and the possible underlying molecular basis. We created BRVO in rats using laser photocoagulation. The animals were then randomly divided into 4 groups to evaluate the effect of minocycline: group A: minocycline 45 mg/kg intraperitoneal injection (i.p.), group B: minocycline 90 mg/kg i.p., group C: normal saline i.p., group D: sham injection. Fundus photography and fluorescein angiography (FA) were conducted. The changes in thickness of retinal layers were measured with optical coherence tomography (OCT) in vivo. We found that retinal edema occurred predominantly in the inner retinal layers. Intraperitoneal administration of minocycline significantly ameliorated retinal edema in the early stage of BRVO. We performed Full field Electroretinography (ffERG) to evaluate retinal function and found that the reduction of b wave amplitude decreased in the combined maximal response. The expressional levels of apoptosis related genes (Bax, Bcl-2) and inflammation related genes (IL-1 β, TNF α, MCP-1 and CCR2) were measured by real-time PCR, the results showed that

  1. Organotypic retinal explant cultures as in vitro alternative for diabetic retinopathy studies.

    PubMed

    Valdés, Joaquín; Trachsel-Moncho, Laura; Sahaboglu, Ayse; Trifunović, Dragana; Miranda, María; Ueffing, Marius; Paquet-Durand, François; Schmachtenberg, Oliver

    2016-01-01

    Diabetic retinopathy (DR) is a major cause of vision loss and one of the most common and debilitating complications of diabetes. Research to prevent DR is hindered by a lack of experimental model systems that faithfully reproduce the disease pathology, in particular for type 2 diabetes, which requires prolonged disease progression in animals to develop some hallmarks of DR. Here, we introduce an alternative in vitro model system for DR, based on serum-free, organotypic rodent retinal explant cultures, which allow physiological and pharmacological manipulation of the retina for up to two weeks under tightly controlled conditions. Retinal explant cultures have the advantage of isolating direct neuronal consequences of diabetic conditions from indirect systemic effects mediated via the retinal vasculature or the immune system. Exposed to conditions emulating type 1 or type 2 diabetes, retinal explants displayed elevated cell death rates among inner retinal neurons as well as photoreceptors, with a particularly strong loss of cone photoreceptors. Our results support a direct impact of diabetic conditions on retinal neurons and may help explain color vision defects observed in DR patients. This serum-free in vitro DR model avoids the animal suffering of established DR models and reduces the overall number of animals needed for such research. It should prove useful to study the mechanisms of neuronal cell death caused by DR and to screen for potential future DR treatments.

  2. The angiotensin II receptor antagonist candesartan cilexetil (TCV-116) ameliorates retinal disorders in rats.

    PubMed

    Nagisa, Y; Shintani, A; Nakagawa, S

    2001-07-01

    The results of the EUCLID trial (EURODIAB Controlled Trial of Lisinopril in Insulin-dependent Diabetes Mellitus) highlighted the importance of the renin-angiotensin system in the pathogenesis of diabetic retinopathy. Candesartan cilexetil (TCV-116), a potent angiotensin II (AII) receptor antagonist, has beneficial effects on hypertension as well as on heart, renal and cerebrovascular disease. We aimed to evaluate the effectiveness of candesarten cilexetil in ameliorating retinal disorders induced by hyperglycaemia. We compared retinal vascular endothelial growth factor (VEGF) mRNA expression and the latencies of retinal oscillatory potentials in TCV-116-treated and control groups of stroke-prone spontaneously hypertensive rats with streptozocin (STZ)-induced diabetes. Retinal VEGF mRNA expression was significantly higher and the latencies of oscillatory potentials were significantly elongated in STZ-treated spontaneously hypertensive rats compared with a non-treated spontaneously hypertensive rat group matched for age. These changes were dependent on hyperglycaemia but independent of hypertension. Treatment with TCV-116 (3 mg/kg) significantly diminished retinal VEGF mRNA expression and the latencies of oscillatory potential peaks, but had no effect on plasma glucose concentrations. These results suggest that TCV-116 is effective in preventing the development of diabetic retinopathy already in the early stages.

  3. Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.

    PubMed

    Carr, Amanda-Jayne; Vugler, Anthony A; Hikita, Sherry T; Lawrence, Jean M; Gias, Carlos; Chen, Li Li; Buchholz, David E; Ahmado, Ahmad; Semo, Ma'ayan; Smart, Matthew J K; Hasan, Shazeen; da Cruz, Lyndon; Johnson, Lincoln V; Clegg, Dennis O; Coffey, Pete J

    2009-12-03

    Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

  4. Zingiber officinale attenuates retinal microvascular changes in diabetic rats via anti-inflammatory and antiangiogenic mechanisms

    PubMed Central

    Dongare, Shirish; Mathur, Rajani; Saxena, Rohit; Mathur, Sandeep; Agarwal, Renu; Nag, Tapas C.; Srivastava, Sushma; Kumar, Pankaj

    2016-01-01

    Purpose Diabetic retinopathy is a common microvascular complication of long-standing diabetes. Several complex interconnecting biochemical pathways are activated in response to hyperglycemia. These pathways culminate into proinflammatory and angiogenic effects that bring about structural and functional damage to the retinal vasculature. Since Zingiber officinale (ginger) is known for its anti-inflammatory and antiangiogenic properties, we investigated the effects of its extract standardized to 5% 6-gingerol, the major active constituent of ginger, in attenuating retinal microvascular changes in rats with streptozotocin-induced diabetes. Methods Diabetic rats were treated orally with the vehicle or the ginger extract (75 mg/kg/day) over a period of 24 weeks along with regular monitoring of bodyweight and blood glucose and weekly fundus photography. At the end of the 24-week treatment, the retinas were isolated for histopathological examination under a light microscope, transmission electron microscopy, and determination of the retinal tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and vascular endothelial growth factor (VEGF) levels. Results Oral administration of the ginger extract resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and vascular basement membrane thickness. Improvement in the architecture of the retinal vasculature was associated with significantly reduced expression of NF-κB and reduced activity of TNF-α and VEGF in the retinal tissue in the ginger extract–treated group compared to the vehicle-treated group. Conclusions The current study showed that ginger extract containing 5% of 6-gingerol attenuates the retinal microvascular changes in rats with streptozotocin-induced diabetes through anti-inflammatory and antiangiogenic actions. Although precise molecular targets remain to be determined, 6-gingerol seems to be a potential candidate for further investigation. PMID:27293376

  5. Stimulation of μ-opioid receptors dilates retinal arterioles by neuronal nitric oxide synthase-derived nitric oxide in rats.

    PubMed

    Someya, Eriko; Mori, Asami; Sakamoto, Kenji; Ishii, Kunio; Nakahara, Tsutomu

    2017-03-21

    Opioids contribute to the regulation of cerebral vascular tone. The purpose of this study was to examine the effects of herkinorin, a non-opioid μ-opioid receptor agonist derived from salvinorin A, on blood vessels in the rat retina and to investigate the mechanism underlying the herkinorin-induced retinal vasodilatory response. Ocular fundus images were captured using an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring the diameter of retinal arterioles in the fundus images. Both systemic blood pressure and heart rate were continuously recorded. Herkinorin increased the retinal arteriolar diameter without significantly changing mean blood pressure and heart rate. The retinal vasodilator response to herkinorin was almost completely prevented following treatment with naloxone, a nonselective opioid receptor antagonist and H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective μ-opioid receptor antagonist. N(ω)-nitro-L-arginine methyl ester, a nonselective nitric oxide (NO) synthase inhibitor, or indomethacin, a cyclooxygenase inhibitor, significantly attenuated the herkinorin-induced retinal vasodilator responses. In addition, N(ω)-propyl-L-arginine, an inhibitor of neuronal NO synthase, diminished the herkinorin-induced retinal vasodilator responses. Seven days after an intravitreal injection of N-methyl-d-aspartic acid, loss of inner retinal neurons and abolishment of the retinal vasodilator response to herkinorin were observed. These results suggest that herkinorin dilates rat retinal arterioles through stimulation of retinal μ-opioid receptors. The μ-opioid receptor-mediated retinal vasodilator response is likely mediated by NO generated by neuronal NO synthase. Retinal neurons play an important role in the retinal vasodilator mechanism involving μ-opioid receptors in rats.

  6. Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions.

    PubMed

    Gomes, Francisco L A F; Zhang, Gen; Carbonell, Felix; Correa, José A; Harris, William A; Simons, Benjamin D; Cayouette, Michel

    2011-01-01

    In vivo cell lineage-tracing studies in the vertebrate retina have revealed that the sizes and cellular compositions of retinal clones are highly variable. It has been challenging to ascertain whether this variability reflects distinct but reproducible lineages among many different retinal progenitor cells (RPCs) or is the product of stochastic fate decisions operating within a population of more equivalent RPCs. To begin to distinguish these possibilities, we developed a method for long-term videomicroscopy to follow the lineages of rat perinatal RPCs cultured at clonal density. In such cultures, cell-cell interactions between two different clones are eliminated and the extracellular environment is kept constant, allowing us to study the cell-intrinsic potential of a given RPC. Quantitative analysis of the reconstructed lineages showed that the mode of division of RPCs is strikingly consistent with a simple stochastic pattern of behavior in which the decision to multiply or differentiate is set by fixed probabilities. The variability seen in the composition and order of cell type genesis within clones is well described by assuming that each of the four different retinal cell types generated at this stage is chosen stochastically by differentiating neurons, with relative probabilities of each type set by their abundance in the mature retina. Although a few of the many possible combinations of cell types within clones occur at frequencies that are incompatible with a fully stochastic model, our results support the notion that stochasticity has a major role during retinal development and therefore possibly in other parts of the central nervous system.

  7. Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets

    PubMed Central

    Seiler, M.J.; Aramant, R.B.; Thomas, B.B.; Peng, Q.; Sadda, S.R.; Keirstead, H.S.

    2010-01-01

    The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from E19 rat fetuses expressing human alkaline phosphatase (hPAP), were transplanted to the subretinal space of thirteen S334ter-3 rats with fast retinal degeneration at the age of 0.8 to 1.3 months. Recipients were sacrificed at the age of 1.6 to 11.8 months. Frozen sections were analyzed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for EM. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 8 rats at the age of 5.3 to 11.8 months. - All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between −2.8 and −3.4 log cd/m2. No such responses were found in age-matched S334ter-3 rats without transplant, or in sham surgeries. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells presumably of host origin. Conclusions: Synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration. PMID:20105230

  8. Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.

    PubMed

    Roth, Steven; Dreixler, John C; Mathew, Biji; Balyasnikova, Irina; Mann, Jacob R; Boddapoti, Venkat; Xue, Lai; Lesniak, Maciej S

    2016-06-01

    We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.

  9. Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats

    PubMed Central

    Roth, Steven; Dreixler, John C.; Mathew, Biji; Balyasnikova, Irina; Mann, Jacob R.; Boddapati, Venkat; Xue, Lai; Lesniak, Maciej S.

    2016-01-01

    Purpose We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. Methods Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. Results Eyes injected with hypoxic BMSC–conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. Conclusions The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect. PMID:27367588

  10. Measuring retinal blood flow in rats using Doppler optical coherence tomography without knowing eyeball axial length

    SciTech Connect

    Liu, Wenzhong; Yi, Ji; Chen, Siyu; Jiao, Shuliang; Zhang, Hao F.

    2015-09-15

    Purpose: Doppler optical coherence tomography (OCT) is widely used for measuring retinal blood flow. Existing Doppler OCT methods require the eyeball axial length, in which empirical values are usually used. However, variations in the axial length can create a bias unaccounted for in the retinal blood flow measurement. The authors plan to develop a Doppler OCT method that can measure the total retinal blood flow rate without requiring the eyeball axial length. Methods: The authors measured the retinal blood flow rate using a dual-ring scanning protocol. The small and large scanning rings entered the eye at different incident angles (small ring: 4°; large ring: 6°), focused on different locations on the retina, and detected the projected velocities/phase shifts along the probing beams. The authors calculated the ratio of the projected velocities between the two rings, and then used this ratio to estimate absolute flow velocity. The authors tested this method in both Intralipid phantoms and in vivo rats. Results: In the Intralipid flow phantom experiments, the preset and measured flow rates were consistent with the coefficient of determination as 0.97. Linear fitting between preset and measured flow rates determined the fitting slope as 1.07 and the intercept as −0.28. In in vivo rat experiments, the measured average total retinal blood flow was 7.02 ± 0.31μl/min among four wild-type rats. The authors’ measured flow rates were consistent with results in the literature. Conclusions: By using a dual-ring scanning protocol with carefully controlled incident angle difference between the two scanning rings in Doppler OCT, the authors demonstrated that it is feasible to measure the absolute retinal blood flow without knowing the eyeball axial length.

  11. Photopic ERG negative response from amacrine cell signaling in RCS rat retinal degeneration.

    PubMed

    Machida, Shigeki; Raz-Prag, Dorit; Fariss, Robert N; Sieving, Paul A; Bush, Ronald A

    2008-01-01

    The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity

  12. Co-culture of Retinal and Endothelial Cells Results in the Modulation of Genes Critical to Retinal Neovascularization.

    PubMed

    Kumar, Ravindra; Harris-Hooker, Sandra; Kumar, Ritesh; Sanford, Gary

    2011-11-23

    Neovascularization (angiogenesis) is a multistep process, controlled by opposing regulatory factors, which plays a crucial role in several ocular diseases. It often results in vitreous hemorrhage, retinal detachment, neovascularization glaucoma and subsequent vision loss. Hypoxia is considered to be one of the key factors to trigger angiogenesis by inducing angiogenic factors (like VEGF) and their receptors mediated by hypoxia inducible factor-1 (HIF-1α) a critical transcriptional factor. Another factor, nuclear factor kappa B (NFκB) also regulates many of the genes required for neovascularization, and can also be activated by hypoxia. The aim of this study was to elucidate the mechanism of interaction between HRPC and HUVEC that modulates a neovascularization response. Human retinal progenitor cells (HRPC) and human umbilical vein endothelial cells (HUVEC) were cultured/co-cultured under normoxia (control) (20% O2) or hypoxia (1% O2) condition for 24 hr. Controls were monolayer cultures of each cell type maintained alone. We examined the secretion of VEGF by ELISA and influence of conditioned media on blood vessel growth (capillary-like structures) via an angiogenesis assay. Total RNA and protein were extracted from the HRPC and HUVEC (cultured and co-cultured) and analyzed for the expression of VEGF, VEGFR-2, NFκB and HIF-1α by RT-PCR and Western blotting. The cellular localization of NFκB and HIF-1α were studied by immunofluorescence and Western blotting. We found that hypoxia increased exogenous VEGF expression 4-fold in HRPC with a further 2-fold increase when cultured with HUVEC. Additionally, we found that hypoxia induced the expression of the VEGF receptor (VEGFR-2) for HRPC co-cultured with HUVEC. Hypoxia treatment significantly enhanced (8- to 10-fold higher than normoxia controls) VEGF secretion into media whether cells were cultured alone or in a co-culture. Also, hypoxia was found to result in a 3- and 2-fold increase in NFκB and HIF-1α m

  13. Correlation in the Discharges of Neighboring Rat Retinal Ganglion Cells During Prenatal Life

    NASA Astrophysics Data System (ADS)

    Maffei, Lamberto; Galli-Resta, Lucia

    1990-04-01

    The spontaneous discharges of neighboring retinal ganglion cells were recorded simultaneously in anesthetized prenatal rats between embryonic days 18 and 21. We report here that in the majority of cases the firings of neighboring retinal ganglion cells are strongly correlated during prenatal life. Correlation in the discharges of neighboring cells during development has long been suggested as a way to consolidate synaptic connections with a target cell onto which they converge, a model first proposed by Hebb. Correlation in the activities of neighboring neurons in the retina could be the basis of developmental processes such as refinement of retinotopic maps in the brain and segregation of the inputs from the two eyes.

  14. A rat retinal damage model predicts for potential clinical visual disturbances induced by Hsp90 inhibitors

    SciTech Connect

    Zhou, Dan; Liu, Yuan; Ye, Josephine; Ying, Weiwen; Ogawa, Luisa Shin; Inoue, Takayo; Tatsuta, Noriaki; Wada, Yumiko; Koya, Keizo; Huang, Qin; Bates, Richard C.; Sonderfan, Andrew J.

    2013-12-01

    In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24 h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6 h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6 h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential. - Highlights: • In human trials some Hsp90 inhibitors cause visual disorders, others do not. • Prolonged inhibition of Hsp90 in the rat eye results in photoreceptor cell death. • Retina/plasma ratio and retinal

  15. Three-dimensional Optical Coherence Tomography Imaging of Retinal Sheet Implants in Live Rats

    PubMed Central

    Seiler, Magdalene J.; Rao, Bin; Aramant, Robert B.; Yu, Lingfeng; Wang, Qiang; Kitayama, Eric; Pham, Sylvia; Yan, Fengrong; Chen, Zhongping; Keirstead, Hans S.

    2010-01-01

    Purpose To obtain three-dimensional images from retinal transplants in live animals and evaluate the placement and structural quality of the transplants. Methods Donor retinal sheets were isolated from E19 fetuses of transgenic rats expressing human alkaline phosphatase (hPAP), and transplanted to the subretinal space of 19–56 d old S334ter-3 rat recipients with fast retinal degeneration (average age at surgery 32d). A total of 143 rats were imaged 1 day – 2.8 months after surgery, using a Fourier domain optical coherence tomography (FDOCT) system, with an axial resolution of 3.5 micron. The CCD A-line integration time was set at 200 μs for better visualization of degenerated retina. After targeting the transplant area, 139 or 199 consecutive slices were scanned. Projection images and movies of the retinal transplant area were computed and later compared with histology. Results OCT scans identified 137 of 141 transplants as a thickening of the degenerated retina. OCT indicated the laminar structure of the transplants and surgical defects, such as RPE/choroid damage with an accuracy rate between 83 and 99%. Three-dimensional projections showed the transplant position in the retina in relation to the optic disc. Histology of transplants by hPAP and hematoxylin-eosin staining was correlated with the OCT results. Conclusions Optical Coherence Tomography is an excellent tool to image retinal layers in a live rat. This procedure helps to evaluate the placement and quality of the transplants in the living eye. PMID:20219535

  16. Human organotypic retinal cultures (HORCs) as a chronic experimental model for investigation of retinal ganglion cell degeneration.

    PubMed

    Osborne, Andrew; Hopes, Marina; Wright, Phillip; Broadway, David C; Sanderson, Julie

    2016-02-01

    There is a growing need for models of human diseases that utilise native, donated human tissue in order to model disease processes and develop novel therapeutic strategies. In this paper we assessed the suitability of adult human retinal explants as a potential model of chronic retinal ganglion cell (RGC) degeneration. Our results confirmed that RGC markers commonly used in rodent studies (NeuN, βIII Tubulin and Thy-1) were appropriate for labelling human RGCs and followed the expected differential expression patterns across, as well as throughout, the macular and para-macular regions of the retina. Furthermore, we showed that neither donor age nor post-mortem time (within 24 h) significantly affected the initial expression levels of RGC markers. In addition, the feasibility of using human post mortem donor tissue as a long-term model of RGC degeneration was determined with RGC protein being detectable up to 4 weeks in culture with an associated decline in RGC mRNA and significant, progressive, apoptotic labelling of NeuN(+) cells. Differences in RGC apoptosis might have been influenced by medium compositions indicating that media constituents could play a role in supporting axotomised RGCs. We propose that using ex vivo human explants may prove to be a useful model for testing the effectiveness of neuroprotective strategies.

  17. The Toxic Effect of ALLN on Primary Rat Retinal Neurons.

    PubMed

    Li, Na; Shang, Lei; Wang, Shu-Chao; Liao, Lv-Shuang; Chen, Dan; Huang, Ju-Fang; Xiong, Kun

    2016-10-01

    N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 μM, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 μM but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 μM. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 μM after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 μM, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.

  18. Neonatal systemic inflammation in rats alters retinal vessel development and simulates pathologic features of retinopathy of prematurity.

    PubMed

    Hong, Hye Kyoung; Lee, Hyun Ju; Ko, Jung Hwa; Park, Ji Hyun; Park, Ji Yeon; Choi, Chang Won; Yoon, Chang-Hwan; Ahn, Seong Joon; Park, Kyu Hyung; Woo, Se Joon; Oh, Joo Youn

    2014-05-15

    Alteration of retinal angiogenesis during development leads to retinopathy of prematurity (ROP) in preterm infants, which is a leading cause of visual impairment in children. A number of clinical studies have reported higher rates of ROP in infants who had perinatal infections or inflammation, suggesting that exposure of the developing retina to inflammation may disturb retinal vessel development. Thus, we investigated the effects of systemic inflammation on retinal vessel development and retinal inflammation in neonatal rats. To induce systemic inflammation, we intraperitoneally injected 100 μl lipopolysaccharide (LPS, 0.25 mg/ml) or the same volume of normal saline in rat pups on postnatal days 1, 3, and 5. The retinas were extracted on postnatal days 7 and 14, and subjected to assays for retinal vessels, inflammatory cells and molecules, and apoptosis. We found that intraperitoneal injection of LPS impaired retinal vessel development by decreasing vessel extension, reducing capillary density, and inducing localized overgrowth of abnormal retinal vessels and dilated peripheral vascular ridge, all of which are characteristic findings of ROP. Also, a large number of CD11c+ inflammatory cells and astrocytes were localized in the lesion of abnormal vessels. Further analysis revealed that the number of major histocompatibility complex (MHC) class IIloCD68loCD11bloCD11chi cells in the retina was higher in LPS-treated rats compared to controls. Similarly, the levels of TNF-α, IL-1β, and IL-12a were increased in LPS-treated retina. Also, apoptosis was increased in the inner retinal layer where retinal vessels are located. Our data demonstrate that systemic LPS-induced inflammation elicits retinal inflammation and impairs retinal angiogenesis in neonatal rats, implicating perinatal inflammation in the pathogenesis of ROP.

  19. [Ameliorative effects on retinal disorder in diabetic SHRSP (stroke-prone spontaneously hypertensive rat)].

    PubMed

    Nagisa, Yasutaka; Shintani, Asae; Nakagawa, Shizue

    2002-10-01

    The results of the EUCLID highlighted the importance of the renin-angiotensin system in the pathogenesis of diabetic retinopathy. We aimed to evaluate the effectiveness of candesartan cilexetil(TCV-116), a potent angiotensin II receptor antagonist, in ameliorating retinal disorders in stroke-prone spontaneously hypertensive rats(SHRSP) with storeptozotocin(STZ)-induced diabetes. Retinal VEGF mRNA expression was significantly higher and the latencies of oscillatory potentials were significantly elongated in STZ-treated SHRSP compared with a non-treated SHRSP group matched for age. Treatment with TCV-116(3 mg/kg) significantly diminished retinal VEGF mRNA expression and the latencies of oscillatory potentials, but had no effect on plasma glucose concentrations. These results suggest that TCV-116 is effective in preventing the development of diabetic retinopathy already in the early stages.

  20. Neuroprotective effects of antibodies on retinal ganglion cells in an adolescent retina organ culture.

    PubMed

    Bell, Katharina; Wilding, Corina; Funke, Sebastian; Perumal, Natarajan; Beck, Sabine; Wolters, Dominik; Holz-Müller, Jana; Pfeiffer, Norbert; Grus, Franz H

    2016-10-01

    Glaucoma, a neurodegenerative disease, is characterized by a progressive loss of retinal ganglion cells (rgc). Up- and down-regulated autoantibody immunoreactivities in glaucoma patients have been demonstrated. Previous studies showed protective effects of down-regulated antibodies [gamma (γ)-synuclein and glial fibrillary acidic protein [GFAP]) on neuroretinal cells. The aim of this study was to test these protective antibody effects on rgc in an organ culture model and to get a better understanding of cell-cell interactions of the retina in the context of the protective effect. We used an adolescent retinal organ culture (pig) with an incubation time of up to 4 days. Retinal explants were incubated with different antibodies for 24 h (anti-GFAP, anti-γ-synuclein and anti-myoglobin antibody as a control). Brn3a and TUNEL staining were performed. We also conducted glutamine synthetase staining and quantification of the retinal explants. Mass spectrometry analyses were performed as well as protein analyses via microarray. We detected a continuous decrease of rgc/mm in the retinal explants throughout the 4 days of incubation with increased TUNEL rgc staining. Immunohistochemical analyses showed a protective effect of anti-γ-synuclein (increased rgc/mm of 41%) and anti-GFAP antibodies (increased rgc/mm of 37%). Mass spectrometric, microarray and immunohistochemical analyses demonstrated Müller cell involvement and decreased endoplasmic reticulum stress response in the antibody-treated retinae. We could detect that the tested antibodies have a protective effect on rgc which seems to be the result of reduced stress levels in the retina as well as a shift of glutamine synthetase localization in the endfeet of the Müller cells towards the inner retinal layer. Loss of retinal ganglion cells (rgc) in glaucoma leads to blindness. Several antibodies are down-regulated in glaucoma patients. Our aim was to test if these antibodies have a protective effect of rgc in a

  1. Pharmacological evidence for the presence of functional beta(3)-adrenoceptors in rat retinal blood vessels.

    PubMed

    Mori, Asami; Miwa, Tomoyo; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2010-08-01

    The aim of this study was to examine whether stimulation of beta(3)-adrenoceptors dilates rat retinal blood vessels and how diabetes affects the vasodilator responses. Images of ocular fundus were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate (HR) were continuously recorded. The beta(3)-adrenoceptor agonist CL316243 (0.3-10 microg/kg/min, i.v.) increased diameter of retinal arterioles (at 10 microg/kg/min, a 31% increase) and decreased mean blood pressure (at 10 microg/kg/min, a 21% decrease) in a dose-dependent manner. CL316243 produced a small but significant increase in HR (at 10 microg/kg/min, a 9% increase). Both SR59230A (1 mg/kg, i.v.) and L-748337 (50 microg/kg, i.v.), beta(3)-adrenoceptor antagonists, significantly prevented CL316243-induced retinal vasodilator responses. Similar observations were made with another beta(3)-adrenoceptor agonist, BRL37344. The beta(2)-adrenoceptor agonist salbutamol also increased diameter of retinal arterioles (at 10 microg/kg/min, a 43% increase), whereas the drug produced greater decrease in blood pressure (at 10 microg/kg/min, a 46% decrease) and increase in HR (at 10 microg/kg/min, a 16% increase), compared with beta(3)-adrenoceptor agonists. The retinal vasodilator responses to CL316243 and BRL37344 observed under blockade of beta(1)/beta(2)-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 microg/kg/min infusion) were unaffected 2 weeks after induction of diabetes by the combination of streptozotocin treatment and D: -glucose feeding. On the other hand, the vasodilator responses to salbutamol of retinal arterioles were significantly reduced in diabetic rats. These results suggest that stimulation of beta(3)-adrenoceptors causes the vasodilation of retinal arterioles in vivo and the vasodilator responses are

  2. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

    PubMed Central

    Sonoda, Shozo; Spee, Christine; Barron, Ernesto; Ryan, Stephen J; Kannan, Ram; Hinton, David R

    2009-01-01

    We provide our detailed, standardized in vitro protocol for culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized, functional monolayer. Disruption of polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease, and in vitro evaluation of novel therapies. The protocol, which takes approximately 6 weeks, describes the culture of RPE from human fetal donor eyes, and the differentiation of these cells into a polarized monolayer with high transepithelial resistance, and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells, and culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye. PMID:19373231

  3. Palmitoylethanolamide treatment reduces retinal inflammation in streptozotocin-induced diabetic rats.

    PubMed

    Paterniti, Irene; Di Paola, Rosanna; Campolo, Michela; Siracusa, Rosalba; Cordaro, Marika; Bruschetta, Giuseppe; Tremolada, Gemma; Maestroni, Anna; Bandello, Francesco; Esposito, Emanuela; Zerbini, Gianpaolo; Cuzzocrea, Salvatore

    2015-12-15

    Although the pathogenesis of diabetic retinopathy (DR) is still insufficiently understood, new evidences indicate 'retinal inflammation' as an important player in the pathogenesis of the complication. Accordingly, common sets of upregulated inflammatory cytokines are found in serum, vitreous and aqueous samples obtained from subjects with DR, and these cytokines can have multiple interactions to impact the pathogenesis of the disease. Thus, based on previously published data, we investigated the effects of Palmitoylethanolamide (PEA), an endogenous lipid amide that belongs to the N-acyl-ethanolamines family, on DR in streptozotocin (STZ)-induced diabetic rats. PEA (10mg/kg) was administered orally daily starting 3 days after the iv administration of STZ. The rats were killed 15 and 60day later and eyes were enucleated to evaluate, through immunohistochemical analysis, the key inflammatory events involved in the breakdown of blood retinal barrier (BRB). Immunohistochemical analysis confirmed the presence of VEGF, ICAM-1, nitrotyrosine (a marker of peroxynitrite), and tight junctions in the retina of STZ-treated rats. Of interest, the extent of injury was significantly reduced after treatment with PEA. Altogether, this study provides the first evidence that PEA attenuates the degree of inflammation while preserving the blood-retinal barrier in rats with experimental DR.

  4. Channelrhodopsin-2 gene transduced into retinal ganglion cells restores functional vision in genetically blind rats.

    PubMed

    Tomita, Hiroshi; Sugano, Eriko; Isago, Hitomi; Hiroi, Teru; Wang, Zhuo; Ohta, Emi; Tamai, Makoto

    2010-03-01

    To test the hypothesis that transduction of the channelrhodopsin-2 (ChR2) gene, a microbial-type rhodopsin gene, into retinal ganglion cells of genetically blind rats will restore functional vision, we recorded visually evoked potentials and tested the experimental rats for the presence of optomotor responses. The N-terminal fragment of the ChR2 gene was fused to the fluorescent protein Venus and inserted into an adeno-associated virus to make AAV2-ChR2V. AAV2-ChR2V was injected intravitreally into the eyes of 6-month-old dystrophic RCS (rdy/rdy) rats. Visual function was evaluated six weeks after the injection by recording visually evoked potentials (VEPs) and testing optomotor responses. The expression of ChR2V in the retina was investigated histologically. We found that VEPs could not be recorded from 6-month-old dystrophic RCS rats that had not been injected with AAV2-ChR2V. In contrast, VEPs were elicited from RCS rats six weeks after injection with AAV2-ChR2V. The VEPs were recorded at stimulation rates <20Hz, which was the same as that of normal rats. Optomotor responses were also significantly better after the AAV2-ChR2V injection. Expression of ChR2V was observed mainly in the retinal ganglion cells. These findings demonstrate that visual function can be restored in blind rats by transducing the ChR2V gene into retinal ganglion cells.

  5. Measurement of retinal blood flow in the rat by combining Doppler Fourier-domain optical coherence tomography with fundus imaging

    NASA Astrophysics Data System (ADS)

    Werkmeister, René M.; Vietauer, Martin; Knopf, Corinna; Fürnsinn, Clemens; Leitgeb, Rainer A.; Reitsamer, Herbert; Gröschl, Martin; Garhöfer, Gerhard; Vilser, Walthard; Schmetterer, Leopold

    2014-10-01

    A wide variety of ocular diseases are associated with abnormalities in ocular circulation. As such, there is considerable interest in techniques for quantifying retinal blood flow, among which Doppler optical coherence tomography (OCT) may be the most promising. We present an approach to measure retinal blood flow in the rat using a new optical system that combines the measurement of blood flow velocities via Doppler Fourier-domain optical coherence tomography and the measurement of vessel diameters using a fundus camera-based technique. Relying on fundus images for extraction of retinal vessel diameters instead of OCT images improves the reliability of the technique. The system was operated with an 841-nm superluminescent diode and a charge-coupled device camera that could be operated at a line rate of 20 kHz. We show that the system is capable of quantifying the response of 100% oxygen breathing on the retinal blood flow. In six rats, we observed a decrease in retinal vessel diameters of 13.2% and a decrease in retinal blood velocity of 42.6%, leading to a decrease in retinal blood flow of 56.7%. Furthermore, in four rats, the response of retinal blood flow during stimulation with diffuse flicker light was assessed. Retinal vessel diameter and blood velocity increased by 3.4% and 28.1%, respectively, leading to a relative increase in blood flow of 36.2%;. The presented technique shows much promise to quantify early changes in retinal blood flow during provocation with various stimuli in rodent models of ocular diseases in rats.

  6. Measurement of retinal blood flow in the rat by combining Doppler Fourier-domain optical coherence tomography with fundus imaging.

    PubMed

    Werkmeister, René M; Vietauer, Martin; Knopf, Corinna; Fürnsinn, Clemens; Leitgeb, Rainer A; Reitsamer, Herbert; Gröschl, Martin; Garhöfer, Gerhard; Vilser, Walthard; Schmetterer, Leopold

    2014-01-01

    A wide variety of ocular diseases are associated with abnormalities in ocular circulation. As such, there is considerable interest in techniques for quantifying retinal blood flow, among which Doppler optical coherence tomography (OCT) may be the most promising. We present an approach to measure retinal blood flow in the rat using a new optical system that combines the measurement of blood flow velocities via Doppler Fourier-domain optical coherence tomography and the measurement of vessel diameters using a fundus camera-based technique. Relying on fundus images for extraction of retinal vessel diameters instead of OCT images improves the reliability of the technique. The system was operated with an 841-nm superluminescent diode and a charge-coupled device camera that could be operated at a line rate of 20 kHz. We show that the system is capable of quantifying the response of 100% oxygen breathing on the retinal blood flow. In six rats, we observed a decrease in retinal vessel diameters of 13.2% and a decrease in retinal blood velocity of 42.6%, leading to a decrease in retinal blood flow of 56.7%. Furthermore, in four rats, the response of retinal blood flow during stimulation with diffuse flicker light was assessed. Retinal vessel diameter and blood velocity increased by 3.4% and 28.1%, respectively, leading to a relative increase in blood flow of 36.2%. The presented technique shows much promise to quantify early changes in retinal blood flow during provocation with various stimuli in rodent models of ocular diseases in rats.

  7. Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor.

    PubMed

    Kumar, Ravindra; Dutt, Kamla

    2006-01-01

    One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D(2)D(3), D(4) receptors; protein kinase-C alpha (PKCalpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFalpha. Brain-derived neurotrophic factor, which is expressed in vivo in RPE (D407), was

  8. Visual functional effects of constant blue light in a retinal degenerate rat model.

    PubMed

    Thomas, Biju B; Seiler, Magdalene J; Aramant, Robert B; Samant, Deedar; Qiu, Guanting; Vyas, Neil; Arai, Shinichi; Chen, Zhenhai; Sadda, SriniVas R

    2007-01-01

    Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line-3 rat pups (21 days old) were exposed for 5-6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S-antigen, red-green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree(-1) in 38-day-old line 3 rats. With a 0.125 cycles degree(-1) stimulus, the head tracking performance of 80-day-old BL rats were similar to that of 220-day-old no-BL-treated line-3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light-damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.

  9. Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells.

    PubMed

    Portugal, Camila Cabral; Miya, Vivian Sayuri; Calaza, Karin da Costa; Santos, Rochelle Alberto Martins; Paes-de-Carvalho, Roberto

    2009-01-01

    Vitamin C is transported in the brain by sodium vitamin C co-transporter 2 (SVCT-2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT-2 and the uptake and release of [(14)C] ascorbate in chick retinal cells. SVCT-2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT-2 was expressed in cultured retinal neurons, but not in glial cells. [(14)C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium-free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate-stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l-beta-threo-benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [(3)H] D-aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2-Carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7-initroquinoxaline-2,3-dione (DNQX) or (5R,2S)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). However, DNQX, but not MK-801 or 2-amino-5-phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non-NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2-bis (2-aminophenoxy) ethane-N',N',N',N',-tetraacetic acid tetrakis (acetoxy-methyl ester) (BAPTA-AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT-2, and the release can be stimulated by NMDA or non-NMDA glutamate receptors.

  10. Measurement of retinal blood flow rate in diabetic rats: disparity between techniques due to redistribution of flow.

    PubMed

    Leskova, Wendy; Watts, Megan N; Carter, Patsy R; Eshaq, Randa S; Harris, Norman R

    2013-04-26

    Reports of altered retinal blood flow in experimental models of type I diabetes have provided contrasting results, which leads to some confusion as to whether flow is increased or decreased. The purpose of our study was to evaluate early diabetes-induced changes in retinal blood flow in diabetic rats, using two distinctly different methods. Diabetes was induced by injection of streptozotocin (STZ), and retinal blood flow rate was measured under anesthesia by a microsphere infusion technique, or by an index of flow based on the mean circulation time between arterioles and venules. Measurements in STZ rats were compared to age-matched nondiabetic controls. In addition, the retinal distribution of fluorescently-labeled red blood cells (RBCs) was viewed by confocal microscopy in excised flat mounts. Retinal blood flow rate was found to decrease by approximately 33% in the STZ rats compared to controls (P < 0.001) as assessed by the microsphere technique. However, in striking contrast, the mean circulation time through the retina was found to be almost 3× faster in the STZ rats (P < 0.01). This contradiction could be explained by flow redistribution through the superficial vessels of the diabetic retina, with this possibility supported by our observation of significantly fewer RBCs flowing through the deeper capillaries. We conclude that retinal blood flow rate is reduced significantly in the diabetic rat, with a substantial decrease of flow through the capillaries due to shunting of blood through the superficial layer, allowing rapid transit from arterioles to venules.

  11. Electrophysiology Alterations in Primary Visual Cortex Neurons of Retinal Degeneration (S334ter-line-3) Rats

    PubMed Central

    Chen, Ke; Wang, Yi; Liang, Xiaohua; Zhang, Yihuai; Ng, Tsz Kin; Chan, Leanne Lai Hang

    2016-01-01

    The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration. PMID:27225415

  12. Methylprednisolone fails to preserve retinal ganglion cells and visual function after ocular ischemia in rats.

    PubMed

    Dimitriu, Cornelia; Bach, Michael; Lagrèze, Wolf A; Jehle, Thomas

    2008-11-01

    Methylprednisolone (MP) is commonly used to treat traumatic optic neuropathy and optic neuritis, but its benefit in terms of neuronal survival remains controversial. The aim of this study was to investigate the effects of MP on retinal ganglion cell (RGC) survival and visual function after ischemia in rats. Ocular ischemia was induced by elevated intraocular pressure. Rats were treated with NaCl, 1 mg/kg/d, or 30 mg/kg/d intraperitoneal MP over 3 days. RGCs were labeled retrogradely 4 days after ischemia and were quantified 6 days later. Post-ischemic retinal function was assessed by scotopic and photopic electroretinography (ERG). Optic nerve function was investigated on days 4 and 10 after ischemia by visual evoked potentials (VEPs). Compared with nonischemic eyes, ischemia reduced RGCs with NaCl to 47% +/- 3% (mean +/- SEM) and to 46% +/- 3% and 43% +/- 6% with 1 mg/kg/d and 30 mg/kg/d MP. ERG did not differ significantly for any parameter among the three groups. Four days after ischemia, the VEPs of rats receiving any dose of MP were significantly higher than the control. VEPs in both steroid groups fell to control levels 10 days after ischemia. Treatment with MP did not improve RGC survival or retinal function. The VEP showed a short-term benefit because of steroids.

  13. BDNF-treated retinal progenitor sheets transplanted to degenerate rats: improved restoration of visual function.

    PubMed

    Seiler, Magdalene J; Thomas, Biju B; Chen, Zhenhai; Arai, Shinichi; Chadalavada, Sridhar; Mahoney, Melissa J; Sadda, Srinivas R; Aramant, Robert B

    2008-01-01

    The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a "low light" intensity of 1cd/m2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects--either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs--still needs to be elucidated.

  14. Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses.

    PubMed

    Gaddini, Lucia; Balduzzi, Maria; Campa, Alessandro; Esposito, Giuseppe; Malchiodi-Albedi, Fiorella; Patrono, Clarice; Matteucci, Andrea

    2017-09-25

    Retinal tissue can receive incidentalγ-rays exposure during radiotherapy either of tumors of the eye and optic nerve or of head-and-neck tumors, and during medical diagnostic procedures. Healthy retina is therefore at risk of suffering radiation-related side effects and the knowledge of pathophysiological response of retinal cells to ionizing radiations could be useful to design possible strategies of prevention and management of radiotoxicity. In this study, we have exploited an in vitro model (primary rat retinal cell culture) to study an array of biological effects induced on retinal neurons by γ-rays. Most of the different cell types present in retinal tissue - either of the neuronal or glial lineages - are preserved in primary rat retinal cultures. Similar to the retina in situ, neuronal cells undergo in vitro a maturational development shown by the formation of polarized neuritic trees and operating synapses. Since 2 Gy is the incidental dose received by the healthy retina per fraction when the standard treatment is delivered to the brain, retina cell cultures have been exposed to 1 or 2 Gy of γ-rays at different level of neuronal differentiation in vitro: days in vitro (DIV)2 or DIV8. At DIV9, retinal cultures were analysed in terms of viability, apoptosis and characterized by immunocytochemistry to identify alterations in neuronal differentiation. After irradiation at DIV2, MTT assay revealed an evident loss of cell viability and βIII-tubulin immunostaining highlighted a marked neuritic damage, indicating that survived neurons showed an impaired differentiation. Differentiated cultures (DIV8) appeared to be more resistant with respect to undifferentiated, DIV2 cultures, both in terms of cell viability and differentiation. Apoptosis evaluated with TUNEL assay showed that irradiation at both DIV2 and DIV8 induced a significant increase in the apoptotic rate. To further investigate the effects of γ-rays on retinal neurons, we evaluated the expression of

  15. Protective effects of catalase on retinal ischemia/reperfusion injury in rats.

    PubMed

    Chen, Baihua; Tang, Luosheng

    2011-11-01

    Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100mmHg for 1h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6h, 24h, 72 h and 5d after injury. Hydrogen peroxide (H(2)O(2)), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H(2)O(2), MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p<0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be

  16. Insulin treatment normalizes retinal neuroinflammation but not markers of synapse loss in diabetic rats.

    PubMed

    Masser, Dustin R; VanGuilder Starkey, Heather D; Bixler, Georgina V; Dunton, Wendy; Bronson, Sarah K; Freeman, Willard M

    2014-08-01

    Diabetic retinopathy is one of the leading causes of blindness in developed countries, and a majority of patients with type I and type II diabetes will develop some degree of vision loss despite blood glucose control regimens. The effects of different insulin therapy regimens on early metabolic, inflammatory and neuronal retinal disease processes such as retinal neuroinflammation and synapse loss have not been extensively investigated. This study compared 3 months non-diabetic and streptozotocin (STZ)-induced diabetic Sprague Dawley rats. Diabetic rats received either no insulin treatment, systemic insulin treatment beginning after 1 week uncontrolled diabetes (early intervention, 11 weeks on insulin), or after 1.5 months uncontrolled diabetes (late intervention, 6 weeks on insulin). Changes in both whole animal metabolic and retinal inflammatory markers were prevented by early initiation of insulin treatment. These metabolic and inflammatory changes were also normalized by the later insulin intervention. Insulin treatment begun 1 week after diabetes induction ameliorated loss of retinal synapse markers. Synapse markers and presumably synapse numbers were equivalent in uncontrolled diabetes and when insulin treatment began at 1.5 months of diabetes. These findings are in agreement with previous demonstrations that retinal synapses are lost within 1 month of uncontrolled diabetes and suggest that synapses are not regained with glycemic control and restoration of insulin signaling. However, increased expression of metabolic and inflammatory markers associated with diabetes was reversed in both groups of insulin treatment. This study also emphasizes the need for insulin treatment groups in diabetic retinopathy studies to provide a more faithful modeling of the human condition. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Photovoltaic restoration of sight with high visual acuity in rats with retinal degeneration

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Goetz, G.; Lorach, H.; Mandel, Y.; Smith, R.; Boinagrov, D.; Lei, X.; Kamins, T.; Harris, J.; Mathieson, K.; Sher, A.

    2015-03-01

    Patients with retinal degeneration lose sight due to gradual demise of photoreceptors. Electrical stimulation of the surviving retinal neurons provides an alternative route for delivery of visual information. Subretinal photovoltaic arrays with 70μm pixels were used to convert pulsed near-IR light (880-915nm) into pulsed current to stimulate the nearby inner retinal neurons. Network-mediated responses of the retinal ganglion cells (RGCs) could be modulated by pulse width (1-20ms) and peak irradiance (0.5-10 mW/mm2). Similarly to normal vision, retinal response to prosthetic stimulation exhibited flicker fusion at high frequencies, adaptation to static images, and non-linear spatial summation. Spatial resolution was assessed in-vitro and in-vivo using alternating gratings with variable stripe width, projected with rapidly pulsed illumination (20-40Hz). In-vitro, average size of the electrical receptive fields in normal retina was 248+/-59μm - similar to their visible light RF size: 249+/-44μm. RGCs responded to grating stripes down to 67μm using photovoltaic stimulation in degenerate rat retina, and 28μm with visible light in normal retina. In-vivo, visual acuity in normally-sighted controls was 29+/-5μm/stripe, vs. 63+/-4μm/stripe in rats with subretinal photovoltaic arrays, corresponding to 20/250 acuity in human eye. With the enhanced acuity provided by eye movements and perceptual learning in human patients, visual acuity might exceed the 20/200 threshold of legal blindness. Ease of implantation and tiling of these wireless arrays to cover a large visual field, combined with their high resolution opens the door to highly functional restoration of sight.

  18. Chemical stimulation of rat retinal neurons: feasibility of an epiretinal neurotransmitter-based prosthesis

    NASA Astrophysics Data System (ADS)

    Inayat, Samsoon; Rountree, Corey M.; Troy, John B.; Saggere, Laxman

    2015-02-01

    Objective. No cure currently exists for photoreceptor degenerative diseases, which cause partial or total blindness in millions of people worldwide. Electrical retinal prostheses have been developed by several groups with the goal of restoring vision lost to these diseases, but electrical stimulation has limitations. It excites both somas and axons, activating retinal pathways nonphysiologically, and limits spatial resolution because of current spread. Chemical stimulation of retinal ganglion cells (RGCs) using the neurotransmitter glutamate has been suggested as an alternative to electrical stimulation with some significant advantages. However, sufficient scientific data to support developing a chemical-based retinal prosthesis is lacking. The goal of this study was to investigate the feasibility of a neurotransmitter-based retinal prosthesis and determine therapeutic stimulation parameters. Approach. We injected controlled amounts of glutamate into rat retinas from the epiretinal side ex vivo via micropipettes using a pressure injection system and recorded RGC responses with a multielectrode array. Responsive units were identified using a spike rate threshold of 3 Hz. Main results. We recorded both somal and axonal units and demonstrated successful glutamatergic stimulation across different RGC subtypes. Analyses show that exogenous glutamate acts on RGC synapses similar to endogenous glutamate and, unlike electrical prostheses, stimulates only RGC somata. The spatial spread of glutamate stimulation was ˜ 290 μm from the injection site, comparable to current electrical prostheses. Further, the glutamate injections produced spatially differential responses in OFF, ON, and ON-OFF RGC subtypes, suggesting that differential stimulation of the OFF and ON systems may be possible. A temporal resolution of 3.2 Hz was obtained, which is a rate suitable for spatial vision. Significance. We provide strong support for the feasibility of an epiretinal neurotransmitter

  19. Glucocorticoid-induced leucine zipper overexpression inhibits lipopolysaccharide-induced retinal inflammation in rats.

    PubMed

    Gu, Ruiping; Lei, Boya; Shu, Qinmeng; Li, Gang; Xu, Gezhi

    2017-02-24

    Glucocorticoid-induced leucine zipper (GILZ) mediates several effects of glucocorticoids and has important anti-inflammatory properties. Here, we explored the role of GILZ in inhibiting retinal inflammation. Endotoxin-induced uveitis (EIU) was established in rats by intravitreal injection of lipopolysaccharide (LPS). GILZ levels decreased in the EIU retina at 4 h after LPS injection and slowly recovered within 24 h. Retinal GILZ was downregulated by recombinant lentivirus-delivered short-hairpin RNA targeting GILZ (shRNA-GILZ-rLV) and upregulated by recombinant lentivirus-mediated GILZ overexpression (Oe-GILZ-rLV). GILZ silencing attenuated the anti-inflammatory effects of intravitreal injection of triamcinolone acetonide (TA) in the EIU retina, as demonstrated by increased retinal interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1and intercellular cell adhesion molecule-1 expression at 18 h after TA injection. A Bio-Plex cytokine assay and western blotting demonstrated that GILZ overexpression inhibited the effects of LPS, downregulating retinal IL-1β, MCP-1, MIP-1α, and IL-17 and inhibiting LPS-induced activation of the retinal toll-like receptor 4-myeloid differentiation factor 88 signaling pathway. At 48 and 72 h after LPS injection, the clinical score of inflammation was significantly lower in Oe-GILZ-rLV-transfected eyes than in blank-rLV-transfected eyes. Histological examination showed a 67.85% reduction of infiltrating inflammatory cells in the anterior chamber and a 58.97% reduction in vitreous cavity of Oe-GILZ-rLV transfected eyes at 48 h after LPS injection. Taken together, our results suggest that GILZ is a novel therapeutic target for the treatment of retinal inflammatory diseases.

  20. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    PubMed

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  1. Astaxanthin Inhibits Expression of Retinal Oxidative Stress and Inflammatory Mediators in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Yeh, Po-Ting; Huang, Hsin-Wei; Yang, Chung-May; Yang, Wei-Shiung; Yang, Chang-Hao

    2016-01-01

    Purpose We evaluated whether orally administered astaxanthin (AST) protects against oxidative damage in the ocular tissues of streptozotocin (STZ)-induced diabetic rats. Methods and Results Fifty 6-week-old female Wistar rats were randomly assigned to receive an injection of STZ to induce diabetes (n = 40) or to remain uninduced (n = 10). The diabetic rats were randomly selected into four groups and they were separately administered normal saline, 0.6 mg/kg AST, 3 mg/kg AST, or 0.5 mg/kg lutein daily for eight weeks. Retinal functions of each group were evaluated by electroretinography. The expression of oxidative stress and inflammatory mediators in the ocular tissues was then assessed by immunohistochemistry, western blot analysis, ELISA, RT-PCR, and electrophoretic mobility shift assay (EMSA). Retinal functions were preserved by AST and lutein in different levels. Ocular tissues from AST- and lutein-treated rats had significantly reduced levels of oxidative stress mediators (8-hydroxy-2'-deoxyguanosine, nitrotyrosine, and acrolein) and inflammatory mediators (intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and fractalkine), increased levels of antioxidant enzymes (heme oxygenase-1 and peroxiredoxin), and reduced activity of the transcription factor nuclear factor-kappaB (NF-κB). Conclusion The xanthophyll carotenoids AST and lutein have neuroprotective effects and reduce ocular oxidative stress, and inflammation in the STZ diabetic rat model, which may be mediated by downregulation of NF-κB activity. PMID:26765843

  2. In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.

    PubMed

    Bakondi, Benjamin; Lv, Wenjian; Lu, Bin; Jones, Melissa K; Tsai, Yuchun; Kim, Kevin J; Levy, Rachelle; Akhtar, Aslam Abbasi; Breunig, Joshua J; Svendsen, Clive N; Wang, Shaomei

    2016-03-01

    Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.

  3. In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

    PubMed Central

    Bakondi, Benjamin; Lv, Wenjian; Lu, Bin; Jones, Melissa K; Tsai, Yuchun; Kim, Kevin J; Levy, Rachelle; Akhtar, Aslam Abbasi; Breunig, Joshua J; Svendsen, Clive N; Wang, Shaomei

    2016-01-01

    Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (RhoS334) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of RhoS334, which prevented retinal degeneration and improved visual function. PMID:26666451

  4. ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

    PubMed

    Paudel, Sharada; Kim, Yeoun-Hee; Huh, Man-Il; Kim, Song-Ja; Chang, Yongmin; Park, Young Jeung; Lee, Kyoo Won; Jung, Jae-Chang

    2013-04-01

    Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro-form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10-specific siRNA, the level of mature ADAM10 decreased. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X-treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X-treated cultures. N-cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N-cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation.

  5. Lycium barbarum polysaccharides promotes in vivo proliferation of adult rat retinal progenitor cells

    PubMed Central

    Wang, Hua; Lau, Benson Wui-Man; Wang, Ning-li; Wang, Si-ying; Lu, Qing-jun; Chang, Raymond Chuen-Chung; So, Kwok-fai

    2015-01-01

    Lycium barbarum is a widely used Chinese herbal medicine prescription for protection of optic nerve. However, it remains unclear regarding the effects of Lycium barbarum polysaccharides, the main component of Lycium barbarum, on in vivo proliferation of adult ciliary body cells. In this study, adult rats were intragastrically administered low- and high-dose Lycium barbarum polysaccharides (1 and 10 mg/kg) for 35 days and those intragastrically administered phosphate buffered saline served as controls. The number of Ki-67-positive cells in rat ciliary body in the Lycium barbarum polysaccharides groups, in particular low-dose Lycium barbarum polysaccharides group, was significantly greater than that in the phosphate buffered saline group. Ki-67-positive rat ciliary body cells expressed nestin but they did not express glial fibrillary acidic protein. These findings suggest that Lycium barbarum polysaccharides can promote the proliferation of adult rat retinal progenitor cells and the proliferated cells present with neuronal phenotype. PMID:26889185

  6. Failure of ascorbate to protect against broadband blue light-induced retinal damage in rat.

    PubMed

    Wu, J; Chen, E; Söderberg, P G

    1999-10-01

    Excessive generation of free radicals due to light absorption is proposed as the most likely mechanism for photochemical retinal damage. The observed reduction of green light-induced retinal injury after ascorbate treatment is believed to be an antioxidative effect. The aim of the present study was to evaluate the possible protection of ascorbate against blue light-induced photoreceptor damage. Cyclic light-reared albino rats were injected intraperitoneally with either ascorbate (1 mg/g body weight) or, as placebo, physiological saline 24 h before and just prior to exposure to blue light. After 20-22 h of dark adaptation, two groups of the rats were exposed in pairs to the blue light (400-480 nm) for 6 h at an average irradiance of 0.7 W/m(2) in the cage. Six days after light exposure, all rats were killed and retinal samples were analyzed. Diffuse blue light irradiation resulted in an uneven distribution of damage in the retina. As judged from the pathological changes in the retina irradiated, no microscopic difference was observed between the two groups. The preserved thickness of the outer nuclear layer was on average 61.3% in the ascorbate-treated and 66.4% in the placebo-treated group. The photoreceptor loss was not significantly different between the two groups. The ascorbate did not protect the retina from blue-light induced damage. This favors the assumption that the mechanisms for blue light-induced retinal damage might differ from that for green light.

  7. In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina.

    PubMed

    Lee, Joo Yong; Hwang, Yoonha; Kim, Ji Hyun; Kim, Yu Sun; Jung, Bok Kyoung; Kim, Pilhan; Lee, Heuiran

    2016-06-01

    The purpose of this study was to evaluate longitudinal gene expression patterns by retinal imaging using a modified custom-built confocal laser-scanning microscope in experimental rats after intravitreal injection of recombinant adeno-associated virus 2 (rAAV2-green fluorescent protein [GFP]). Ten 9-week-old Wistar rats were divided into two groups: experimental group (group 1) that received a rAAV2-GFP intravitreal injection and control group (group 2) that received a vehicle. After anesthesia using a Zoletil intraperitoneal injection, 8 μL rAAV2-GFP in group 1 or vehicle in group 2 was injected intravitreally using a 33-G Hamilton syringe. In vivo fluorescence retinal images were acquired under anesthesia at 2, 4, 6, and 13 days after rAAV or vehicle delivery. Differences in GFP fluorescence were identified starting from day 2 after the intravitreal injection of rAAV2-GFP in group 1. Between days 4 and 6, the intensity and area of fluorescence in the retina began to increase and peaked at day 13. Based on the pattern of GFP expression, the axon of the nerve fiber layer ganglion cell was identified. In group 2, eyes treated with the vehicle showed a small amount of autofluorescence in a limited area for up to 2 weeks, with no increase in intensity during this period. In vivo retinal imaging confirmed gene expression within 2 weeks after the intravitreal injection of rAAV2-GFP. Gene transfer and expression in the rat retina occurs quickly in 2 days and appears to peak within 2 weeks of gene delivery. In vivo retinal imaging may be a useful noninvasive tool to continuously monitor gene expression in the retina over time.

  8. Tempol protects against intravitreous indocyanine green-induced retinal damage in rats.

    PubMed

    Thaler, Sebastian; Voykov, Bogomil; Willmann, Gabriel; Fiedorowicz, Michal; Rejdak, Robert; Gekeler, Florian; May, C Albrecht; Schatz, Andreas; Schuettauf, Frank

    2012-11-01

    Indocyanine green (ICG) has been widely used as a vital dye for macular surgery. However, ICG can be toxic to retinal cells. Here we evaluate whether tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a free radical scavenger, can protect against ICG-induced retinal damage in rats. Brown Norway rats received intravitreal injections of ICG 0.5 % or BSS as controls. Tempol (20 mg/kg BW) or PBS as a control was administered intraperitoneally 24 h and 30 min before ICG and once daily for 7 consecutive days. Tempol was detected in the retina using electron paramagnetic resonance (EPR) spectroscopy. One week after ICG injections, the effects of tempol on retinal toxicity were assessed by retinal ganglion cell (RGC) back-labeling and by light microscopy. Electroretinography (ERG) was performed after 1 and 2 weeks. ICG administration reduced RGC numbers by 17 % (1,943 ± 45 vs. 2,342 ± 31 RGCs/mm(2)). Tempol treatment rescued RGCs in a significant manner (2,258 ± 36, p < 0.01) and diminished morphological changes detected by light microscopy. ICG-injected eyes showed a significant reduction of ERG potentials only in PBS-treated animals (V(max) 530 ± 145 µV vs. 779 ± 179 µV, p = 0.0052), but not in the tempol-treated group. Tempol significantly attenuates ICG-induced toxicity in rat retinas and may therefore be considered for further evaluation as accompanying treatment in ICG-assisted chromovitrectomy.

  9. Differential Expression of Three T-Type Calcium Channels in Retinal Bipolar Cells in Rat

    PubMed Central

    Hu, Caiping; Bi, Anding; Pan, Zhuo-Hua

    2009-01-01

    Retinal bipolar cells convey visual information from photoreceptors to retinal third-order neurons, amacrine and ganglion cells, with graded potentials through diversified cell types. To understand the possible role of voltage-dependent T-type Ca2+ currents in retinal bipolar cells, we investigated the pharmacological and biophysical properties of T-type Ca2+ currents in acutely dissociated retinal cone bipolar cells from rat using whole-cell patch-clamp recordings. We observed a broad group of cone bipolar cells with prominent T-type Ca2+ currents (T-rich) and another group with prominent L-type Ca2+ currents (L-rich). Based on the pharmacological and biophysical properties of the T-type Ca2+ currents, T-rich cone bipolar cells could be divided into three subgroups. Each subgroup appeared to express a single dominant T-type Ca2+ channel subunit. The T-type calcium currents could generate low threshold regenerative potentials or spikes. Our results suggest that T-type Ca2+ channels may play an active and distinct signaling role in second-order neurons of the visual system, in contrast to the common signaling by L-rich bipolar cells. PMID:19275782

  10. Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures.

    PubMed

    Silva, Thayane Martins; França, Guilherme Rapozeiro; Ornelas, Isis Moraes; Loiola, Erick Correia; Ulrich, Henning; Ventura, Ana Lucia Marques

    2015-06-01

    When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y1 are involved in the growth of glial cells. UTPγS but not ADPβS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA(+)) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.

  11. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    PubMed

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Light-dependent changes in outer retinal water diffusion in rats in vivo

    PubMed Central

    Bissig, David

    2012-01-01

    Purpose To test the hypothesis that in rats, intraretinal light-dependent changes on diffusion-weighted magnetic resonance imaging (MRI) in vivo are consistent with known retinal layer-specific physiology. Methods In male Sprague-Dawley rats, retinal morphology (thickness, extent, surface area, volume) and intraretinal profiles of the apparent diffusion coefficient (ADC, i.e., water mobility) parallel and perpendicular to the optic nerve were measured in vivo using quantitative MRI methods during light and dark stimulation. Results The parallel ADC in the posterior half of the avascular, photoreceptor-dominated outer retina was significantly higher in light than dark, and this pattern was reversed (dark>light) in the anterior outer retina. The perpendicular ADC in the posterior outer retina was similar in light and dark, but was significantly higher in dark than light in the anterior outer retina. No light-dark changes in the inner retina were noted. Conclusions We identified light-dependent intraretinal diffusion changes that reflected established stimulation-based changes in outer retinal hydration. These findings are expected to motivate future applications of functional diffusion-based MRI in blinding disorders of the outer retina. PMID:23129976

  13. Long-term Characterization of Retinal Degeneration in Royal College of Surgeons Rats Using Spectral-Domain Optical Coherence Tomography

    PubMed Central

    Ryals, Renee C.; Andrews, Michael D.; Datta, Shreya; Coyner, Aaron S.; Fischer, Cody M.; Wen, Yuquan; Pennesi, Mark E.; McGill, Trevor J.

    2017-01-01

    Purpose Prospective treatments for age-related macular degeneration and inherited retinal degenerations are commonly evaluated in the Royal College of Surgeons (RCS) rat before translation into clinical application. Historically, retinal thickness obtained through postmortem anatomic assessments has been a key outcome measure; however, utility of this measurement is limited because it precludes the ability to perform longitudinal studies. To overcome this limitation, the present study was designed to provide a baseline longitudinal quantification of retinal thickness in the RCS rat by using spectral-domain optical coherence tomography (SD-OCT). Methods Horizontal and vertical linear SD-OCT scans centered on the optic nerve were captured from Long-Evans control rats at P30, P60, P90 and from RCS rats between P17 and P90. Total retina (TR), outer nuclear layer+ (ONL+), inner nuclear layer (INL), and retinal pigment epithelium (RPE) thicknesses were quantified. Histologic sections of RCS retina obtained from P21 to P60 were compared to SD-OCT images. Results In RCS rats, TR and ONL+ thickness decreased significantly as compared to Long-Evans controls. Changes in INL and RPE thickness were not significantly different between control and RCS retinas. From P30 to P90 a subretinal hyperreflective layer (HRL) was observed and quantified in RCS rats. After correlation with histology, the HRL was identified as disorganized outer segments and the location of accumulated debris. Conclusions Retinal layer thickness can be quantified longitudinally throughout the course of retinal degeneration in the RCS rat by using SD-OCT. Thickness measurements obtained with SD-OCT were consistent with previous anatomic thickness assessments. This study provides baseline data for future longitudinal assessment of therapeutic agents in the RCS rat. PMID:28253400

  14. Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

    PubMed

    Bray, A F; Cevallos, R R; Gazarian, K; Lamas, M

    2014-11-07

    Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain

  15. Retinal changes in rats flown on Cosmos 936 - A cosmic ray experiment

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Corbett, R.; Turnbill, C.; Black, S.; Dayhoff, D.; Mcgourty, J.; Lee, R.; Harrison, G.; Savik, L.

    1980-01-01

    Ten rats, five centrifuged during flight to simulate gravity and five stationary in flight and experiencing hypogravity, orbited the Earth. No differences were noted between flight-stationary and flight-centrifuged animals, but changes were seen between these two groups and ground controls. Morphological alterations were observed comparable to those in the experiment flown on Cosmos 782 and to the retinal cells exposed to high-energy particles at Berkeley. Affected cells in the outer nuclear layer showed swelling, clearing of cytoplasm, and disruption of the membranes. Tissue channels were again found, similar to those seen on 782. After space flight, preliminary data indicated an increase in cell size in montages of the nuclear layer of both groups of flight animals. This experiment shows that weightlessness and environmental conditions other than cosmic radiation do not contribute to the observed damage of retinal cells.

  16. A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness

    NASA Astrophysics Data System (ADS)

    Maya-Vetencourt, José Fernando; Ghezzi, Diego; Antognazza, Maria Rosa; Colombo, Elisabetta; Mete, Maurizio; Feyen, Paul; Desii, Andrea; Buschiazzo, Ambra; di Paolo, Mattia; di Marco, Stefano; Ticconi, Flavia; Emionite, Laura; Shmal, Dmytro; Marini, Cecilia; Donelli, Ilaria; Freddi, Giuliano; Maccarone, Rita; Bisti, Silvia; Sambuceti, Gianmario; Pertile, Grazia; Lanzani, Guglielmo; Benfenati, Fabio

    2017-06-01

    The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

  17. A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness

    PubMed Central

    Antognazza, Maria Rosa; Colombo, Elisabetta; Mete, Maurizio; Feyen, Paul; Desii, Andrea; Buschiazzo, Ambra; Di Paolo, Mattia; Di Marco, Stefano; Ticconi, Flavia; Emionite, Laura; Shmal, Dmytro; Marini, Cecilia; Donelli, Ilaria; Freddi, Giuliano; Maccarone, Rita; Bisti, Silvia; Sambuceti, Gianmario; Pertile, Grazia; Lanzani, Guglielmo; Benfenati, Fabio

    2017-01-01

    The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of Retinitis pigmentosa. Electrophysiological and behavioral analyses reveal a prosthesis-dependent recovery of light-sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness. PMID:28250420

  18. Retinal changes in rats flown on Cosmos 936 - A cosmic ray experiment

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Corbett, R.; Turnbill, C.; Black, S.; Dayhoff, D.; Mcgourty, J.; Lee, R.; Harrison, G.; Savik, L.

    1980-01-01

    Ten rats, five centrifuged during flight to simulate gravity and five stationary in flight and experiencing hypogravity, orbited the Earth. No differences were noted between flight-stationary and flight-centrifuged animals, but changes were seen between these two groups and ground controls. Morphological alterations were observed comparable to those in the experiment flown on Cosmos 782 and to the retinal cells exposed to high-energy particles at Berkeley. Affected cells in the outer nuclear layer showed swelling, clearing of cytoplasm, and disruption of the membranes. Tissue channels were again found, similar to those seen on 782. After space flight, preliminary data indicated an increase in cell size in montages of the nuclear layer of both groups of flight animals. This experiment shows that weightlessness and environmental conditions other than cosmic radiation do not contribute to the observed damage of retinal cells.

  19. A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness.

    PubMed

    Maya-Vetencourt, José Fernando; Ghezzi, Diego; Antognazza, Maria Rosa; Colombo, Elisabetta; Mete, Maurizio; Feyen, Paul; Desii, Andrea; Buschiazzo, Ambra; Di Paolo, Mattia; Di Marco, Stefano; Ticconi, Flavia; Emionite, Laura; Shmal, Dmytro; Marini, Cecilia; Donelli, Ilaria; Freddi, Giuliano; Maccarone, Rita; Bisti, Silvia; Sambuceti, Gianmario; Pertile, Grazia; Lanzani, Guglielmo; Benfenati, Fabio

    2017-03-06

    The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

  20. Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats.

    PubMed

    Fu, Yan; Hou, Baoke; Weng, Chuanhuang; Liu, Weiping; Dai, Jiaman; Zhao, Congjian; Yin, Zheng Qin

    2017-04-21

    Following retinal degeneration, retinal remodeling can cause neuronal microcircuits to undergo structural alterations, which particularly affect the dendrites of bipolar cells. However, the mechanisms and functional consequences of such changes remain unclear. Here, we used Royal College of Surgeon (RCS) rats as a model of retinal degeneration, to study structural changes in rod bipolar cells (RBCs) and the underlying mechanisms of these changes. We found that, with retinal degeneration, RBC dendrites extended into the outer nuclear layer (ONL) of the retina, and the ectopic dendrites formed synapses with the remaining photoreceptors. This ectopic neuritogenesis was associated with brain-derived neurotrophic factor (BDNF) - expression of which was negatively regulated by miR-125b-5p. Overexpression of miR-125b-5p in the retinae of RCS rats diminished RBC ectopic dendrites, and compromised the b-wave of the flash electroretinogram (ERG). In contrast, down-regulation of miR-125b-5p (or exogenous BDNF treatment) increased RBC ectopic dendrites, and improved b-wave. Furthermore, we showed that the regulation of ectopic neuritogenesis by BDNF occurred via the downstream modulation of the TrkB-CREB signaling pathway. Based on these findings, we conclude that ectopic dendrites are likely to be providing functional benefits and that, in RCS rats, miR-125b-5p regulates ectopic neuritogenesis by RBCs through modulation of the BDNF-TrkB-CREB pathway. This suggests that therapies that reduce miR-125b-5p expression could be beneficial in human retinal degenerative disease.

  1. Changes in Retinal Morphology, Electroretinogram and Visual Behavior after Transient Global Ischemia in Adult Rats

    PubMed Central

    Zhao, Ying; Yu, Bo; Xiang, Yong-Hui; Han, Xin-Jia; Xu, Ying; So, Kwok-Fai; Xu, An-Ding; Ruan, Yi-Wen

    2013-01-01

    The retina is a light-sensitive tissue of the central nervous system that is vulnerable to ischemia. The pathological mechanism underlying retinal ischemic injury is not fully understood. The purpose of this study was to investigate structural and functional changes of different types of rat retinal neurons and visual behavior following transient global ischemia. Retinal ischemia was induced using a 4-vessel occlusion model. Compared with the normal group, the number of βIII-tubulin positive retinal ganglion cells and calretinin positive amacrine cells were reduced from 6 h to 48 h following ischemia. The number of recoverin positive cone bipolar cells transiently decreased at 6 h and 12 h after ischemia. However, the fluorescence intensity of rhodopsin positive rod cells and fluorescent peanut agglutinin positive cone cells did not change after reperfusion. An electroretinogram recording showed that the a-wave, b-wave, oscillatory potentials and the photopic negative response were completely lost during ischemia. The amplitudes of the a- and b-waves were partially recovered at 1 h after ischemia, and returned to the control level at 48 h after reperfusion. However, the amplitudes of oscillatory potentials and the photopic negative response were still reduced at 48 h following reperfusion. Visual behavior detection showed there was no significant change in the time spent in the dark chamber between the control and 48 h group, but the distance moved, mean velocity in the black and white chambers and intercompartmental crosses were reduced at 48 h after ischemia. These results indicate that transient global ischemia induces dysfunction of retinal ganglion cells and amacrine cells at molecular and ERG levels. However, transient global ischemia in a 17 minute duration does not appear to affect photoreceptors. PMID:23776500

  2. Glial reaction to photoelectric dye-based retinal prostheses implanted in the subretinal space of rats.

    PubMed

    Tamaki, Takayuki; Matsuo, Toshihiko; Hosoya, Osamu; Tsutsui, Kimiko M; Uchida, Tetsuya; Okamoto, Kazuo; Uji, Akihito; Ohtsuki, Hiroshi

    2008-01-01

    We have designed a new type of retinal prosthesis using polyethylene films coupled with photoelectric dye molecules that absorb light and convert photon energy to electric potentials. An extruded-blown film of high-density polyethylene was used as the original polyethylene film. Recrystallized film was made by recrystallization from the melting of the original polyethylene film. A photoelectric dye,2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to the two types of polyethylene films through amide linkages. Samples of the original dye-coupled film, the dye-coupled recrystallized film, and the dye-uncoupled plain film were implanted in the subretinal space of normal adult rats. Frozen sections were cut from the eyes enucleated at 1 week or 1 month and were either stained with hematoxylin and eosin, stained immunohistochemically for glial fibrillary acidic protein (GFAP), or processed for in situ apoptosis detection. The results revealed that retinal tissue damage was negligible with no inflammatory cells and few apoptotic cells. GFAP was significantly up-regulated in retinal sites with the implantation of all types of polyethylene films at 1 week, compared with the adjacent retinal sites (P < 0.005, analysis of variance). The GFAP up-regulation was also present at 1 month for the plain film and dye-coupled recrystallized film (P < 0.05). Glial cell encirclement around the films increased significantly between 1 week and 1 month (P = 0.023, two-factor analysis of variance) but was not significantly different among the three types of polyethylene films (P = 0.4531). These results showed evidence of glial reactions to the photoelectric dye-coupled polyethylene films implanted into the subretinal space of rat eyes and also proved their basic biological safety.

  3. Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model.

    PubMed

    Danias, John; Shen, Fran; Kavalarakis, Manolis; Chen, Bin; Goldblum, David; Lee, Kevin; Zamora, Maria-Florencia; Su, YanLing; Brodie, Scott E; Podos, Steven M; Mittag, Thom

    2006-02-01

    Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP

  4. Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model.

    PubMed Central

    Danias, John; Shen, Fran; Kavalarakis, Manolis; Chen, Bin; Goldblum, David; Lee, Kevin; Zamora, Maria-Florencia; Su, YanLing; Brodie, Scott E; Podos, Steven M; Mittag., Thom

    2006-01-01

    Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar(N=5) and Brown-Norway(B-N)(N=7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats(N=7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. A group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs from contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared with contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (>30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP

  5. [Effect of premature birth on retinal vascular development in the neonatal rat].

    PubMed

    Yang, Xiang-min; Li, Rong; Wang, Yu-sheng; Chu, Zhao-jie; Gao, Xiang

    2013-08-01

    To study the effects of premature birth on the development of rat retinal vasculature. Experimental study. Sixty pregnant Sprague-Dawley rats were divided into four groups: bacterial lipopolysaccharide-induced preterm group (LPS group), RU-486 induced preterm group (RP group), cesarean section induced preterm group (CP group), and the normal delivery rats as the control group. The weight of rats from each group was recorded until postnatal day 21. On postnatal day 4, 7, 10 and 14 (P4, P7, P10 and P14), the retina of right eye was dissected and whole-mounted. Each premature group was divided into two subgroups based on the number of rats in each litter, the small subgroup (6-8 rats per litter, group 1) and the large subgroup (14-18 rats per litter, group 2). The development of retinal vascularization process was observed on P4, P7 and P10 (n = 6).Independent t test, one-way ANOVA and LSD-t test were used to analyzed the results. The weight of premature rats in LPS, CP and RP groups was significantly lower than that in the normal group within postnatal 21 days (LSD-t test: all P < 0.05). On the P4 and P7 in LPS group, the proportions of retinal superficial vascularized area of newborn rats [(0.47 ± 0.02) % ,(0.63 ± 0.04)%] were less than that in the normal group [(0.57 ± 0.04) % , (0.74 ± 0.05)%] (t4 d = 6.427, P 4 d = 0.000;t7 d = 5.111, P 7 d = 0.000). On the P4 and P7 in RP group, this proportions [(0.49 ± 0.04) %,(0.65 ± 0.04)%] were less than that in the normal group [(0.57 ± 0.04) %, (0.74 ± 0.05)%] (t4 d = 4.469, P 4 d = 0.000;t7 d = 2.491, P 7 d = 0.022). On P4, P7 and P10 in CP group, this proportions [(0.49 ± 0.05) %, (0.61 ± 0.05) %, (0.94 ± 0.03)%] were also less than that in the normal group[ (0.57 ± 0.04) %, (0.74 ± 0.05) %, (0.97 ± 0.02)%] (t4 d = 4.044, P 4 d = 0.001;t7 d = 6.011, P 7 d = 0.000; t 10 d = 2.331, P 10 d = 0.030). Retinal superficial vascularization completed on P14 in all groups. On the P4 and P7 in LPS group, the

  6. Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina

    PubMed Central

    Nieto, Paula S.; Valdez, Diego J.; Acosta-Rodríguez, Victoria A.; Guido, Mario E.

    2011-01-01

    The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca2+mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell. PMID:22022612

  7. Protective effects of triptolide on retinal ganglion cells in a rat model of chronic glaucoma

    PubMed Central

    Yang, Fan; Wang, Dongmei; Wu, Lingling; Li, Ying

    2015-01-01

    Purpose To study the effects of triptolide, a Chinese herb extract, on retinal ganglion cells (RGCs) in a rat model of chronic glaucoma. Methods Eighty Wistar rats were randomly divided into triptolide group (n=40) and normal saline (NS) group (n=40). Angle photocoagulation was used to establish the model of glaucoma, with right eye as laser treated eye and left eye as control eye. Triptolide group received triptolide intraperitoneally daily, while NS group received NS. Intraocular pressure (IOP), anti-CD11b immunofluorescent stain in retina and optic nerve, RGCs count with Nissel stain and microglia count with anti-CD11b immunofluorescence stain in retina flat mounts, retinal tumor necrosis factor (TNF)-α mRNA detection by reverse transcription–polymerase chain reaction, and double immunofluorescent labeling with anti-TNF-α and anti-CD11b in retinal frozen section were performed. Results Mean IOP of the laser treated eyes significantly increased 3 weeks after photocoagulation (P<0.05), with no statistical difference between the two groups (P>0.05). RGCs survival in the laser treated eyes was significantly improved in the triptolide group than the NS group (P<0.05). Microglia count in superficial retina of the laser treated eyes was significantly less in the triptolide group (30.40±4.90) than the NS group (35.06±7.59) (P<0.05). TNF-α mRNA expression in the retina of the laser treated eyes in the triptolide group decreased by 60% compared with that in the NS group (P<0.01). The double immunofluorescent labeling showed that TNF-α was mainly distributed around the microglia. Conclusion Triptolide improved RGCs survival in this rat model of chronic glaucoma, which did not depend on IOP decrease but might be exerted by inhibiting microglia activities and reducing TNF-α secretion. PMID:26604697

  8. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    SciTech Connect

    Padilla, S.S.; Lyerly, D.P. )

    1989-12-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with (35S)methionine and (3H)fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure.

  9. Epiretinal transplantation of human bone marrow mesenchymal stem cells rescues retinal and vision function in a rat model of retinal degeneration.

    PubMed

    Tzameret, Adi; Sher, Ifat; Belkin, Michael; Treves, Avraham J; Meir, Amilia; Nagler, Arnon; Levkovitch-Verbin, Hani; Rotenstreich, Ygal; Solomon, Arieh S

    2015-09-01

    Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28 days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6 weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20 weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection.

  10. Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.

    PubMed

    Adler, R; Jerdan, J; Hewitt, A T

    1985-11-01

    The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

  11. Electrophysiological properties of rat retinal Müller (glial) cells in postnatally developing and in pathologically altered retinae.

    PubMed

    Felmy, F; Pannicke, T; Richt, J A; Reichenbach, A; Guenther, E

    2001-05-01

    Retinal glial Müller cells are characterized by dominant K(+) conductances. The cells may undergo changes of their membrane currents during ontogeny and gliosis as described in rabbit and man. Although the rat retina is often used in physiological experiments, the electrophysiology of rat Müller cells is less well studied. The aim of the present study was to characterize their membrane currents in postnatal development and in two models of retinal degeneration. Freshly isolated cells were subjected to whole-cell patch clamp recordings. During the first 4 weeks after birth of rats, their Müller cells displayed an increase in all membrane currents, particularly in the inward currents elicited at hyperpolarizing potentials. The decrease of the membrane resistance from more than 760 MOmega to less than 50 MOmega was accompanied by a shift of the zero current potential from about -20 mV to -80 mV, similar as earlier observed in developing rabbit Müller cells. These developmental changes were found in pigmented Brown Norway rats as well as in rats with inherited retinal dystrophy (RCS rats). Moreover, an infection of Lewis rats with the Borna disease virus caused substantial neuroretinal degeneration but did not result in a strong reduction of inward currents and of the zero current potential of the Müller cells. Thus, rat Müller cells fail to change their basic membrane properties in two different models of retinal pathology. This is in contrast to human and rabbit Müller cells, which have been shown to undergo dramatic changes of their membrane physiology in response to retinal diseases and injuries.

  12. The Extract of Aster Koraiensis Prevents Retinal Pericyte Apoptosis in Diabetic Rats and Its Active Compound, Chlorogenic Acid Inhibits AGE Formation and AGE/RAGE Interaction

    PubMed Central

    Kim, Junghyun; Jo, Kyuhyung; Lee, Ik-Soo; Kim, Chan-Sik; Kim, Jin Sook

    2016-01-01

    Retinal capillary cell loss is a hallmark of early diabetic retinal changes. Advanced glycation end products (AGEs) are believed to contribute to retinal microvascular cell loss in diabetic retinopathy. In this study, the protective effects of Aster koraiensis extract (AKE) against damage to retinal vascular cells were investigated in streptozotocin (STZ)-induced diabetic rats. To examine this issue further, AGE accumulation, nuclear factor-kappaB (NF-κB) and inducible nitric oxide synthase (iNOS) were investigated using retinal trypsin digests from streptozotocin-induced diabetic rats. In the diabetic rats, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling)-positive retinal microvascular cells were markedly increased. Immunohistochemical studies revealed that AGEs were accumulated within the retinal microvascular cells, and this accumulation paralleled the activation of NF-κB and the expression of iNOS in the diabetic rats. However, AKE prevented retinal microvascular cell apoptosis through the inhibition of AGE accumulation and NF-κB activation. Moreover, to determine the active compounds of AKE, two major compounds, chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were tested in an in vitro assay. Among these compounds, chlorogenic acid significantly reduced AGE formation as well as AGE/RAGE (receptor for AGEs) binding activity. These results suggest that AKE, particularly chlorogenic acid, is useful in inhibiting AGE accumulation in retinal vessels and exerts a preventive effect against the injuries of diabetic retinal vascular cells. PMID:27657123

  13. Sinomenine inhibits activation of rat retinal microglia induced by advanced glycation end products.

    PubMed

    Wang, Ai Ling; Li, Zhengbin; Yuan, Ming; Yu, Albert C H; Zhu, Xiu'An; Tso, Mark O M

    2007-12-05

    Diabetic retinopathy involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retina injury following an inflammatory stimulus. Cytokines, released by activated microglia, regulate the influx of inflammatory cells to the damaged area. Thus, therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Sinomenine, an alkaloid isolated from the stem and root of Sinomenium acutum, has long been recognized as an anti-inflammatory drug for rheumatoid arthritis and also inhibits macrophage activation. In this study, we activated retinal microglia in culture with advanced glycation end products (AGEs) treatment and attempted to determine whether sinomenine could reduce the production of cytokines from the activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF alpha, IL-1 beta and IL-6, were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) both in the presence and absence of AGEs. The effect of sinomenine on levels of reactive oxygen species (ROS) and the nuclear translocation of NF-kB p65 were studied with a laser confocal scanning microscope. AGEs treatment induced a significant release of TNF alpha, IL-1 beta, and IL-6 from retinal microglia. Sinomenine could inhibit release of these cytokines. Sinomenine attenuated ROS production in a dose-dependent fashion and reduced the nuclear translocation of NF-kB p65 in AGEs-activated retinal microglia in culture.

  14. The effects of taurine on vigabatrin, high light intensity and mydriasis induced retinal toxicity in the pigmented rat.

    PubMed

    Rasmussen, Allan D; Truchot, Nathalie; Pickersgill, Nigel; Thale, Zia Irene; Rosolen, Serge G; Botteron, Catherine

    2015-01-01

    The overall purpose of this study was to establish a model that may be used for examining the effect of Vigabatrin-induced retinal toxicity in pigmented rats, and subsequently examine the possible effects of taurine on the retinal toxicity. In the first part of the study, pigmented Long Evans rats were subjected to combinations of induced mydriasis, low/high light intensities (40/2000 lx) and oral administration of near-MTD (Maximum Tolerated Dose) doses (200 mg/kg/day) of Vigabatrin for up to 6 weeks. The combination of mydriasis and high light intensity applied to Long Evans rats resulted in retinal damage that was increased by the administration of Vigabatrin. In the second part of the study Long Evans rats were subjected to combinations of induced mydriasis and high/low light intensity (40/2000 lx) while being orally administered low (30 mg/kg/day) or high (200 mg/kg/day) doses of Vigabatrin for up to 6 weeks. In addition, selected groups of animals were administered taurine via the drinking water (20 mg/ml), resulting in systemic taurine concentrations of approximately threefold the endogenous concentration. The combined results of the studies demonstrate that retinal damage can be induced in pigmented animals when combining mydriasis and high light intensity. Retinal damage was functionally evaluated by electroretinography (ERG), then confirmed by histopathology. While depending on mydriasis and high light intensity, administration of Vigabatrin increased the retinal toxicity and resulted in the formation of rosette-like structures in the retina in a dose-related manner. Administration of taurine did not alleviate the Vigabatrin-induced retinal toxicity, as demonstrated either functionally by ERG or morphologically, although systemic concentrations of 3-fold the endogenous levels were reached, and it was thus not possible to demonstrate a protective effect of taurine in these pigmented animals. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. Serum albumin induces osmotic swelling of rat retinal glial cells.

    PubMed

    Löffler, Silvana; Wurm, Antje; Kutzera, Franziska; Pannicke, Thomas; Krügel, Katja; Linnertz, Regina; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2010-03-04

    Edema in the ischemic neural tissue develops by increased vascular permeability associated with extravasation of albumin, and by glial swelling. Here, we show that bovine serum albumin acutely administered to slices of the rat retina causes swelling of glial somata under hypoosmotic conditions. The effect of albumin was dose-dependent, with half-maximal and maximal effects at 10 nM and 1 microM, respectively, and was mediated by activation of transforming growth factor-beta receptor type II, oxidative stress, and the production of arachidonic acid and prostaglandins. Albumin-induced glial swelling was prevented by glutamate and purinergic receptor agonists. The data suggest that serum albumin may induce glial swelling in the presence of osmotic gradients.

  16. Treatment of adult neural progenitor cells prior to transplantation affects graft survival and integration in a neonatal and adult rat model of selective retinal ganglion cell depletion.

    PubMed

    Mellough, Carla B; Cui, Qi; Harvey, Alan R

    2007-01-01

    We tested whether microenvironmental changes surrounding apoptotic neural degeneration, cellular pre-treatment and timing of transplant can influence the survival and differentiation of transplanted cells. This was done by transplanting adult hippocampal precursor cells (AHPCs) into normal and retinal ganglion cell (RGC) depleted rat retinae. Apoptotic RGC death was induced in neonates by removal of the contralateral superior colliculus (SC) and in adults by unilateral optic nerve transection, with or without a peripheral nerve (PN) graft. AHPCs were transplanted 24 h after SC ablation, or 5, 7 or 14 days following optic nerve (ON) transection. Hosts received untreated grafts, or grafts treated by co-culture with embryonic retinal explants or the neuropeptide somatostatin. AHPCs integrated within all neonatal and 65% of adult retinae. Greater numbers of AHPCs were observed within the ganglion cell layer (GCL) in SC lesioned hosts. Explant co-culture induced proliferation of grafted AHPCs within host retinae. Somatostatin-treatment resulted in reduced overall engraftment but increased integration within the GCL. In lesioned adults, greatest GCL engraftment was observed following 7 or 14 day grafts. Some AHPCs in the inner retina expressed neuronal antigens and extended processes into the ON. These data indicate that various factors can influence the behaviour of grafted cells and work towards encouraging the functional restoration of retinal circuitry.

  17. Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor.

    PubMed

    de Almeida-Pereira, Luana; Magalhães, Camila Feitosa; Repossi, Marinna Garcia; Thorstenberg, Maria Luiza Prates; Sholl-Franco, Alfred; Coutinho-Silva, Robson; Ventura, Ana Lucia Marques; Fragel-Madeira, Lucianne

    2016-08-24

    Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y1 receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPβ-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60 % at P5 rats. Furthermore, immunolabeling against P2Y1 receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptors, reduced cell proliferation by approximately 20 % in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57(KIP2) and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y1 receptors regulating transition from G1 to S phase of the cell cycle.

  18. Comparison between PACAP- and enriched environment-induced retinal protection in MSG-treated newborn rats.

    PubMed

    Kiss, Peter; Atlasz, Tamas; Szabadfi, Krisztina; Horvath, Gabor; Griecs, Monika; Farkas, Jozsef; Matkovits, Attila; Toth, Gabor; Lubics, Andrea; Tamas, Andrea; Gabriel, Robert; Reglodi, Dora

    2011-01-10

    Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors occur throughout the nervous system, including the retina. PACAP exerts diverse actions in the eye: it influences ocular blood flow, contraction of the ciliary muscle, and has retinoprotective effects. This effect has been proven in different models of retinal degeneration. We have previously shown that PACAP protects against monosodium-glutamate (MSG)-induced damage in neonatal rats. The beneficial effects of enriched environment, another neuroprotective strategy, have long been known. Environmental enrichment has been shown to decrease different neuronal injuries. It also influences the development of the visual system. We have recently demonstrated that significant neuroprotection can be achieved in MSG-induced retinal degeneration in animals kept in an enriched environment. Combination of neuroprotective strategies often results in increased protection. Therefore, the aim of the present study was to compare the two neuroprotective strategies alone and in combination therapy. We found that both PACAP and environmental enrichment led to a similar degree of retinal protection, but the two treatments together did not lead to increased protection: their effects were not additive.

  19. A new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression

    PubMed Central

    Seiler, Magdalene J.; Aramant, Robert B.; Jones, Melissa K.; Ferguson, Dave L.; Bryda, Elizabeth C.

    2015-01-01

    Purpose The goal of this study was to develop an immunodeficient rat model of retinal degeneration (RD nude rats) that will not reject transplanted human cells. Methods SD-Tg(S334ter)3Lav females homozygous for a mutated mouse rhodopsin transgene were mated with NTac:NIH-Whn (NIH nude) males homozygous for the Foxn1rnu allele. Through selective breeding, a new stock, SD-Foxn1 Tg(S334ter)3Lav (RD nude) was generated such that all animals were homozygous for the Foxn1rnu allele and either homo- or hemizygous for the S334ter transgene. PCR-based assays for both the Foxn1rnu mutation and the S334ter transgene were developed for accurate genotyping. Immunodeficiency was tested by transplanting sheets of hESC-derived neural progenitor cells to the subretinal space of RD nude rats, and, as a control, NIH nude rats. Rats were killed between 8 and 184 days after surgery, and eye sections were analyzed for human, neuronal, and glial markers. Results After transplantation to RD nude and to NIH nude rats, hESC-derived neural progenitor cells differentiated to neuronal and glial cells, and migrated extensively from the transplant sheets throughout the host retina. Migration was more extensive in RD nude than in NIH nude rats. Already 8 days after transplantation, donor neuronal processes were found in the host inner plexiform layer. In addition, host glial cells extended processes into the transplants. The host retina showed the same photoreceptor degeneration pattern as in the immunocompetent SD-Tg(S334ter)3Lav rats. Recipients survived well after surgery. Conclusions This new rat model is useful for testing the effect of human cell transplantation on the restoration of vision without interference of immunosuppression. PMID:24817311

  20. Enzymatic conversion of all-trans-. beta. -carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa

    SciTech Connect

    Lakshman, M.R.; Mychkovsky, I.; Attlesey, M. )

    1989-12-01

    Enzymatic conversion of all-trans-{beta}-carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (i) the product gave rise to its O-ethyloxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristics of authentic retinal O-ethyloxime. High-pressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound; (ii) the mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime; (iii) the specific activity of the enzymatically formed ({sup 14}C)retinal O-ethyloxime remained constant even after repeated crystallization; (iv) the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of {beta}-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal.

  1. Gender-Dependent Effects of Enriched Environment and Social Isolation in Ischemic Retinal Lesion in Adult Rats

    PubMed Central

    Kiss, Peter; Szabadfi, Krisztina; Horvath, Gabor; Tamas, Andrea; Farkas, Jozsef; Gabriel, Robert; Reglodi, Dora

    2013-01-01

    Exposure to an enriched environment has been shown to have many positive effects on brain structure and function. Numerous studies have proven that enriched environment can reduce the lesion induced by toxic and traumatic injuries. Impoverished environment, on the other hand, can have deleterious effects on the outcome of neuronal injuries. We have previously shown that enriched conditions have protective effects in retinal injury in newborn rats. It is well-known that the efficacy of neuroprotective strategies can depend on age and gender. The aim of the present study, therefore, was to examine the effects of environmental enrichment and social isolation in retinal ischemia. We used bilateral common carotid artery occlusion to induce retinal hypoperfusion in adult Wistar rats of both genders. Groups were housed in standard, enriched or impoverished conditions. Impoverished environment was induced by social isolation. Retinas were processed for histological analysis after two weeks of survival. In the present study, we show that (1) enriched environment has protective effects in adult ischemic retinal lesion, while (2) impoverished environment further increases the degree of ischemic injury, and (3) that these environmental effects are gender-dependent: females are less responsive to the positive effects of environmental enrichment and more vulnerable to retinal ischemia in social isolation. In summary, our present study shows that the effects of both positive and negative environmental stimuli are gender-dependent in ischemic retinal lesions. PMID:23921682

  2. iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study

    PubMed Central

    Westenskow, Peter D.; Bucher, Felicitas; Bravo, Stephen; Kurihara, Toshihide; Feitelberg, Daniel; Paris, Liliana P.; Aguilar, Edith; Lin, Jonathan H.; Friedlander, Martin

    2016-01-01

    Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE) cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD), a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE) viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 2 1/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes. PMID:26880994

  3. Retinal Afferent Ingrowth to Neocortical Transplants in the Adult Rat Superior Colliculus is due to the Regeneration of Damaged Axons

    PubMed Central

    Ross, D. T.; Das, G. D.

    1994-01-01

    Retinal afferent ingrowth to embryonic neural transplants in the adult rat superior colliculus may represent either sprouting of intact axons or the regeneration of transected axons. If ingrowth represents regeneration of damaged retinofugai axons, then lesions that axotomize more retinofugal axons at the transplantation site should induce greater retinal afferent ingrowth. Alternately, if ingrowth represents terminal or collateral sprouting of intact retinofugal axons at or near the transplant/host optic layer interface, then the magnitude of retinal afferent ingrowth should be directly related to the total area of this interface. To test between these two hypotheses surgical knife wounds were made either parallel (in the sagittal plane) or perpendicular (in the transverse plane) to the course of axons in the stratum opticum, embryonic neocortical tissue was transplanted at the coordinates of these tectal slits, and retinal afferent ingrowth visualized 1-90 days after surgery using anterogradely transported HRP. A zone of traumatic reaction (ztr) in the optic layers was seen in every case, characterized by hypertrophied axons and swollen terminal clubs at 1 day. Between 30 and 90 days the damaged retinofugal axons in the zone formed dense fascicles and neuroma-like tangles. Retinal afferent ingrowth occurred only across transplant interface regions with the ztr. The magnitude of ingrowth was directly related to the area of the ztr interface and not the total optic layer interface area. Retinal afferent ingrowth appears to reflect the intrinsic regenerative capacity of adult mammalian retinal ganglion cells and not sprouting of undamaged axons. PMID:7703292

  4. Profound re-organization of cell surface proteome in equine retinal pigment epithelial cells in response to in vitro culturing.

    PubMed

    Szober, Christoph M; Hauck, Stefanie M; Euler, Kerstin N; Fröhlich, Kristina J H; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A

    2012-10-31

    The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

  5. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

    PubMed Central

    Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich, Kristina J. H.; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A.

    2012-01-01

    The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies. PMID:23203049

  6. In the Early Stages of Diabetes, Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity.

    PubMed

    Osorio-Paz, Ixchel; Uribe-Carvajal, Salvador; Salceda, Rocío

    2015-01-01

    In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in ex vivo retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal ex vivo retinas, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO2 production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO2 production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)(-1) and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress.

  7. Novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis

    PubMed Central

    Link, Dietmar; Strohmaier, Clemens; Seifert, Bernd U.; Riemer, Thomas; Reitsamer, Herbert A.; Haueisen, Jens; Vilser, Walthard

    2011-01-01

    We present a novel non-invasive and non-contact system for reflex-free retinal imaging and dynamic retinal vessel analysis in the rat. Theoretical analysis was performed prior to development of the new optical design, taking into account the optical properties of the rat eye and its specific illumination and imaging requirements. A novel optical model of the rat eye was developed for use with standard optical design software, facilitating both sequential and non-sequential modes. A retinal camera for the rat was constructed using standard optical and mechanical components. The addition of a customized illumination unit and existing standard software enabled dynamic vessel analysis. Seven-minute in-vivo vessel diameter recordings performed on 9 Brown-Norway rats showed stable readings. On average, the coefficient of variation was (1.1 ± 0.19) % for the arteries and (0.6 ± 0.08) % for the veins. The slope of the linear regression analysis was (0.56 ± 0.26) % for the arteries and (0.15 ± 0.27) % for the veins. In conclusion, the device can be used in basic studies of retinal vessel behavior. PMID:22076270

  8. In the Early Stages of Diabetes, Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity

    PubMed Central

    Osorio-Paz, Ixchel; Uribe-Carvajal, Salvador; Salceda, Rocío

    2015-01-01

    In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in ex vivo retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal ex vivo retinas, increasing glucose concentrations from 5.6mM to 30mM caused a four-fold increase in glucose accumulation and CO2 production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO2 production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)-1 and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress. PMID:25951172

  9. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

    PubMed

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-04-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

  10. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

    PubMed Central

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-01-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration. PMID:27116668

  11. Increased vascular density and vitreo-retinal membranes accompany vascularization of the pigment epithelium in the dystrophic rat retina.

    PubMed

    Caldwell, R B; Roque, R S; Solomon, S W

    1989-09-01

    Observations of vascularization of the retinal pigment epithelium (RPE) and formation of vitreo-retinal membranes (VRMs) in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy suggest that vascular proliferation occurs in this model. To test this hypothesis, we studied the progression of vascular changes in RCS and age-matched control rats using quantitative light microscope morphometry and electron microscopy. At 2 weeks, prior to photoreceptor degeneration, the dystrophic retina is comparable with the control. By 2 months, extensive degeneration of photoreceptor cells results in significant thinning of the dystrophic retina as compared with the control. Signs of vascular degeneration are evident at the electron microscope level--"ghost" vessels consisting of acellular basal lamina surrounded by amorphous electron-dense material; degenerating endothelial cells and pericytes; and abnormal deposits of extracellular matrix (ECM) material around blood vessels. Vascular degeneration is accompanied by glial changes in the form of necrotic perivascular glial processes and abnormal ECM deposits among the altered Muller cell processes. At 2-4 months in the dystrophic retina, numbers of vessel profiles in dystrophic retinas are decreased as compared with controls. However, vascular degeneration is overshadowed by the formation of numerous capillary tufts within the RPE layer, which together with retinal thinning results in increased vessel density. Between 4-12 months, the retinal thickness diminishes further, vascularization of the RPE increases, vitreo-retinal membranes are formed, and vascular density increases. In summary, following an initial period of vascular degeneration, vascularization of the RPE is accompanied by an increase in retinal vessel density and by the formation of vitreo-retinal membranes.

  12. Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

    PubMed

    Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

    2010-07-15

    In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

  13. Contralateral retinal dopamine decrease and melatonin increase in progression of hemiparkinsonium rat.

    PubMed

    Meng, Tao; Zheng, Zhi-Hong; Liu, Ting-Ting; Lin, Ling

    2012-05-01

    Both dopamine (DA) and melatonin (MLT) are abundant neuromodulators located in vertebrate retina. The retinal DA deficiency and variations in MLT levels have been linked to Parkinson's disease (PD). No studies have investigated the ipsilateral and contralateral DA and MLT in retina and their relationships in 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats. We established PD rat model by unilateral injection of 6-OHDA into the right substantia nigra and the right medial forebrain bundle. Eye tissue was collected and the levels of MLT and DA were measured twice daily at 10:00 and 22:00. The concentrations of DA and its metabolites, 3,4-dihydroxyphenylacetic (DOPAC) and homovanillic acid (HVA), as well as MLT were determined by HPLC. The results show that DA levels in the eye contralateral to the side of a unilateral intracerebral 6-OHDA lesion significantly decreased (P < 0.001). Both the ratios of DOPAC/DA and HVA/DA were increased in comparison with the vehicle groups after 3 weeks post-lesion. The concentrations of MLT at 10:00 and 22:00 in both eyes were distinctly increased compared with the vehicle groups (P < 0.05). The change of DA and its metabolites, as well as MLT appeared to correlate well with the rotation behavior of rats. These findings suggest that rats receive a unilateral intracerebral injection of 6-OHDA that mainly causes the contralateral eye destruction of DA-containing neurons. Increased retinal MLT level probably is associated with the progression of PD.

  14. Comparative analysis of three purification protocols for retinal ganglion cells from rat.

    PubMed

    Gao, Fengjuan; Li, Tingting; Hu, Jianyan; Zhou, Xujiao; Wu, Jihong; Wu, Qiang

    2016-01-01

    To make comparative analyses of the common three purification protocols for retinal ganglion cells (RGCs), providing a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments. Rat RGCs were isolated and purified using three methods, including two-step immunopanning (TIP) separation, two-step immunopanning-magnetic (TIPM) separation, and flow cytometric (FC) separation. Immunocytochemical staining, quantitative real-time PCR, flow cytometry, electrophysiology, and Cell Counting Kit-8 (CCK-8) analyses were performed to compare the purity, yield, and viability of the RGCs. The RGC yields from the TIP, TIPM, and FC methods were 24.60±15.98 × 10(4), 5.28±4.42 × 10(4), and 5.4±2.7 × 10(3) per retina, respectively. We easily controlled the relative purity of the RGCs with the FC method and even reached 100% of the maximum expected purity. However, the RGC purity was only 80.97±5.45% and 95.41±3.23% using the TIP and TIPM methods, respectively. The contaminant cells were mainly large, star-shaped, glial fibrillary acidic protein (GFAP)-positive astrocytes and small, round, syntaxin 1-positive amacrine cells with multiple short neurites. The RGCs purified with FC could not be cultured successively in our study; however, the TIP-RGCs survived more than 20 days with good viability, while the TIPM-RGCs survived less than 9 days. The three protocols for purifying the RGCs each had its own pros and cons. The RGCs isolated by the TIP method exhibited the highest viability and yield but had low purity. The purity of the RGCs isolated with the FC method could reach approximately 100% but had a low yield and cell viability. The TIPM method was reliable and produced RGCs with considerable purity, yield, and viability. This study provides a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments.

  15. Low-Oxygen Culture Conditions Extend the Multipotent Properties of Human Retinal Progenitor Cells

    PubMed Central

    Tucker, Budd A.; Young, Michael J.

    2014-01-01

    Purpose: Development of an effective cell-based therapy is highly dependent upon having a reproducible cell source suitable for transplantation. One potential source, isolated from the developing fetal neural retina, is the human retinal progenitor cell (hRPC). One limiting factor for the use of hRPCs is their in vitro expansion limit. As such, the aim of this study was to determine whether culturing hRPCs under 3% O2 would support their proliferative capacity while maintaining multipotency. Methods: To determine the effect of low oxygen on the ability of hRPCs to self-renew, rates of proliferation and apoptosis, telomerase activity, and expression of proliferative, stemness, and differentiation markers were assessed for hRPCs cultured in 3% and 20% oxygen conditions. Results: Culture under 3% oxygen increases the proliferation rate and shifts the proliferation limit of hRPCs to greater 40 divisions. This increased capacity for proliferation is correlated with an upregulation of Ki67, CyclinD1, and telomerase activity and a decrease in p53 expression and apoptosis. Increased expression of cMyc, Klf4, Oct4, and Sox2 in 3% O2 is correlated with stabilization of both HIF1α and HIF2α. The eye field development markers Pax6, Sox2, and Otx2 are present in hRPCs up to passage 16 in 3% O2. Following in vitro differentiation hRPCs expanded in the 3% O2 were able to generate specialized retinal cells, including rods and cones. Conclusions: Low-oxygen culture conditions act to maintain both multipotency and self-renewal properties of hRPCs in vitro. The extended expansion limits permit the development of a clinical-grade reagent for transplantation. PMID:24320879

  16. Rat retina shows robust circadian expression of clock and clock output genes in explant culture.

    PubMed

    Buonfiglio, Daniella C; Malan, André; Sandu, Cristina; Jaeger, Catherine; Cipolla-Neto, José; Hicks, David; Felder-Schmittbuhl, Marie-Paule

    2014-01-01

    Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.

  17. Activated adult microglia influence retinal progenitor cell proliferation and differentiation toward recoverin-expressing neuron-like cells in a co-culture model.

    PubMed

    Xu, Yunhe; Balasubramaniam, Balini; Copland, David A; Liu, Jian; Armitage, M John; Dick, Andrew D

    2015-07-01

    Microglia contribute to immune homeostasis of the retina, and thus act as a potential regulator determining successful repair or retinal stem cell transplantation. We investigated the interaction between human microglia and retinal progenitor cells in cell co-culture to further our exploration on developing a new therapeutic strategy for retinal degeneration. Microglia and retinal progenitor cultures were developed using CD11b(+) and CD133(+), respectively, from adult donor retina. Microglia activation was developed using interferon-gamma and lipopolysaccharide. Retinal progenitor differentiation was analysed in co-culture with or without microglial activation. Retinal progenitor proliferation was analysed in presence of conditioned medium from activated microglia. Phenotype and function of adult human retinal cell cultures were examined using cell morphology, immunohistochemistry and real-time PCR. By morphology, neuron-like cells generated in co-culture expressed photoreceptor marker recoverin. Neurospheres derived from retinal progenitor cells showed reduced growth in the presence of conditioned medium from activated microglia. Delayed retinal progenitor cell migration and reduced cellular differentiation was observed in co-cultures with activated microglia. In independent experiments, activated microglia showed enhanced mRNA expression of CXCL10, IL-27, IL-6, and TNF-alpha compared to controls. Adult human retina retains retinal progenitors or potential to reprogram cells to then proliferate and differentiate into neuron-like cells in vitro. Human microglia support retinal progenitor differentiation into neuron-like cells, but such capacity is altered following microglial activation. Modulating microglia activity is a potential approach to promote retinal repair and facilitate success of stem-cell transplantation.

  18. Prenatal Hypoxia Is Associated with Long-Term Retinal Dysfunction in Rats

    PubMed Central

    Bourque, Stephane L.; Kuny, Sharee; Reyes, Laura M.; Davidge, Sandra T.; Sauvé, Yves

    2013-01-01

    Background Intra-uterine growth restriction (IUGR) has been associated with increased predisposition to age-related complications. We tested the hypothesis that rat offspring models of IUGR would exhibit exacerbated, age-related retinal dysfunction. Methods Female Sprague-Dawley rats (maintained at 11.5% O2 from gestational day 15 to 21 to induce IUGR) and control offspring (maintained at 21% O2 throughout pregnancy) had retinal function assessed at 2 months (young) and 14 months of age (aged) with electroretinogram (ERG) recordings. Retinal anatomy was assessed by immunofluorescence. Results Deficits in rod-driven retina function were observed in aged IUGR offspring, as evidenced by reduced amplitudes of dark-adapted mixed a-wave Vmax (by 49.3%, P<0.01), b-wave Vmax (by 42.1%, P<0.001) and dark-adapted peak oscillatory potentials (by 42.3%, P<0.01). In contrast to the rod-driven defects specific to aged IUGR offspring, light adapted ERG recordings revealed cone defects in young animals, that were stationary until old age. At 2 months, IUGR offspring had amplitude reductions for both b-wave (Vmax by 46%, P<0.01) and peak oscillatory potential (Vmax by 38%, P<0.05). Finally, defects in cone-driven responses were further confirmed by reduced maximal photopic flicker amplitudes at 2 (by 42%, P<0.001) and 14 months (by 34%, P = 0.06) and critical flicker fusion frequencies at 14 months (Control: 42±1 Hz, IUGR: 35±2 Hz, P<0.05). These functional changes were not paralleled by anatomical losses in IUGR offspring retinas. Conclusions These data support that the developing retina is sensitive to stressors, and that pathways governing cone- and rod-driven function differ in their susceptibilities. In the case of prenatal hypoxia, cone- and rod-driven dysfunction manifest at young and old ages, respectively. We must, therefore, take into account the specific impact that fetal programming might exert on age-related retinal dystrophies when considering related diagnoses

  19. High-fat diet enhanced retinal dehydrogenase activity, but suppressed retinol dehydrogenase activity in liver of rats.

    PubMed

    Zhang, Mian; Liu, Can; Hu, Meng-yue; Zhang, Ji; Xu, Ping; Li, Feng; Zhong, Ze-yu; Liu, Li; Liu, Xiao-dong

    2015-04-01

    Evidence has shown that hyperlipidemia is associated with retinoid dyshomeostasis. In liver, retinol is mainly oxidized to retinal by retinol dehydrogenases (RDHs) and alcohol dehydrogenases (ADHs), further converted to retinoic acid by retinal dehydrogenases (RALDHs). The aim of this study was to investigate whether high-fat diet (HFD) induced hyperlipidemia affected activity and expression of hepatic ADHs/RDHs and RALDHs in rats. Results showed that retinol levels in liver, kidney and adipose tissue of HFD rats were significantly increased, while plasma retinol and hepatic retinal levels were markedly decreased. HFD rats exhibited significantly downregulated hepatic ADHs/RDHs activity and Adh1, Rdh10 and Dhrs9 expression. Oppositely, hepatic RALDHs activity and Raldh1 expression were upregulated in HFD rats. In HepG2 cells, treatment of HFD rat serum inhibited ADHs/RDHs activity and induced RALDHs activity. Among the tested abnormally altered components in HFD rat serum, cholesterol reduced ADHs/RDHs activity and RDH10 expression, while induced RALDHs activity and RALDH1 expression in HepG2 cells. Contrary to the effect of cholesterol, cholesterol-lowering agent pravastatin upregulated ADHs/RDHs activity and RDH10 expression, while suppressed RALDHs activity and RALDH1 expression. In conclusion, hyperlipidemia oppositely altered activity and expression of hepatic ADHs/RDHs and RALDHs, which is partially due to the elevated cholesterol levels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  20. KR-31378, a potassium-channel opener, induces the protection of retinal ganglion cells in rat retinal ischemic models.

    PubMed

    Choi, Anho; Choi, Jun-Sub; Yoon, Yone-Jung; Kim, Kyung-A; Joo, Choun-Ki

    2009-04-01

    KR-31378 is a newly developed K(ATP)-channel opener. To investigate the ability of KR-31378 to protect retinal ganglion cells (RGC), experiments were conducted using two retinal ischemia models. Retinal ischemia was induced by transient high intraocular pressure (IOP) for acute ischemia and by three episcleral vein occlusion for chronic retinal ischemia. KR-31378 was injected intraperitoneally and administered orally in the acute and chronic ischemia models, respectively. Under the condition of chronic ischemia, RGC density in the KR-31378-treated group was statistically higher than that in the non-treated group, and IOP was reduced. In the acute retinal ischemia model, 90% of RGC were degenerated after one week in non-treated retina, but, RGC in KR-31378-treated retina were protected from ischemic damage in a dose-dependent manner and showed inhibited glial fibrillary acidic protein (GFAP) expression. Furthermore, the KR-31378 protective effect was inhibited by glibenclamide treatment in acute ischemia. These findings indicate that systemic KR-31378 treatment may protect against ischemic injury-induced ganglion cell loss in glaucoma.

  1. Anti-diabetic drug metformin dilates retinal blood vessels through activation of AMP-activated protein kinase in rats.

    PubMed

    Mori, Asami; Ishikawa, Eriko; Amano, Tomoyo; Sakamoto, Kenji; Nakahara, Tsutomu

    2017-03-05

    The aim of this study was to examine whether metformin, a biguanide anti-hyperglycemic drug, dilates retinal blood vessels in rats. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo and diameters of retinal blood vessels were measured. Both systemic blood pressure and heart rate were continuously recorded. Metformin (0.01-0.3mg/kg/min) increased diameters of retinal blood vessels in a dose-dependent manner. This retinal vasodilator effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase. Similar results were obtained with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR, 0.01-1mg/kg/min). Neither metformin nor AICAR exerted significant effect on mean blood pressure and heart rate. However, a significant pressor response to AICAR was observed upon inhibition of NO synthase. These results suggest that metformin dilates retinal blood vessels through activation of AMPK, and NO plays an important role in the retinal vasodilator response following AMPK activation. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Early retinal blood vessel growth in normal and growth restricted rat pups raised in oxygen and room air.

    PubMed

    Dhaliwal, C A; Wade, J; Gillespie, T; Aspinall, P; McIntosh, N; Fleck, B W

    2011-11-01

    Premature infants are born with incompletely vascularised retinas and are at a risk of developing retinopathy of prematurity (ROP). Rate of prenatal and postnatal body growth is important in the pathogenesis of ROP. The aim of this study was to develop a physiology-based rat model in order to study the effect of growth restriction and oxygen on early retinal vascular development. Rat mothers were fed either a normal (18% casein) or low (9% casein) protein diet (to cause pup growth restriction) from the last week of gestation. After birth, mother and pups were placed in either room air or a specialised oxygen chamber that delivered a rapidly fluctuating hyperoxic oxygen profile. The oxygen profile was based on that from a premature infant who developed severe ROP. On day 14, retinas were dissected, flat-mounted and stained using biotinylated lectin. Images were captured by confocal microscopy. The avascular areas of the retinas were measured and compared. Growth restricted rat pups had significantly larger retinal avascular areas than 'normally grown' rat pups (Mann-Whitney U test, p<0.001). Growth restricted rat pups raised in fluctuating oxygen had significantly larger retinal avascular areas than growth restricted rat pups raised in room air (Mann-Whitney U test, p=0.001). The authors have developed a novel model for ROP that involves inducing both intrauterine and postnatal growth restriction and also exposes neonatal rat pups to fluctuating oxygen. This physiology-based model can be used to study the effects of growth, nutrition and oxygen on early retinal vascular development.

  3. 17β-estradiol ameliorates light-induced retinal damage in Sprague-Dawley rats by reducing oxidative stress.

    PubMed

    Wang, Shaolan; Wang, Baoying; Feng, Yan; Mo, Mingshu; Du, Fangying; Li, Hongbo; Yu, Xiaorui

    2015-01-01

    Oxidative stress is considered as a major cause of light-induced retinal neurodegeneration. The protective role of 17β-estradiol (βE2) in neurodegenerative disorders is well known, but its underlying mechanism remains unclear. Here, we utilized a light-induced retinal damage model to explore the mechanism by which βE2 exerts its neuroprotective effect. Adult male and female ovariectomized (OVX) rats were exposed to 8,000 lx white light for 12 h to induce retinal light damage. Electroretinogram (ERG) assays and hematoxylin and eosin (H&E) staining revealed that exposure to light for 12 h resulted in functional damage to the rat retina, histological changes, and retinal neuron loss. However, intravitreal injection (IVI) of βE2 significantly rescued this impaired retinal function in both female and male rats. Based on the level of malondialdehyde (MDA) production (a biomarker of oxidative stress), an increase in retinal oxidative stress followed light exposure, and βE2 administration reduced this light-induced oxidative stress. Quantitative reverse-transcriptase (qRT)-PCR indicated that the messenger RNA (mRNA) levels of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were downregulated in female OVX rats but were upregulated in male rats after light exposure, suggesting a gender difference in the regulation of these antioxidant enzyme genes in response to light. However, βE2 administration restored or enhanced the SOD and Gpx expression levels following light exposure. Although the catalase (CAT) expression level was insensitive to light stimulation, βE2 also increased the CAT gene expression level in both female OVX and male rats. Further examination indicated that the antioxidant proteins thioredoxin (Trx) and nuclear factor erythroid 2-related factor 2 (Nrf2) are also involved in βE2-mediated antioxidation and that the cytoprotective protein heme oxygenase-1 (HO-1) plays a key role in the endogenous defense mechanism

  4. Protective effect of clusterin on rod photoreceptor in rat model of retinitis pigmentosa

    PubMed Central

    Baral, Erika; Yu, Wan-Qing; Craft, Cheryl Mae

    2017-01-01

    Retinitis Pigmentosa (RP) begins with the death of rod photoreceptors and is slowly followed by a gradual loss of cones and a rearrangement of the remaining retinal neurons. Clusterin is a chaperone protein that protects cells and is involved in various pathophysiological stresses, including retinal degeneration. Using a well-established transgenic rat model of RP (rhodopsin S334ter), we investigated the effects of clusterin on rod photoreceptor survival. To investigate the role of clusterin in S334ter-line3 retinas, Voronoi analysis and immunohistochemistry were used to evaluate the geometry of rod distribution. Additionally, immunoblot analysis, Bax activation, STAT3 and Akt phosphorylation were used to evaluate the pathway involved in rod cell protection. In this study, clusterin (10μg/ml) intravitreal treatment produced robust preservation of rod photoreceptors in S334ter-line3 retina. The mean number of rods in 1mm2 was significantly greater in clusterin injected RP retinas (postnatal (P) 30, P45, P60, & P75) than in age-matched saline injected RP retinas (P<0.01). Clusterin activated Akt, STAT3 and significantly reduced Bax activity; in addition to inducing phosphorylated STAT3 in Müller cells, which suggests it may indirectly acts on photoreceptors. Thus, clusterin treatment may interferes with mechanisms leading to rod death by suppressing cell death through activation of Akt and STAT3, followed by Bax suppression. Novel insights into the pathway of how clusterin promotes the rod cell survival suggest this treatment may be a potential therapeutic strategy to slow progression of vision loss in human RP. PMID:28767729

  5. The effect of intraocular and intracranial pressure on retinal structure and function in rats

    PubMed Central

    Zhao, Da; He, Zheng; Vingrys, Algis J; Bui, Bang V; Nguyen, Christine T O

    2015-01-01

    An increasing number of studies indicate that the optic nerve head of the eye is sensitive not only to changes in intraocular pressure (IOP), but also to intracranial pressure (ICP). This study examines changes to optic nerve and retinal structure in a rat model in response to a range of IOP and ICP levels using optical coherence tomography. Furthermore, we examine the functional sequelae of these structural changes by quantifying the effect of pressure changes on the electroretinogram. IOP elevation (10–90 mmHg) induces progressive deformation of the optic nerve head and retinal surface (P < 0.05), compression of the retina (P < 0.05) and bipolar cell (b-wave), and retinal ganglion cell (scotopic threshold response) dysfunction (P < 0.05). Simultaneously altering ICP (−5 to 30 mmHg) modifies these IOP-induced responses, with lower ICP (−5 mmHg) exacerbating and higher ICP (15–30 mmHg) ameliorating structural and functional deficits. Thus, the balance between IOP and ICP (optic nerve pressure gradient, ONPG = IOP − ICP) plays an important role in optic nerve integrity. Structural and functional parameters exhibit a two-phase relationship to ONPG, with structural changes being more sensitive to ONPG modification (threshold = −0.6 to 11.3 mmHg) compared with functional changes (threshold = 49.7–54.6 mmHg). These findings have implications for diseases including glaucoma, intracranial hypertension, and long-term exposure to microgravity. PMID:26290528

  6. Neurofilament expression in cultured rat adenohypophysial cells.

    PubMed

    Quintanar, J L; Salinas, E

    2001-01-01

    The aim of the present work was to investigate in cultured rat adenohypophysial cells: a) the presence of neurofilaments of 200 kDa (NF-H), b) the effect of thyroid hormone (T(3)) and thyrotropin releasing hormone (TRH) on the expression of NF-H and c) the possible role of NF-H on thyrotropin (TSH) secretion. The presence of NF-H was observed by immunocytochemistry in cultured rat adenohypophysial cells. The exposure to T(3) for 12 h produced a significant increase in NF-H expression; whereas incubation with TRH or T(3)+TRH resulted in no change. The cells treated with T(3) or TRH or T(3)+TRH for 24 h showed no alteration. However, incubation for 48 h with TRH or T(3)+TRH caused significant decrease in NF-H expression. Incubation with NF-H antibodies produced a significant inhibition of calcium-induced TSH release in digitonin-permeabilized adenohypophysial cells. These results provide evidence that NF-H is present in cultured rat adenohypophysial cells, and that T(3) and TRH can modify NF-H expression. It can be suggested that in cultured adenohypophysial cells, NF-H may play a role in the secretory process.

  7. Filter-cultured ARPE-19 cells as outer blood-retinal barrier model.

    PubMed

    Mannermaa, Eliisa; Reinisalo, Mika; Ranta, Veli-Pekka; Vellonen, Kati-Sisko; Kokki, Heidi; Saarikko, Anni; Kaarniranta, Kai; Urtti, Arto

    2010-07-11

    Retinal pigment epithelium (RPE) regulates drug transfer between posterior eye segment and blood circulation, but there is no established RPE cell model for drug delivery studies. We evaluated ARPE-19 filter culture model for this purpose. Passive permeability of 6-carboxyfluorescein, betaxolol and FITC-dextran (40kDa) and active transport of 6-carboxyfluorescein, sodium fluorescein, rhodamine 123, cyclosporine A and digoxin in ARPE-19 model were investigated and compared with isolated bovine RPE-choroid tissue. In addition, barrier properties, and mRNA expression of RPE-specific and melanogenesis-related genes (RPE65, VMD2, CRALBP, OTX-2, MITF-A, TRP-1, tyrosinase) were measured in various culture conditions. The filter grown ARPE-19 cell model showed reasonable barrier properties (TER close to 100Omegacm(2)), but its permeability was slightly higher than that of isolated bovine RPE/choroid specimens. In active transport studies the ARPE-19 model mimics qualitatively the permeability profile of bovine RPE-choroid, but ARPE-19 model underestimates the importance of active transport relative to passive diffusion. Long-term filter-cultured ARPE-19 cells expressed various RPE-specific and melanogenesis-related genes at higher levels than the ARPE-19 cells cultured short-term in flasks. ARPE-19 model can be used to study drug permeation processes in the RPE.

  8. Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films.

    PubMed

    Lu, L; Garcia, C A; Mikos, A G

    1998-01-01

    Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.

  9. A modified chronic ocular hypertension rat model for retinal ganglion cell neuroprotection.

    PubMed

    Zhong, Lichun

    2013-09-01

    This study aimed to modify a chronic ocular hypertension (OHT) rat model to screen for potential compounds to protect retinal ganglion cells (RGCs) from responding to increased intraocular pressure (IOP). A total of 266 rats were prepared and randomly grouped according to different time-points, namely, weeks 3, 8, 16, and 24. Rats were sedated and eye examination was performed to score as the corneal damage on a scale of 1 to 4. The OHT rat model was created via the injection of a hypertonic saline solution into the episcleral veins once weekly for two weeks. OHT was identified when the IOP at week 0 was [Symbol: see text] 6 mmHg than that at week -2 for the same eye. Viable RGCs were labeled by injecting 4% FluoroGold. Rats were sacrificed, and the eyes were enucleated and fixed. The fixed retinas were dissected to prepare flat whole-mounts. The viable RGCs were visualized and imaged. The IOP (mean ± SD) was calculated, and data were analyzed by the paired t-test and one-way ANOVA. The OHT model was created in 234 of 266 rats (87.97%), whereas 32 rats (12.03%) were removed from the study because of the absence of IOP elevation (11.28%) and/or corneal damage scores over 4 (0.75%). IOP was elevated by as much as 81.35% for 24 weeks. The average IOP was (16.68 ± 0.98) mmHg in non-OHT eyes (n = 234), but was (27.95 ± 0.97) mmHg in OHTeyes (n = 234). Viable RGCs in the OHT eyes were significantly decreased in a time-dependent manner by 29.41%, 38.24%, 55.32%, and 59.30% at weeks 3, 8, 16, and 24, respectively, as compared to viable RGCs in the non-OHT eyes (P < 0.05). The OHT model was successfully created in 88% of the rats. The IOP in the OHT eyes was elevated by approximately 81% for 24 weeks. The number of viable RGCs was decreased by 59% of the rats in a time-dependent manner. The modified OHT model may provide an effective and reliable method for screening drugs to protect RGCs from glaucoma.

  10. Distribution of melanosomes across the retinal pigment epithelium of a hooded rat: implications for light damage

    SciTech Connect

    Howell, W.L.; Rapp, L.M.; Williams, T.P.

    1982-02-01

    Distribution of melanosomes across the retinal pigment epithelium of hooded rats (Long-Evans) is studied at the light microscopic and electron microscopic levels. This distribution is shown to be nonuniform: more melanosomes exist in the periphery than elsewhere and, importantly, there are very few melanosomes in a restricted area of the central portion of the superior hemisphere compared with the corresponding part of the inferior hemisphere. The region with fewest melanosomes is precisely the one that is highly susceptible to light damage. Because this region is the same in both pigmented and albino eyes, the paucity of melanin in this region is not the cause of its great sensitivity to light damage. Nor does light cause the nonuniform distribution of melanin. A possible explanation, involving a proposed vestigial tapetum, is given in order to explain the correlation of melanosome counts and sensitivity to light damage.

  11. Retinal safety of intravitreal rtPA in healthy rats and under excitotoxic conditions

    PubMed Central

    Daruich, Alejandra; Parcq, Jérôme; Delaunay, Kimberley; Naud, Marie-Christine; Le Rouzic, Quentin; Picard, Emilie; Crisanti, Patricia; Vivien, Denis; Berdugo, Marianne

    2016-01-01

    Purpose Intravitreal recombinant tissue plasminogen activator (rtPA) is used off-label for the surgical management of submacular hemorrhage, a severe complication of neovascular age-related macular degeneration. rtPA is approved for coronary and cerebral thrombolysis. However, in ischemic stroke rtPA is known to increase excitotoxic neural cell death by interacting with the N-methyl-D-aspartate (NMDA) receptor. We therefore investigated the retinal toxicity of rtPA in healthy rats and in a model of NMDA-induced retinal excitotoxicity. Methods First, rtPA at three different doses (2.16 µg/5 µl, 0.54 µg/5 µl, and 0.27 µg/5 µl) or vehicle (NaCl 0.9%) was injected intravitreally in healthy rat eyes. Electroretinograms (ERGs) were performed at 24 h or 7 days. Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptotic retinal ganglion cells (RGCs) were counted on flatmounted retinas at 24 h or 7 days. Next, NMDA + vehicle or NMDA + rtPA (0.27 µg/5 µl) was injected intravitreally to generate excitotoxic conditions. Apoptotic annexin V-FITC-labeled RGCs and surviving Brn3a-labeled RGCs were quantified on flatmounted retinas and radial sections, 18 h after treatment. Results In healthy rat eyes, the number of apoptotic RGCs was statistically significantly increased 24 h after the administration of rtPA at the highest dose (2.16 µg/5 µl; p = 0.0250) but not at the lower doses of 0.54 and 0.27 µg/5 µl (p = 0.36 and p = 0.20), compared to vehicle. At day 7, there was no difference in the apoptotic RGC count between the rtPA- and vehicle-injected eyes (p = 0.70, p = 0.52, p = 0.11). ERG amplitudes and implicit times were not modified at 24 h or 7 days after injection of any tested rtPA doses, compared to the baseline. Intravitreal administration of NMDA induced RGC death, but under these excitotoxic conditions, coadministration of rtPA did not increase the number of dead RGCs (p = 0.70). Similarly, the number of surviving RGCs on the flatmounted retinas and

  12. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

    PubMed

    Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

    2017-02-24

    An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP](mi2004) zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

  13. Hypothermia Prevents Retinal Damage Generated by Optic Nerve Trauma in the Rat.

    PubMed

    Rey-Funes, Manuel; Larrayoz, Ignacio M; Contartese, Daniela S; Soliño, Manuel; Sarotto, Anibal; Bustelo, Martín; Bruno, Martín; Dorfman, Verónica B; Loidl, César F; Martínez, Alfredo

    2017-07-31

    Ocular and periocular traumatisms may result in loss of vision. Hypothermia provides a beneficial intervention for brain and heart conditions and, here, we study whether hypothermia can prevent retinal damage caused by traumatic neuropathy. Intraorbital optic nerve crush (IONC) or sham manipulation was applied to male rats. Some animals were subjected to hypothermia (8 °C) for 3 h following surgery. Thirty days later, animals were subjected to electroretinography and behavioral tests. IONC treatment resulted in amplitude reduction of the b-wave and oscillatory potentials of the electroretinogram, whereas the hypothermic treatment significantly (p < 0.05) reversed this process. Using a descending method of limits in a two-choice visual task apparatus, we demonstrated that hypothermia significantly (p < 0.001) preserved visual acuity. Furthermore, IONC-treated rats had a lower (p < 0.0001) number of retinal ganglion cells and a higher (p < 0.0001) number of TUNEL-positive cells than sham-operated controls. These numbers were significantly (p < 0.0001) corrected by hypothermic treatment. There was a significant (p < 0.001) increase of RNA-binding motif protein 3 (RBM3) and of BCL2 (p < 0.01) mRNA expression in the eyes exposed to hypothermia. In conclusion, hypothermia constitutes an efficacious treatment for traumatic vision-impairing conditions, and the cold-shock protein pathway may be involved in mediating the beneficial effects shown in the retina.

  14. Inhibition of Adult Rat Retinal Ganglion Cells by D1-type Dopamine Receptor Activation

    PubMed Central

    Hayashida, Yuki; Rodríguez, Carolina Varela; Ogata, Genki; Partida, Gloria J.; Oi, Hanako; Stradleigh, Tyler W.; Lee, Sherwin C.; Colado, Anselmo Felipe; Ishida, Andrew T.

    2011-01-01

    The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D1a-receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections, and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D1-type receptors, SCH-23390 reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking Ih. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (INa) amplitude and tetrodotoxin, at doses that reduced INa as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability, and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the pre- and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing. PMID:19940196

  15. Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat.

    PubMed

    Duisit, Ghislaine; Conrath, Hervé; Saleun, Sylvie; Folliot, Sebastien; Provost, Nathalie; Cosset, François-Loïc; Sandrin, Virginie; Moullier, Philippe; Rolling, Fabienne

    2002-10-01

    The purpose of our study was to evaluate lentiviral vector-mediated rat retinal transduction using simian immunodeficiency virus (SIV) pseudotyped with envelope proteins from vesicular stomatitis virus G glycoprotein (VSV-G), Mokola virus G protein (MK-G), amphotropic murine leukemia virus envelope (4070A-Env), influenza A virus hemagglutinin (HA), lymphocytic choriomeningitis virus G protein (LCMV-G), and RD114 retrovirus envelope (RD114-Env). The six pseudotyped lentivirus vectors carried CMV-driven green fluorescent protein (GFP) or beta-galactosidase (beta-gal) reporter genes. Intravitreal and subretinal injections of each pseudotyped recombinant SIV were performed in cohorts of Wistar rats. Our results showed that no transgene expression was detected after intravitreal injection of each pseudotyped SIV vector. Also, no transduction could be detected following subretinal injection of RD114 pseudotyped SIV vectors. However, selective transduction of retinal pigment epithelium (RPE) cells was repeatedly obtained after subretinal delivery of VSV-G, MK-G, 4070A-Env, HA, and LCMV-G pseudotyped SIV. GFP expression was maximum as soon as 4 days postadministration for VSV-G, MK-G, 4070A-Env, and HA pseudotypes, with no evidence of pseudotransduction for VSV-G. Maximum transgene expression was observed 3 weeks postinjection for LCMV-6. Importantly, HA and VSV-G pseudotyped SIV lead to such a high level of transgene expression that GFP-related toxicity occurred. Therefore, when a high level of GFP synthesis is achieved, replacement of enhanced GFP (egfp, Aequorea victoria) by a low-toxicity GFP (Renilla reniformis) cDNA is necessary to allow long-term expression.

  16. Light-induced retinal degeneration causes a transient downregulation of melanopsin in the rat retina.

    PubMed

    García-Ayuso, Diego; Galindo-Romero, Caridad; Di Pierdomenico, Johnny; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta; Villegas Pérez, María P

    2017-08-01

    In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m(+)RGCs) and the expression of the melanopsin protein in the retina. The m(+)RGCs may be more resistant than other RGCs to lesion, but the effects of an acute light exposure in this population are unknown. Albino rats were exposed to white light (3000 lux) continuously for 48 h and processed 0, 3, 7 or 30 days after light exposure (ALE). Whole-mounted retinas were immunodetected with antibodies against melanopsin, Brn3a, and rhodopsin to study the populations of m(+)RGC, Brn3a(+)RGC and rods (which are the most abundant photoreceptors in the rat retina). Three days ALE there was substantial rod loss in an arciform area of the superior retina and with time this loss expanded in the form of rings all throughout the retina. Light exposure did not affect the number of Brn3a(+)RGCs but diminished the numbers of m(+)RGCs. Immediately ALE there was a significant decrease in the mean number of immunodetected m(+)RGCs that was more marked in the superior retina. Later, the number of m(+)RGCs increased progressively and reached normal values one month ALE. Western blot analysis showed that melanopsin expression down-regulates shortly ALE and recovers thereafter, in accordance with the anatomical data. This study demonstrates that there is a transient downregulation of melanopsin expression in the RGCs during the first month ALE. Further studies would be needed to clarify the long-term effect of light exposure on the m(+)RGC population. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Retinal glutamate transporter changes in experimental glaucoma and after optic nerve transection in the rat.

    PubMed

    Martin, Keith R G; Levkovitch-Verbin, Hana; Valenta, Danielle; Baumrind, Lisa; Pease, Mary Ellen; Quigley, Harry A

    2002-07-01

    High levels of glutamate can be toxic to retinal ganglion cells. Effective buffering of extracellular glutamate by retinal glutamate transporters is therefore important. This study was conducted to investigate whether glutamate transporter changes occur with two models of optic nerve injury in the rat. Glaucoma was induced in one eye of 35 adult Wistar rats by translimbal diode laser treatment to the trabecular meshwork. Twenty-five more rats underwent unilateral optic nerve transection. Two glutamate transporters, GLAST (EAAT-1) and GLT-1 (EAAT-2), were studied by immunohistochemistry and quantitative Western blot analysis. Treated and control eyes were compared 3 days and 1, 4, and 6 weeks after injury. Optic nerve damage was assessed semiquantitatively in epoxy-embedded optic nerve cross sections. Trabecular laser treatment resulted in moderate intraocular pressure (IOP) elevation in all animals. After 1 to 6 weeks of experimental glaucoma, all treated eyes had significant optic nerve damage. Glutamate transporter changes were not detected by immunohistochemistry. Western blot analysis demonstrated significantly reduced GLT-1 in glaucomatous eyes compared with control eyes at 3 days (29.3% +/- 6.7%, P = 0.01), 1 week (55.5% +/- 13.6%, P = 0.02), 4 weeks (27.2% +/- 10.1%, P = 0.05), and 6 weeks (38.1% +/- 7.9%, P = 0.01; mean reduction +/- SEM, paired t-tests, n = 5 animals per group, four duplicate Western blot analyses per eye). The magnitude of the reduction in GLT-1 correlated significantly with mean IOP in the glaucomatous eye (r(2) = 0.31, P = 0.01, linear regression). GLAST was significantly reduced (33.8% +/- 8.1%, mean +/- SEM) after 4 weeks of elevated IOP (P = 0.01, paired t-test, n = 5 animals per group). In contrast to glaucoma, optic nerve transection resulted in an increase in GLT-1 compared with the control eye (P = 0.01, paired t-test, n = 15 animals). There was no significant change in GLAST after transection. GLT-1 and GLAST were significantly

  18. Iron and other elements (Cu, Zn, Ca) contents in retina of rats during development and hereditary retinal degeneration

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Llabador, Y.; Devès, G.; Vesvres, M. H.; Simonoff, M.; Yefimova, M.; Courtois, Y.; Jeanny, J. C.

    2001-07-01

    The retina as well as other tissues needs iron to survive, but modifications in iron metabolism have also been suggested to contribute to cerebral neurodegenerative diseases. Our study was intended to investigate iron distribution in the retina of normal rats and Royal College of Surgeons (RCS) rats affected by hereditary degeneration of the photoreceptors at different developmental stages (35, 45 and 55 days after birth). Iron (Fe) distribution was determined by proton induced X-ray emission (PIXE) microanalysis on retinal sections and compared to other tissues (cornea, liver, spleen) and to other elements (Cu, Zn, Ca). Elemental concentrations were determined in different retinal layers especially the photoreceptors, which are progressively altered and disappear in the RCS rats. Iron is unevenly distributed throughout the rat retina. The highest concentration is observed in the choroid and the retinal pigmented epithelium and in the inner segments of photoreceptors. Iron content is lower in the outer segments but still significant. It increases during both the development and the disease at the level of the segments. This last localised iron increase can result in an overproduction of free radicals and be correlated with the photoreceptor cell loss. The distributions of other elements (Cu, Zn, Ca) revealed interesting temporal progressions.

  19. Effect(s) of preterm birth on normal retinal vascular development and oxygen-induced retinopathy in the neonatal rat.

    PubMed

    Li, Rong; Yang, Xiangmin; Wang, Yusheng; Chu, Zhaojie; Liu, Tao; Zhu, Tong; Gao, Xiang; Ma, Zhen

    2013-12-01

    Maturity is a critical factor in the pathogenesis of retinopathy of prematurity (ROP). One widely used method for studying this condition is that of oxygen-induced retinopathy (OIR). The general conditions of an OIR term animal, both at the time of birth and following birth, differ from those of the preterm infant. This, to simulate preterm conditions and to provide a basis for further studies on ROP, we investigated the effect(s) of preterm birth on retinal vascularization using the neonatal rat. Sprague-Dawley (SD) rats were delivered preterm by caesarean section on the day 19 of gestation. Term pups were used as controls. On the day of birth, preterm and term pups were housed under conditions of room air or cyclic oxygen. Retinas of pups housed in room air on days 4, 7, 10, 14, 18 and 22, as well as pups housed in oxygen on days 14, 18, and 22 were whole-mounted and stained with isolectin-B4. On day 18, cross-sections of the retina were cut and stained with hematoxylin and eosin for the identification of preretinal neovascular tufts. Images of avascular and neovascular areas were compared using light and fluorescence microscopy. Preterm pups had significantly larger avascular retinal areas than term rats on the various postnatal days. After exposure to cyclic oxygen, preterm pups demonstrated significantly larger avascular (days 14 and 18) and neovascular areas (day 18) compared with term rats. On day 22, residual retinopathy of preterm pups was greater than that of term pups. Preterm birth of rats, which are comparable in their physiology to humans, had negative effects on retinal vascularization. The impaired retinal vascular development and subsequent vasoproliferation resulting from hyperoxia in preterm pups is more severe and enduring.

  20. Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition

    PubMed Central

    Xing, Yao; Cui, Li-Jun; Kang, Qian-Yan

    2016-01-01

    Background: Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. Methods: RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48–72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3. Results: CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05). Conclusion: Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation. PMID:27364798

  1. Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments.

    PubMed

    Hernández, María; Urcola, J Haritz; Vecino, Elena

    2008-05-01

    The aim of the present study is to evaluate the neuroprotective effect of two antiglaucomatous substances, regardless of their hypotensive effect in the eye. Brimonidine, which does not reduce IOP when administered intraperitoneally, and latanoprost, which has a renowned hypotensive effect topically. We examined rat retinal ganglion cell (RGC) survival and size distribution in experimental glaucoma in response to different glaucomatous agents. IOP was elevated by episcleral vein cauterization (EVC) prior to the application of different treatments: (I) PBS application (control group), (II) intraperitoneal administration of brimonidine (a general hypotensive agent), (III) topical application of latanoprost (an ocular hypotensive agent), and (IV) latanoprost combined with brimonidine. After 12 weeks, RGCs were retrogradely labeled with fluorogold and RGC density was analyzed. EVC caused a significant increase (42%) in IOP in each group before drug treatment. After 12weeks of EVC, RGC survival in control vs. EVC rats was 78.9+/-3.2%. No IOP reduction was observed in brimonidine injected rats, but RGC survival at 12 weeks was total (103.7+/-2.7%). In latanoprost treated rats, IOP dropped by around 22% and 94.7+/-3.7% of the RGC population survived. Finally in the latanoprost+brimonidine combined group, IOP was significantly reduced by 25% and 94.4+/-2.2% of RGCs survived. Surprisingly, whereas EVC led to a 6% increase in RGC soma size, brimonidine treatment was associated with a 9% reduction in the soma size of RGCs at 12 weeks. We conclude that brimonidine exerts a neuroprotective effect via a mechanism which is independent of IOP reduction. These findings indicate that cell survival in glaucoma may be enhanced by neuroprotective strategies which are independent of IOP reduction. No synergistic neuroprotective effect was observed when both treatments were applied simultaneously.

  2. HUMAN RETINAL ENDOTHELIAL CELLS AND ASTROCYTES CULTURED ON 3-D SCAFFOLDS FOR OCULAR DRUG DISCOVERY AND DEVELOPMENT.

    PubMed

    Beharry, Kay D; Cai, Charles L; Valencia, Gloria B; Lazzaro, Douglas; Valencia, Arwin M; Salomone, Fabrizio; Aranda, Jacob V

    2017-09-15

    Topical ocular ketorolac improves the outcomes of severe retinopathy of prematurity and when administered with systemic caffeine, decreases the severity of oxygen-induced retinopathy. We tested the hypothesis that co-cultures of human retinal endothelial cells (HRECs) and human retinal astrocytes (HRAs) on 3-dimensional (3-D) hydrogel scaffolds is a more representative biomimetic paradigm of the blood-retinal-barrier (BRB) than 2-D cultures, and should be utilized for preclinical drug discovery and development. Mono- and co-cultures of HRECs and HRAs were treated with standard doses of ketorolac, ibuprofen, and/or caffeine, and exposed to hyperoxia, intermittent hypoxia (IH), or normoxia on 2-D surfaces or 3-D biodegradable hydrogel scaffolds (AlgiMatrix or Geltrex). Media and cells were collected at 72hours post treatment for arachidonic acid metabolites. Cells cultured on 3-D scaffolds exhibited less oxidative stress and variability in drug responses. HRAs enhanced the responses of HRECs to drugs and changes in oxygen environment. PGE2 and PGI2 were the predominant prostanoids produced in response to IH, reflecting COX-2 immunoreactivity. We conclude that HRECs and HRAs co-cultured on 3-D scaffolds may recapitulate drug responses of the dynamic BRB and therefore should be implemented for preclinical ocular drug discovery and development. Published by Elsevier Inc.

  3. Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

    PubMed Central

    Wileman, S.; Booth, N.; Moore, N.; Redmill, B.; Forrester, J.; Knott, R.

    2000-01-01

    BACKGROUND/AIMS—Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β in retinal angiogenesis in the context of diabetes mellitus.
METHODS—Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-β (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay.
RESULTS—Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05).
CONCLUSIONS—These data demonstrate that TGF-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

 PMID:10729302

  4. Green tea catechins are potent anti-oxidants that ameliorate sodium iodate-induced retinal degeneration in rats

    PubMed Central

    Yang, Yaping; Qin, Yong Jie; Yip, Yolanda W. Y.; Chan, Kwok Ping; Chu, Kai On; Chu, Wai Kit; Ng, Tsz Kin; Pang, Chi Pui; Chan, Sun On

    2016-01-01

    Green tea extracts exhibit anti-oxidative and anti-inflammatory actions in different disease conditions. We hypothesized that green tea extract and its catechin constituents ameliorate sodium iodate-induced retinal degeneration in rats by counteracting oxidative stress. In this study, adult Sprague-Dawley rats were intravenously injected with a single dose of sodium iodate. Green tea extract (GTE; Theaphenon-E) or combinations of its catechin constituents, including (−)-epigallocatechin gallate (EGCG), were administered intra-gastrically before injection. Live imaging analysis using confocal scanning laser ophthalmoscopy and spectral-domain optical coherence tomography showed a progressive increase of degenerating profile across the retinal surface and decrease in thickness of outer nuclear layer (ONL) at Day-14 of post-injection. These lesions were significantly ameliorated by Theaphenon-E and catechin combinations with EGCG. Catechins with exclusion of EGCG did not show obvious protective effect. Histological analyses confirmed that Theaphenon-E and catechins containing EGCG protect the retina by reducing ONL disruption. Retinal protective effects were associated with reduced expression of superoxide dismutase, glutathione peroxidase and caspase-3, and suppression of 8-iso-Prostaglandin F2α generation in the retina. In summary, GTE and its catechin constituents are potent anti-oxidants that offer neuroprotection to the outer retinal degeneration after sodium iodate insult, among which EGCG is the most active constituent. PMID:27383468

  5. Combined transplantation of human mesenchymal stem cells and human retinal progenitor cells into the subretinal space of RCS rats.

    PubMed

    Qu, Linghui; Gao, Lixiong; Xu, Haiwei; Duan, Ping; Zeng, Yuxiao; Liu, Yong; Yin, Zheng Qin

    2017-03-15

    Retinitis pigmentosa (RP) is one of hereditary retinal diseases characterized by the loss of photoreceptors. Cell transplantation has been clinically applied to treat RP patients. Human retinal progenitor cells (HRPCs) and human bone marrow-derived mesenchymal stem cells (HBMSCs) are the two commonly and practically used stem cells for transplantation. Since combined transplantation could be a promising way to integrate the advantages of both stem cell types, we transplanted HRPCs and HBMSCs into the subretinal space (SRS) of Royal College of Surgeons (RCS) rats. We report that HRPCs/HBMSCs combined transplantation maintains the electroretinogram results much better than HRPCs or HBMSCs single transplantations. The thickness of outer nuclear layer also presented a better outcome in the combined transplantation. Importantly, grafted cells in the combination migrated better, both longitudinally and latitudinally, than single transplantation. The photoreceptor differentiation of grafted cells in the retina of RCS rats receiving combined transplantation also showed a higher ratio than single transplantation. Finally, activation of microglia and the gliosis of Müller cells were more effectively suppressed in combined transplantation, indicating better immunomodulatory and anti-gliosis effects. Taken together, combining the transplantation of HRPCs and HBMSCs is a more effective strategy in stem cell-based therapy for retinal degenerative diseases.

  6. Green tea catechins are potent anti-oxidants that ameliorate sodium iodate-induced retinal degeneration in rats.

    PubMed

    Yang, Yaping; Qin, Yong Jie; Yip, Yolanda W Y; Chan, Kwok Ping; Chu, Kai On; Chu, Wai Kit; Ng, Tsz Kin; Pang, Chi Pui; Chan, Sun On

    2016-07-07

    Green tea extracts exhibit anti-oxidative and anti-inflammatory actions in different disease conditions. We hypothesized that green tea extract and its catechin constituents ameliorate sodium iodate-induced retinal degeneration in rats by counteracting oxidative stress. In this study, adult Sprague-Dawley rats were intravenously injected with a single dose of sodium iodate. Green tea extract (GTE; Theaphenon-E) or combinations of its catechin constituents, including (-)-epigallocatechin gallate (EGCG), were administered intra-gastrically before injection. Live imaging analysis using confocal scanning laser ophthalmoscopy and spectral-domain optical coherence tomography showed a progressive increase of degenerating profile across the retinal surface and decrease in thickness of outer nuclear layer (ONL) at Day-14 of post-injection. These lesions were significantly ameliorated by Theaphenon-E and catechin combinations with EGCG. Catechins with exclusion of EGCG did not show obvious protective effect. Histological analyses confirmed that Theaphenon-E and catechins containing EGCG protect the retina by reducing ONL disruption. Retinal protective effects were associated with reduced expression of superoxide dismutase, glutathione peroxidase and caspase-3, and suppression of 8-iso-Prostaglandin F2α generation in the retina. In summary, GTE and its catechin constituents are potent anti-oxidants that offer neuroprotection to the outer retinal degeneration after sodium iodate insult, among which EGCG is the most active constituent.

  7. Mead acid supplementation does not rescue rats from cataract and retinal degeneration induced by N-methyl-N-nitrosourea.

    PubMed

    Emoto, Yuko; Yoshizawa, Katsuhiko; Hamazaki, Kei; Kinoshita, Yuichi; Yuki, Michiko; Yuri, Takashi; Kawashima, Hiroshi; Tsubura, Airo

    2015-01-01

    Fatty acids and their derivatives play a role in the response to ocular disease. Our current study investigated the effects of dietary mead acid (MA, 5,8,11-eicosatrienoic acid) supplementation on N-methyl-N-nitrosourea (MNU)-induced cataract and retinal degeneration in Sprague-Dawley rats. Experiment 1 was designed to inhibit cataract formation, with the dams fed a 2.4% MA or basal (<0.01% MA) diet during lactational periods. On postnatal day 7, male pups received a single intraperitoneal (ip) injection of 50 mg/kg MNU or vehicle. Lens opacity and morphology were examined 7 and 14 days after the MNU injection. Experiment 2 was designed to inhibit retinal degeneration and was performed with female postweaning rats. In this experiment, dams were fed the 2.4% MA or basal diet during the lactational periods. Thereafter, the female pups were continuously fed the same diets during their postweaning periods. On postnatal day 21 (at weaning), pups received a single ip injection of 50 mg/kg MNU. Retinal morphology was examined 7 days after the MNU injection. In experiment 3, six-week-old female rats were fed the 2.4% MA or basal diet starting at one week before the MNU injection and were then continuously fed the same diets until sacrifice. Rats at 7 weeks of age were given a single ip injection of 40 mg/kg MNU, and the retina was then examined morphologically one week after the MNU injection. In experiment 1, mature cataract was found in all of the MNU-treated groups, with or without MA supplementation. In experiments 2 and 3, atrophy of both the peripheral and central outer retina occurred in all rats exposed to MNU, with or without MA supplementation, respectively. The severities of the cataracts and retinal atrophy in the rats were similar regardless of MA supplementation. Dietary mead acid, which is used as a substitute in essential fatty acid deficiency in the body, does not modify MNU-induced cataract and retinal degeneration in rat models.

  8. Cannabinoid receptor agonists modulate calcium channels in rat retinal Müller cells.

    PubMed

    Yang, W; Li, Q; Wang, S-Y; Gao, F; Qian, W-J; Li, F; Ji, M; Sun, X-H; Miao, Y; Wang, Z

    2016-01-28

    While activation of cannabinoid CB1 receptor (CB1R) regulates a variety of retinal neuronal functions by modulating ion channels in these cells, effect of activated cannabinoid receptors on Ca(2+) channels in retinal Müller cells is still largely unknown. In the present work we show that three subunits of T-type Ca(2+) channels, CaV3.1, CaV3.2 and CaV3.3, as well as one subunit of L-type Ca(2+) channels, CaV1.2, were expressed in rat Müller cells by immunofluorescent staining. Consistently, nimodipine- and mibefradil-sensitive Na(+) currents through L- and T-type Ca(2+) channels could be recorded electrophysiologically. The cannabinoid receptor agonist WIN55212-2 significantly suppressed Ca(2+) channel currents, mainly the T-type one, in acutely isolated rat Müller cells in a dose-dependent manner, with an IC50 of 3.98μM. The WIN55212-2 effect was not blocked by AM251/SR141716, specific CB1R antagonists. Similar suppression of the currents was observed when anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endogenous ligands of cannabinoid receptors, were applied. Moreover, even though CB2 receptors (CB2Rs) were expressed in rat Müller cells, the effects of WIN55212-2 and 2-AG on Ca(2+) channel currents were not blocked by AM630, a selective CB2R antagonist. However, the effect of AEA could be partially rescued by AM630. These results suggest that WIN55212-2 and 2-AG receptor-independently suppressed the Ca(2+) channel currents in Müller cells, while AEA suppressed the currents partially through CB2Rs. The existence of receptor-dependent and -independent mechanisms suggests that cannabinoids may modulate Müller cell functions through multiple pathways. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. Number and spatial distribution of intrinsically photosensitive retinal ganglion cells in the adult albino rat.

    PubMed

    Galindo-Romero, C; Jiménez-López, M; García-Ayuso, D; Salinas-Navarro, M; Nadal-Nicolás, F M; Agudo-Barriuso, M; Villegas-Pérez, M P; Avilés-Trigueros, M; Vidal-Sanz, M

    2013-03-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs - identified by their Brn3a expression - and the population of ipRGCs - identified by melanopsin immunodetection - thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard deviation of ipRGCs in the albino rat is 2047 ± 309. Their distribution in the retina seems to be complementary to that of Brn3a(+)RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.

  10. Schwann cells promote the survival of rat retinal ganglion cells after optic nerve section.

    PubMed Central

    Maffei, L; Carmignoto, G; Perry, V H; Candeo, P; Ferrari, G

    1990-01-01

    Schwann cells (SCs) are known to play an important role for the regeneration of mammalian peripheral nerves. Their effect is likely due to the production of neuronotrophic and/or supportive factors. Here we study the effect of intraocular transplant of SCs on the survival of rat retinal ganglion cells (RGCs) after the intracranial section of the optic nerve. SCs were injected intraocularly in adult hooded rats. Surviving RGCs were retrogradely labeled with horseradish peroxidase applied to the proximal stump of the optic nerve. Results show that intraocular transplants of SCs promote the survival of a large number of RGCs for periods as long as 9 and 14 weeks after optic nerve section. In experimental retinae, surviving RGCs were 2- to 8-fold more numerous than in controls. This finding suggests that SCs are the source of factors that promote the survival of RGCs. Nerve growth factor is produced by SCs, and the intraocular injection of nerve growth factor has been previously shown to promote RGC survival. The rescuing effect of SCs on RGCs is greater than that obtained by intraocular injection of nerve growth factor. This greater effect may be due to the action of other neurotrophic factors produced by SCs or by transplanted SCs producing NGF in a sustained fashion. Images PMID:2308946

  11. Retinal ganglion cell survival and axon regeneration after optic nerve injury in naked mole-rats.

    PubMed

    Park, Kevin K; Luo, Xueting; Mooney, Skyler J; Yungher, Benjamin J; Belin, Stephane; Wang, Chen; Holmes, Melissa M; He, Zhigang

    2017-02-01

    In the adult mammalian central nervous system (CNS), axonal damage often triggers neuronal cell death and glial activation, with very limited spontaneous axon regeneration. In this study, we performed optic nerve injury in adult naked mole-rats, the longest living rodent, with a maximum life span exceeding 30 years, and found that injury responses in this species are quite distinct from those in other mammalian species. In contrast to what is seen in other mammals, the majority of injured retinal ganglion cells (RGCs) survive with relatively high spontaneous axon regeneration. Furthermore, injured RGCs display activated signal transducer and activator of transcription-3 (STAT3), whereas astrocytes in the optic nerve robustly occupy and fill the lesion area days after injury. These neuron-intrinsic and -extrinsic injury responses are reminiscent of those in "cold-blooded" animals, such as fish and amphibians, suggesting that the naked mole-rat is a powerful model for exploring the mechanisms of neuronal injury responses and axon regeneration in mammals. J. Comp. Neurol. 525:380-388, 2017. © 2016 Wiley Periodicals, Inc.

  12. Neuroprotective effect of astaxanthin against rat retinal ganglion cell death under various stresses that induce apoptosis and necrosis.

    PubMed

    Yamagishi, Reiko; Aihara, Makoto

    2014-01-01

    Astaxanthin is a type of carotenoid known to have strong antioxidant effects. The purpose of this study was to investigate whether astaxanthin confers a neuroprotective effect against glutamate stress, oxidative stress, and hypoxia-induced apoptotic or necrotic cell death in primary cultures of rat retinal ganglion cells (RGCs). Purified rat RGCs were exposed to three kinds of stressors induced by 25 μM glutamate for 72 h, B27 medium without an antioxidant for 4 h, and a reduced oxygen level of 5% for 12 h. Each assay was repeated 12 times, with or without 1 nM, 10 nM, and 100 nM astaxanthin. The number of live RGCs was then counted using a cell viability assay. RGC viability in each condition was evaluated and compared with controls. In addition, we measured apoptosis and DNA damage. We found that under glutamate stress, RGC viability was reduced to 58%. Cultures with 1 nM, 10 nM, and 100 nM astaxanthin showed an increase in RGC viability of 63%, 74%, and 84%, respectively. Under oxidative stress, RGC viability was reduced to 40%, and astaxanthin administration resulted in increased viability of 43%, 50%, and 67%, respectively. Under hypoxia, RGC viability was reduced to 66%, and astaxanthin administration resulted in a significant increase in viability to 67%, 77%, and 93%, respectively. These results indicate that 100 nM astaxanthin leads to a statistically significant increase in RGC viability under the three kinds of stressors tested, compared to controls (Dunnett's test, p<0.05). The apoptotic activity of RGCs under glutamate stress increased to 32%, but was reduced to 15% with 100 nM astaxanthin administration. Glutamate stress led to a 58% increase in DNA damage, which was reduced to 43% when cultured with 100 nM astaxanthin. Thus, 100 nM astaxanthin showed a statistically significant reduction in apoptosis and DNA damage in RGCs (Wilcoxon rank-sum test, p<0.05). Our results suggest that astaxanthin has a neuroprotective effect against RGC death induced by

  13. Neuroprotective effect of astaxanthin against rat retinal ganglion cell death under various stresses that induce apoptosis and necrosis

    PubMed Central

    Yamagishi, Reiko

    2014-01-01

    Purpose Astaxanthin is a type of carotenoid known to have strong antioxidant effects. The purpose of this study was to investigate whether astaxanthin confers a neuroprotective effect against glutamate stress, oxidative stress, and hypoxia-induced apoptotic or necrotic cell death in primary cultures of rat retinal ganglion cells (RGCs). Methods Purified rat RGCs were exposed to three kinds of stressors induced by 25 μM glutamate for 72 h, B27 medium without an antioxidant for 4 h, and a reduced oxygen level of 5% for 12 h. Each assay was repeated 12 times, with or without 1 nM, 10 nM, and 100 nM astaxanthin. The number of live RGCs was then counted using a cell viability assay. RGC viability in each condition was evaluated and compared with controls. In addition, we measured apoptosis and DNA damage. Results We found that under glutamate stress, RGC viability was reduced to 58%. Cultures with 1 nM, 10 nM, and 100 nM astaxanthin showed an increase in RGC viability of 63%, 74%, and 84%, respectively. Under oxidative stress, RGC viability was reduced to 40%, and astaxanthin administration resulted in increased viability of 43%, 50%, and 67%, respectively. Under hypoxia, RGC viability was reduced to 66%, and astaxanthin administration resulted in a significant increase in viability to 67%, 77%, and 93%, respectively. These results indicate that 100 nM astaxanthin leads to a statistically significant increase in RGC viability under the three kinds of stressors tested, compared to controls (Dunnett’s test, p<0.05). The apoptotic activity of RGCs under glutamate stress increased to 32%, but was reduced to 15% with 100 nM astaxanthin administration. Glutamate stress led to a 58% increase in DNA damage, which was reduced to 43% when cultured with 100 nM astaxanthin. Thus, 100 nM astaxanthin showed a statistically significant reduction in apoptosis and DNA damage in RGCs (Wilcoxon rank-sum test, p<0.05). Conclusions Our results suggest that astaxanthin has a

  14. Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a rat model of retinal dystrophy.

    PubMed

    Tzameret, Adi; Sher, Ifat; Belkin, Michael; Treves, Avraham J; Meir, Amilia; Nagler, Arnon; Levkovitch-Verbin, Hani; Barshack, Iris; Rosner, Mordechai; Rotenstreich, Ygal

    2014-01-01

    Vision incapacitation and blindness associated with retinal degeneration affect millions of people worldwide. Cell based therapy and specifically transplantation of human adult bone marrow-derived stem cells (hBM-MSCs) present possible treatment strategy. Subretinal transplantation of human or rat BM-MSCs was shown previously to improve retinal function in Royal College Surgeons (RCS) rats. In those studies cells were transplanted via a transscleral-transchoroidal approach, creating a localized subretinal bleb. Limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection site. Here we describe a new surgical method for subretinal transplantation that facilitates uniform distribution of transplanted cells as a thin layer along most of the subretinal space. We assessed the therapeutic effect of hBM-MSCs on RCS rats when transplanted either subretinally or intravitreally. We also examined whether a second transplantation can prolong the therapeutic effect. A cell suspension of 2.5 × 10(6) cells in 5 μl was injected subretinally or intravitreally in RCS rats at 28 days postnatal. In the subretinal group, hBM-MSCs were transplanted posterior to the limbus in the superotemporal part of the eye through a longitudinal triangular scleral tunnel reaching the choroid. In the intravitreal group, the cells were injected into the superotemporal part of the vitreous cavity. In cross sections of subretinally transplanted eyes, removed 2 h following transplantation, hBM-MSCs were distributed as a near-homogenous thin layer along most of the subretinal space. In some animals the cells were also detected in the choroid. In the intravitreal injection group, hBM-MSCs were clustered in the vitreous cavity. Transplanted cells could be detected up to 2 weeks after transplantation but not at later time points. Retinal function and structure were assessed by electroretinogram (ERG) and histology analysis, respectively. Six

  15. Effect of Microtubule Disruption on Dynamics of Acidic Organelles in the Axons of Primary Cultured Retinal Ganglion Cells.

    PubMed

    Miyake, Seiji; Takihara, Yuji; Yokota, Satoshi; Takamura, Yoshihiro; Inatani, Masaru

    2017-09-22

    Axonal transport is fundamental to autophagy in neuronal cells. To understand its biological significance in various conditions, it is necessary to monitor the process of autophagy. However, monitoring methods are often limited to static analyses, such as protein expression and histological observations. Autophagy has multistep process and is highly dynamic; therefore, additional techniques are necessary to study autophagy. In this study, we quantified the dynamics of autophagy-related organelle transport under conditions of dynamic instability and catastrophic disruption of microtubules using in vitro live imaging. Retinal ganglion cells (RGCs) were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning. After 7 days of culture, acidic organelles were stained by LysoTracker. Dynamics of acidic organelles was quantified using kymographs. Colchicine was used to induce microtubule disruption. Movement of acidic organelles was observed at five time points: before, and at 6, 24, 72, and 120 h after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to determine cell viability. The status of axonal transport of acidic organelles (n = 363) from 27 RGCs was classified into four categories: anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). Six hours after the induction of microtubule disruption in 14 of 27 RGCs, almost all acidic organelles (n = 236) were stationary. All acidic components had completely stopped moving 24 h later. At 72 h after stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGCs. Finally, the remaining RGCs became positive for EthD-1. In the control (13 of 27 RGCs), axonal transport was maintained for 120 h and EthD-1-positive RGCs were not observed. Almost all acidic organelles were transported retrogradely along the axon, which was inhibited by colchicine. Understanding the dynamics of acidic organelles may provide useful parameters for

  16. Zerumbone, a Phytochemical of Subtropical Ginger, Protects against Hyperglycemia-Induced Retinal Damage in Experimental Diabetic Rats

    PubMed Central

    Tzeng, Thing-Fong; Liou, Shorong-Shii; Tzeng, Yu-Cheng; Liu, I-Min

    2016-01-01

    Diabetic retinopathy (DR), the most ordinary and specific microvascular complication of diabetes, is a disease of the retina. Zerumbone (ZER) is a monocyclic sesquiterpene compound, and based on reports, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The aim of the current study is to evaluate the protective effect of zerumbone against DR in streptozotocin (STZ)-induced diabetic rats. STZ-diabetic rats were treated with ZER (40 mg/kg) once a day orally for 8 weeks. ZER administration significantly (p < 0.05) lowered the levels of plasma glucose (32.5% ± 5.7% lower) and glycosylated hemoglobin (29.2% ± 3.4% lower) in STZ-diabetic rats. Retinal histopathological observations indicated that disarrangement and reduction in thickness of retinal layers were reversed in ZER-treated diabetic rats. ZER downregulated both the elevated levels of advanced glycosylated end products (AGEs) and the higher levels of the receptors for AGEs (RAGE) in retinas of diabetic rats. What’s more, ZER significantly (p < 0.05) ameliorated diabetes-induced upregulation of tumor necrosis factor-α, interleukin (IL)-1 and IL-6. ZER also attenuated overexpression of vascular endothelial growth factor and intercellular adhesion molecule-1, and suppressed activation of nuclear factor (NF)-κB and apoptosis in the retinas of STZ-diabetic rats. Our results suggest ZER possesses retinal protective effects, which might be associated with the blockade of the AGEs/RAGE/NF-κB pathway and its anti-inflammatory activity. PMID:27463726

  17. Zerumbone, a Phytochemical of Subtropical Ginger, Protects against Hyperglycemia-Induced Retinal Damage in Experimental Diabetic Rats.

    PubMed

    Tzeng, Thing-Fong; Liou, Shorong-Shii; Tzeng, Yu-Cheng; Liu, I-Min

    2016-07-25

    Diabetic retinopathy (DR), the most ordinary and specific microvascular complication of diabetes, is a disease of the retina. Zerumbone (ZER) is a monocyclic sesquiterpene compound, and based on reports, it is the predominant bioactive compound from the rhizomes of Zingiber zerumbet. The aim of the current study is to evaluate the protective effect of zerumbone against DR in streptozotocin (STZ)-induced diabetic rats. STZ-diabetic rats were treated with ZER (40 mg/kg) once a day orally for 8 weeks. ZER administration significantly (p < 0.05) lowered the levels of plasma glucose (32.5% ± 5.7% lower) and glycosylated hemoglobin (29.2% ± 3.4% lower) in STZ-diabetic rats. Retinal histopathological observations indicated that disarrangement and reduction in thickness of retinal layers were reversed in ZER-treated diabetic rats. ZER downregulated both the elevated levels of advanced glycosylated end products (AGEs) and the higher levels of the receptors for AGEs (RAGE) in retinas of diabetic rats. What's more, ZER significantly (p < 0.05) ameliorated diabetes-induced upregulation of tumor necrosis factor-α, interleukin (IL)-1 and IL-6. ZER also attenuated overexpression of vascular endothelial growth factor and intercellular adhesion molecule-1, and suppressed activation of nuclear factor (NF)-κB and apoptosis in the retinas of STZ-diabetic rats. Our results suggest ZER possesses retinal protective effects, which might be associated with the blockade of the AGEs/RAGE/NF-κB pathway and its anti-inflammatory activity.

  18. Progressive Early Breakdown of Retinal Pigment Epithelium Function in Hyperglycemic Rats

    PubMed Central

    Desjardins, Danielle M.; Yates, Phil W.; Dahrouj, Mohammad; Liu, Yueying; Crosson, Craig E.; Ablonczy, Zsolt

    2016-01-01

    Purpose Diabetic macular edema (DME), an accumulation of fluid in the subretinal space, is a significant cause of vision loss. The impact of diabetes on the breakdown of the inner blood–retina barrier (BRB) is an established event that leads to DME. However, the role of the outer BRB in ocular diabetes has received limited attention. We present evidence that the breakdown of normal RPE function in hyperglycemia facilitates conditions conducive to DME pathogenesis. Methods Brown Norway rats (130–150 g) were injected intraperitoneally with streptozotocin (STZ; 60 mg/kg) to induce hyperglycemia. After 4 weeks, Evans blue (EB) dye was injected intravenously to determine whether there was leakage of albumin into the retina. Subretinal saline blebs (0.5–1 μL) were placed 4 and 9 weeks after STZ injection, and time-lapse optical coherence tomography tracked the resorption rate. In a subset of rats, intravitreal bevacizumab, a humanized monoclonal antibody targeted to VEGF, was given at 5 weeks and resorption was measured at 9 weeks. Results The ability of the RPE to transport fluid was reduced significantly after 4 and 9 weeks of hyperglycemia with a reduction of over 67% at 9 weeks. No EB dye leakage from inner retinal vessels was measured in hyperglycemic animals compared to control. The intravitreal administration of bevacizumab at week 5 significantly increased the rate of fluid transport in rats subjected to hyperglycemia for 9 weeks. Conclusions These results demonstrate that chronic hyperglycemia altered RPE fluid transport, in part dependent on the actions of VEGF. These results support the idea that RPE dysfunction is an early event associated with hyperglycemia that contributes to fluid accumulation in DME. PMID:27191823

  19. Retinal ganglion cell protection with geranylgeranylacetone, a heat shock protein inducer, in a rat glaucoma model.

    PubMed Central

    Caprioli, Joseph; Ishii, Yoko; Kwong, Jacky M K

    2003-01-01

    PURPOSE: To study the effects of geranylgeranylacetone (GCA) on the expression of inducible (HSP72) and constitutive (HSC70) heat shock proteins (HSPs) on retinal ganglion cells (RGCs) in a rat model of glaucoma. METHODS: Adult Wistar rats were given intraperitoneal injections of GGA, 200 mg/kg daily. Western blot analysis and immunohistochemical staining for HSP72 and HSC70 were performed after 1, 3, and 7 days of GGA administration. After 7 days of GGA pretreatment, intraocular pressure (IOP) was elevated unilaterally by repeated trabecular argon laser photocoagulation 5 days after intracameral injection of india ink. After the first laser photocoagulation, CGA was given twice a week. RGC survival was evaluated after 5 weeks of IOP elevation. Immunohistochemistry and TdT-mediated biotin-dUTP nick end labeling (TUNEL) were performed after 1 week of IOP elevation. Quercetin, an inhibitor of HSP expression, was also administered to a separate group. RESULTS: There was increased expression of HSP72 in RGCs at 3 and 7 days after GGA administration, but HSC70 was unchanged. After 5 weeks of IOP elevation, there was 27% +/- 6% loss of RGCs. The administration of GGA significantly reduced the loss of RGCs, lessened optic nerve damage, decreased the number of TUNEL-positive cells in the RGC layer, and increased HSP72. Quercetin administration abolished these protective effects. CONCLUSIONS: These results demonstrate that systemic administration of GGA protects RGCs from glaucomatous damage in a rat model and suggest a novel pathway for netroprotection for patients with glaucoma. PMID:14971562

  20. Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture.

    PubMed

    Miura, Yoko; Klettner, Alexa; Noelle, Bernhard; Hasselbach, Heike; Roider, Johann

    2010-01-01

    To evaluate the changes of morphological and functional characteristics of the retinal pigment epithelium (RPE)-choroid perfusion culture during cultivation. PorcineRPE-choroid tissue was cultivated in a perfusion tissue culture system. After the indicated times, histology, immunolocalization of collagen IV and von Willebrand factor, RPE cell viability with calcein-AM, TUNEL assay and occludin immunolocalization of RPE cells were examined. The tissue was treated with selective RPE treatment laser after different time periods and the wound healing response was characterized. Vascular endothelial growth factor secretion was measured by enzyme-linked immunosorbent assay. On day 8, prominent morphological degenerative changes of RPE cells were observed in histology. According to the immunohistochemistry for collagen IV, the Bruch's membrane did not display any obvious decomposition until day 8. Von Willebrand factor staining decreased during cultivation, especially at the choriocapillaris. Calcein-AM staining and TUNEL assay displayed the increase of apoptotic changes in only a minority of the cells on day 4, but in many cells on day 8. Occludin delocalization was observed on day 8. Selective RPE treatment laser-produced wounds were completely closed by monolayer RPE when wounded on fresh and 3-day-old cultures, but not when wounded on 6-day-old cultures. Vascular endothelial growth factor secretion was stable between days 2 and 5, but increased after that. Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance. Copyright 2009 S. Karger AG, Basel.

  1. Human retinal development in an in situ whole eye culture system.

    PubMed

    Engelsberg, Karl; Ghosh, Fredrik

    2011-01-01

    Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6-7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7-14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells

  2. Glutamate stimulation of retinal ganglion cells in normal and s334ter-4 rat retinas: a candidate for a neurotransmitter-based retinal prosthesis.

    PubMed

    Finlayson, Paul G; Iezzi, Raymond

    2010-07-01

    PURPOSE. To investigate the suitability of glutamate as a potential agent for a neurotransmitter-based retinal prosthesis. METHODS. Retinal ganglion cells (RGCs) from P35-70 albino Sprague-Dawley (normal) and P60-254 S334ter-4 (photoreceptor degeneration) rats were recorded extracellularly in flattened eye cup preparations, to assess their responses to glutamate, applied locally via micropipettes. RESULTS. Brief local application of glutamate effectively excited RGCs in both normal and degenerated retinas. Epiretinal surface application of glutamate was less likely to excite RGCs than was subsurface application (20 microm below the epiretinal surface). Glutamate evoked RGC firing rates, and the response patterns were similar for epiretinal surface and subsurface applications. Subsurface application of 2 mM glutamate effectively excited cells within 130 microm of the ejection sites. Response latencies averaged 281 ms and were significantly longer for OFF RGCs than for ON RGCs in normal retinas (P = 0.025). Suppression of activity was observed at shorter latencies ( approximately 100 ms) after glutamate application in most of the spontaneously active RGCs. Responses to each glutamate application were similar, and the duration of activity was directly dependent on the duration of application. RGC responses varied from recurrent high-frequency bursts to sustained firing at rates above 40 spikes/s, in normal and degenerated retinas. Paired, sequential applications of glutamate evoked two distinguishable responses, with interstimulus intervals as low as 200 ms. Overall, RGC response sensitivity to glutamate was similar in normal and degenerated retinas. CONCLUSIONS. Glutamate is an excellent candidate for a neurotransmitter-based retinal prosthesis, as its local application effectively stimulates RGCs with high spatial and temporal resolution.

  3. High-mobility group Box-1 is involved in NMDA-induced retinal injury the in rat retina.

    PubMed

    Sakamoto, Kenji; Mizuta, Aya; Fujimura, Kyosuke; Kurauchi, Yuki; Mori, Asami; Nakahara, Tsutomu; Ishii, Kunio

    2015-08-01

    High-mobility group Box-1 (HMGB1) is known to be released from injured cells and to induce an inflammatory response. Although HMGB1 was reported to mediate ischemia-reperfusion injury of the brain, its role in glutamate excitotoxicity of the retina remains controversial. Here, the authors demonstrated the evidence that HMGB1 is involved in the retinal damage induced by NMDA. Under ketamine/xylazine anesthesia, male Sprague-Dawley rats were subjected to intravitreal injection of NMDA (200 nmol/eye) or HMGB1 protein derived from bovines (5-15 μg/eye). Intravitreal anti-HMGB1 IgY (5 μg/eye) was simultaneously administered with NMDA or HMGB1. Seven days later, animals were killed and 5-μm retinal sections through the optic nerve head were obtained. These specimens were subjected to morphometry. Intravitreal NMDA and HMGB1 protein evoked cell loss in the ganglion cell layer 7 days later. Intravitreal anti-HMGB1 IgY reduced these damages. Anti-HMGB1 IgY reduced the number of 8-hydroxy-deoxyguanosine (8-OHdG)-positive cells induced by intravitreal NMDA. Toll-like receptor 2/4 antagonist peptide, receptor for advanced glycation end-products (RAGE) antagonist peptide, and FPS-ZM1 significantly reduced the retinal damage induced by HMGB1 protein. The results in the present study suggest that HMGB1 is at least in part involved in NMDA-induced retinal injury, and probably induces cell death of retinal ganglion cells with increase of oxidative stress, via activation of toll-like receptor 2/4 and RAGE in the rat retina. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. White Light–Emitting Diodes (LEDs) at Domestic Lighting Levels and Retinal Injury in a Rat Model

    PubMed Central

    Shang, Yu-Man; Wang, Gen-Shuh; Sliney, David; Lee, Li-Ling

    2013-01-01

    Background: Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000–10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model. Objective: We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements. Methods: We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level. Results: H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury. Conclusion: Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich “white” LEDs for general lighting. Citation: Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light–emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269–276; http://dx.doi.org/10.1289/ehp.1307294 PMID:24362357

  5. White light-emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model.

    PubMed

    Shang, Yu-Man; Wang, Gen-Shuh; Sliney, David; Yang, Chang-Hao; Lee, Li-Ling

    2014-03-01

    Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000-10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model. We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements. We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level. H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury. Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich "white" LEDs for general lighting. Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light-emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269-276; http://dx.doi.org/10.1289/ehp.1307294.

  6. The effect of intravitreal vascular endothelial growth factor on inner retinal oxygen delivery and metabolism in rats.

    PubMed

    Blair, Norman P; Wanek, Justin; Teng, Pang-yu; Shahidi, Mahnaz

    2016-02-01

    Vascular endothelial growth factor (VEGF) is stimulated by hypoxia and plays an important role in pathologic vascular leakage and neovascularization. Increased VEGF may affect inner retinal oxygen delivery (DO2) and oxygen metabolism (MO2), however, quantitative information is lacking. We tested the hypotheses that VEGF increases DO2, but does not alter MO2. In 10 rats, VEGF was injected intravitreally into one eye, whereas balanced salt solution (BSS) was injected into the fellow eye, 24 h prior to imaging. Vessel diameters and blood velocities were determined by red-free and fluorescent microsphere imaging, respectively. Vascular PO2 values were derived by phosphorescence lifetime imaging of an intravascular oxyphor. Retinal blood flow, vascular oxygen content, DO2 and MO2 were calculated. Retinal arterial and venous diameters were larger in VEGF-injected eyes compared to control eyes (P < 0.03), however no significant difference was observed in blood velocity (P = 0.21). Thus, retinal blood flow was greater in VEGF-injected eyes (P = 0.007). Retinal vascular PO2 and oxygen content were similar between control and VEGF-injected eyes (P > 0.11), while the arteriovenous oxygen content difference was marginally lower in VEGF-injected eyes (P = 0.05). DO2 was 950 ± 340 and 1380 ± 650 nL O2/min in control and VEGF-injected eyes, respectively (P = 0.005). MO2 was 440 ± 150 and 490 ± 190 nL O2/min in control and VEGF-injected eyes, respectively (P = 0.31). Intravitreally administered VEGF did not alter MO2 but increased DO2, suggesting VEGF may play an offsetting role in conditions characterized by retinal hypoxia.

  7. Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

    PubMed

    Vogler, Stefanie; Grosche, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-10-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

  8. Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

    PubMed

    Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

    2011-03-17

    The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions.

  9. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    PubMed

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway.

  10. Synergistic protective effects of escin and low‑dose glucocorticoids on blood‑retinal barrier breakdown in a rat model of retinal ischemia.

    PubMed

    Zhang, Fenglan; Li, Yuanbin; Zhang, Leiming; Mu, Guoying

    2013-05-01

    Escin, a natural mixture of triterpenoid saponins isolated from the seed of the horse chestnut (Aesculus hippocastanum), has been demonstrated to possess glucocorticoid (GC)‑like anti‑edematous and anti‑inflammatory effects. The aim of the present study was to investigate whether escin exhibits synergistic protective effects on blood‑retinal barrier (BRB) breakdown when combined with GCs in a rat model of retinal ischemia. Low concentrations of escin and triamcinolone acetonide (TA) alone did not affect BRB permeability. However, when administered together, low‑dose escin and TA significantly reduced BRB permeability following ischemia. Furthermore, low‑dose escin and TA alone did not affect the expression of occludin in the ischemic retina; however, when administered together, they significantly increased occludin expression in the ganglion cell layer of the ischemic retina. This indicates that escin and GCs have synergistic protective effects on BRB breakdown and the molecular mechanisms may be correlated with the upregulation of occludin. Therefore, the administration of escin may allow a reduction in the dose of GCs for the treatment of macular edema. The combination of escin with GCs is potentially a beneficial treatment method for BRB breakdown and warrants further investigation.

  11. Controlled delivery of tauroursodeoxycholic acid from biodegradable microspheres slows retinal degeneration and vision loss in P23H rats.

    PubMed

    Fernández-Sánchez, Laura; Bravo-Osuna, Irene; Lax, Pedro; Arranz-Romera, Alicia; Maneu, Victoria; Esteban-Pérez, Sergio; Pinilla, Isabel; Puebla-González, María Del Mar; Herrero-Vanrell, Rocío; Cuenca, Nicolás

    2017-01-01

    Successful drug therapies for treating ocular diseases require effective concentrations of neuroprotective compounds maintained over time at the site of action. The purpose of this work was to assess the efficacy of intravitreal controlled delivery of tauroursodeoxycholic acid (TUDCA) encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres for the treatment of the retina in a rat model of retinitis pigmentosa. PLGA microspheres (MSs) containing TUDCA were produced by the O/W emulsion-solvent evaporation technique. Particle size and morphology were assessed by light scattering and scanning electronic microscopy, respectively. Homozygous P23H line 3 rats received a treatment of intravitreal injections of TUDCA-PLGA MSs. Retinal function was assessed by electroretinography at P30, P60, P90 and P120. The density, structure and synaptic contacts of retinal neurons were analyzed using immunofluorescence and confocal microscopy at P90 and P120. TUDCA-loaded PLGA MSs were spherical, with a smooth surface. The production yield was 78%, the MSs mean particle size was 23 μm and the drug loading resulted 12.5 ± 0.8 μg TUDCA/mg MSs. MSs were able to deliver the loaded active compound in a gradual and progressive manner over the 28-day in vitro release study. Scotopic electroretinografic responses showed increased ERG a- and b-wave amplitudes in TUDCA-PLGA-MSs-treated eyes as compared to those injected with unloaded PLGA particles. TUDCA-PLGA-MSs-treated eyes showed more photoreceptor rows than controls. The synaptic contacts of photoreceptors with bipolar and horizontal cells were also preserved in P23H rats treated with TUDCA-PLGA MSs. This work indicates that the slow and continuous delivery of TUDCA from PLGA-MSs has potential neuroprotective effects that could constitute a suitable therapy to prevent neurodegeneration and visual loss in retinitis pigmentosa.

  12. Controlled delivery of tauroursodeoxycholic acid from biodegradable microspheres slows retinal degeneration and vision loss in P23H rats

    PubMed Central

    Lax, Pedro; Arranz-Romera, Alicia; Maneu, Victoria; Esteban-Pérez, Sergio; Pinilla, Isabel; Puebla-González, María del Mar; Herrero-Vanrell, Rocío

    2017-01-01

    Successful drug therapies for treating ocular diseases require effective concentrations of neuroprotective compounds maintained over time at the site of action. The purpose of this work was to assess the efficacy of intravitreal controlled delivery of tauroursodeoxycholic acid (TUDCA) encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres for the treatment of the retina in a rat model of retinitis pigmentosa. PLGA microspheres (MSs) containing TUDCA were produced by the O/W emulsion-solvent evaporation technique. Particle size and morphology were assessed by light scattering and scanning electronic microscopy, respectively. Homozygous P23H line 3 rats received a treatment of intravitreal injections of TUDCA-PLGA MSs. Retinal function was assessed by electroretinography at P30, P60, P90 and P120. The density, structure and synaptic contacts of retinal neurons were analyzed using immunofluorescence and confocal microscopy at P90 and P120. TUDCA-loaded PLGA MSs were spherical, with a smooth surface. The production yield was 78%, the MSs mean particle size was 23 μm and the drug loading resulted 12.5 ± 0.8 μg TUDCA/mg MSs. MSs were able to deliver the loaded active compound in a gradual and progressive manner over the 28-day in vitro release study. Scotopic electroretinografic responses showed increased ERG a- and b-wave amplitudes in TUDCA-PLGA-MSs-treated eyes as compared to those injected with unloaded PLGA particles. TUDCA-PLGA-MSs-treated eyes showed more photoreceptor rows than controls. The synaptic contacts of photoreceptors with bipolar and horizontal cells were also preserved in P23H rats treated with TUDCA-PLGA MSs. This work indicates that the slow and continuous delivery of TUDCA from PLGA-MSs has potential neuroprotective effects that could constitute a suitable therapy to prevent neurodegeneration and visual loss in retinitis pigmentosa. PMID:28542454

  13. Therapeutic efficacy of melatonin in reducing retinal damage in an experimental model of early type 2 diabetes in rats.

    PubMed

    Salido, Ezequiel M; Bordone, Melina; De Laurentiis, Andrea; Chianelli, Mónica; Keller Sarmiento, María Inés; Dorfman, Damián; Rosenstein, Ruth E

    2013-03-01

    Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25 mg/kg). One day or 3 wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 wk of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a-wave, b-wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.

  14. Heat shock response and thermal resistance in cultured human retinal pigment epithelium.

    PubMed

    Wakakura, M; Foulds, W S

    1993-01-01

    The heat shock response was examined in cultured human retinal pigment epithelium (RPE) using indirect immunofluorescence. Mild head shock (39.5-40 degrees C for 1 hr) caused no changes in cell morphology and cells continued to produce the intermediate filament proteins, cytokeratin (keratin) and vimentin. In addition, cells subjected to mild heat shock demonstrated the presence of a heat shock protein (HSP-90). After severe heat shock (45.5-46 degrees C for 1 hr) most cells showed marked morphological changes and, in addition, HSP-90 and/or stress-induced 40 kDa protein production was significantly enhanced. The expression of vimentin was relatively well preserved whereas that of keratin was markedly reduced. When the more severe grade of heat shock was preceded by mild heat shock 20-24 hr earlier, the subsequent severe heat shock resulted in less marked morphological change than in cells not preconditioned and, in addition, the expression of both vimentin and keratin was relatively well preserved. Mildly heat shocked cells appeared to gain thermal resistance supporting the theory that the concomitant synthetic capacity for HSP and normal cellular proteins contributes to thermal resistance. In doubly heat shocked cells, however, HSP-90 expression was not enhanced. The discrepancy between the expression of HSP and thermal resistance is discussed.

  15. Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

    PubMed

    Rickman, D W

    1999-11-15

    The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

  16. Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells.

    PubMed

    Wu, Wen-Chuan; Wu, Meng-Hsien; Chang, Yo-Chen; Hsieh, Ming-Chu; Wu, Horng-Jiun; Cheng, Kai-Chun; Lai, Yu-Hung; Kao, Ying-Hsien

    2010-08-01

    Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with anti-fungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy.

  17. Retinal origin of electrically evoked potentials in response to transcorneal alternating current stimulation in the rat.

    PubMed

    Foik, Andrzej T; Kublik, Ewa; Sergeeva, Elena G; Tatlisumak, Turgut; Rossini, Paolo M; Sabel, Bernhard A; Waleszczyk, Wioletta J

    2015-02-03

    Little is known about the physiological mechanisms underlying the reported therapeutic effects of transorbital alternating current stimulation (ACS) in vision restoration, or the origin of the recorded electrically evoked potentials (EEPs) during such stimulation. We examined the issue of EEP origin and electrode configuration for transorbital ACS and characterized the physiological responses to CS in different structures of the visual system. We recorded visually evoked potentials (VEPs) and EEPs from the rat retina, visual thalamus, tectum, and visual cortex. The VEPs were evoked by light flashes and EEPs were evoked by electric stimuli delivered by two electrodes placed either together on the same eye or on the eyeball and in the neck. Electrically evoked potentials and VEPs were recorded before and after bilateral intraorbital injections of tetrodotoxin that blocked retinal ganglion cell activity. Tetrodotoxin abolished VEPs at all levels in the visual pathway, confirming successful blockage of ganglion cell activity. Tetrodotoxin also abolished EEPs and this effect was independent of the stimulating electrode configurations. Transorbital electrically evoked responses in the visual pathway, irrespective of reference electrode placement, are initiated by activation of the retina and not by passive conductance and direct activation of neurons in other visual structures. Thus, placement of stimulating electrodes exclusively around the eyeball may be sufficient to achieve therapeutic effects. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

  18. Light-induced retinal damage using different light sources, protocols and rat strains reveals LED phototoxicity.

    PubMed

    Krigel, A; Berdugo, M; Picard, E; Levy-Boukris, R; Jaadane, I; Jonet, L; Dernigoghossian, M; Andrieu-Soler, C; Torriglia, A; Behar-Cohen, F

    2016-12-17

    To save energy, the European directives from the Eco-design of Energy Using Products (2005/32/CE) have recommended the replacement of incandescent lamps by more economic devices such as Light Emitting Diodes (LEDs). However, the emission spectrum of these devices is enriched in blue radiations, known to be potentially dangerous to the retina. Recent studies showed that light exposure contributes to the onset of early stages of age-related macular degeneration (AMD). Here, we investigate, in albinos and pigmented rats, the effects of different exposure protocols. Twenty-four hours exposure at high luminance was compared to a cyclic (dark/light) exposure at domestic levels for 1week and 1month, using different LEDs (Cold-white, blue and green), as well as fluorocompact bulbs and fluorescent tubes. The data suggest that the blue component of the white-LED may cause retinal toxicity at occupational domestic illuminance and not only in extreme experimental conditions, as previously reported. It is important to note that the current regulations and standards have been established on the basis of acute light exposure and do not take into account the effects of repeated exposure. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  19. Retinal Electrophysiological Effects of Intravitreal Bone Marrow Derived Mesenchymal Stem Cells in Streptozotocin Induced Diabetic Rats

    PubMed Central

    Akkoç, Tolga; Eraslan, Muhsin; Şahin, Özlem; Özkara, Selvinaz; Vardar Aker, Fugen; Subaşı, Cansu; Karaöz, Erdal; Akkoç, Tunç

    2016-01-01

    Diabetic retinopathy is the most common cause of legal blindness in developed countries at middle age adults. In this study diabetes was induced by streptozotocin (STZ) in male Wistar albino rats. After 3 months of diabetes, rights eye were injected intravitreally with green fluorescein protein (GFP) labelled bone marrow derived stem cells (BMSC) and left eyes with balanced salt solution (Sham). Animals were grouped as Baseline (n = 51), Diabetic (n = 45), Diabetic+BMSC (n = 45 eyes), Diabetic+Sham (n = 45 eyes), Healthy+BMSC (n = 6 eyes), Healthy+Sham (n = 6 eyes). Immunohistology analysis showed an increased retinal gliosis in the Diabetic group, compared to Baseline group, which was assessed with GFAP and vimentin expression. In the immunofluorescence analysis BMSC were observed to integrate mostly into the inner retina and expressing GFP. Diabetic group had prominently lower oscillatory potential wave amplitudes than the Baseline group. Three weeks after intravitreal injection Diabetic+BMSC group had significantly better amplitudes than the Diabetic+Sham group. Taken together intravitreal BMSC were thought to improve visual function. PMID:27300133

  20. Upregulation of Gem relates to retinal ganglion cells apoptosis after optic nerve crush in adult rats.

    PubMed

    Xu, Fan; Huang, Hui; Wu, Yu; Lu, Lu; Jiang, Li; Chen, Lifei; Zeng, Siming; Li, Li; Li, Min

    2014-10-01

    GTP-binding protein Gem, a member protein of the Ras superfamily, can regulate actin cytoskeleton reorganization mediated by Rho-associated coiled-coil-containing protein kinase (ROCK). One attractive activity of the ROCK is playing a potential role in physiological and pathological process in retinal ganglion cells (RGCs) apoptosis. However, the function of Gem in retina is still with limited understanding. To investigate whether Gem is involved in optic nerve injury, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis indicated that Gem was significantly increased in the retina at the 3rd day after ONC. Meanwhile, double-immunofluorescent staining showed that Gem expression was mainly up-regulated in ganglion cell layer and co-localized with NeuN (a marker of RGCs). Additionally, the co-localizations of Gem/active-caspase-3 and Gem/TUNEL-positive cells were detected in RGCs. Furthermore, the expression of active-caspase-3 and TUNEL-positive cells was parallel with that of Gem. Finally, expression pattern of ROCK family (only ROCK2 but not ROCK1) was increased in the differentiated process, which was collected with the expression of GEM and active-caspase-3. Based on the present results, it is suggested that Gem might play a crucial role in RGCs apoptosis after ONC, which might be involved in ROCK pathway.

  1. Retinitis Pigmentosa

    MedlinePlus

    ... Action You are here Home › Retinal Diseases Listen Retinitis Pigmentosa What is retinitis pigmentosa? What are the symptoms? ... is available? What treatment is available? What is retinitis pigmentosa? Retinitis pigmentosa, also known as RP, refers to ...

  2. Discordant Anatomical, Electrophysiological, and Visual Behavioral Profiles of Retinal Degeneration in Rat Models of Retinal Degenerative Disease

    PubMed Central

    McGill, Trevor J.; Prusky, Glen T.; Douglas, Robert M.; Yasumura, Douglas; Matthes, Michael T.; Lowe, Robert J.; Duncan, Jacque L.; Yang, Haidong; Ahern, Kelly; Daniello, Kate M.; Silver, Byron; LaVail, Matthew M.

    2012-01-01

    Purpose. To assess structural, functional, and visual behavioral relationships in mutant rhodopsin transgenic (Tg) rats and to determine whether early optokinetic tracking (OKT) visual experience, known to permanently elevate visual thresholds in normal rats, can enhance vision in rats with photoreceptor degeneration. Methods. Eight lines of pigmented Tg rats and RCS rats were used in this study. OKT thresholds were tested at single ages (1, 2, 3, 4, and 6 months) in naïve groups of rats, or daily in groups that began at eye-opening (P15) or 10 days later (P25). Electroretinogram (ERG) response amplitudes were recorded after OKT testing, and outer nuclear layer (ONL) thickness measurements were then obtained. Results. OKT thresholds, when measured at a single time point in naïve Tg lines beginning at P30, did not decline until months after significant photoreceptor loss. Daily testing of Tg lines resulted mostly with OKT thresholds inversely related to photoreceptor degeneration, with rapid degenerations resulting in sustained OKT thresholds for long periods despite the rapid photoreceptor loss. Slower degenerations resulted in rapid decline of thresholds, long before the loss of most photoreceptors, which was even more pronounced when daily testing began at eye opening. This amplified loss of function was not a result of testing-induced damage to the rod or cone photoreceptors, as ERG amplitudes and ONL thicknesses were the same as untested controls. Conclusions. The unexpected lack of correlation of OKT testing with photoreceptor degeneration in the Tg rats emphasizes the need in behavioral therapeutic studies for careful analysis of visual thresholds of experimental animals prior to therapeutic intervention. PMID:22899760

  3. Differentiation of Lens Tissue from the Progeny of Chick Retinal Pigment Cells Cultured In Vitro: A Demonstration of a Switch of Cell Types in Clonal Cell Culture

    PubMed Central

    Eguchi, Goro; Okada, T. S.

    1973-01-01

    Clonal cell lines isolated from the pigmented retina of 8.5-day chick embryos initially retained the capacity to form pigment. After several passages, however, many cells lost pigment granules and differentiated into lens-like structures. The lens-specific nature of the structures formed in cultures originally derived from retinal pigment cells was established by both ultrastructural and immunological studies. Images PMID:4576021

  4. Purinergic regulation of high-glucose-induced caspase-1 activation in the rat retinal Müller cell line rMC-1

    PubMed Central

    Trueblood, Katherine E.; Mohr, Susanne

    2011-01-01

    Chronic activation of proinflammatory caspase-1 in the retinas of diabetic animals and patients in vivo and retinal Müller cells in vitro is well documented. In this study we characterized how elevated glucose and extracellular purines contribute to the activation of caspase-1 in a cultured rat Müller cell (rMC-1) model. The ability of high glucose (25 mM, 24 h) to activate caspase-1 was attenuated by either apyrase, which metabolizes extracellular ATP to AMP, or adenosine deaminase (ADA), which metabolizes extracellular adenosine to inosine. This suggested that autocrine stimulation of ATP-sensing P2 receptors and adenosine-sensing P1 receptors may in part mediate the response to high glucose. Exogenous ATP, 5′-N-ethylcarboxamido-adenosine (NECA), a nonselective P1 receptor agonist, or forskolin (FSK) increased caspase-1 activity in rMC-1 cells cultured in control glucose (5 mM) medium. Accumulation of active caspase-1 was also increased by dipyridamole, which suppresses adenosine reuptake. High-glucose stimulation of caspase-1 was attenuated by suramin, a nonselective P2 antagonist, or A2 adenosine receptor antagonists, but not by antagonism of P2X7 ATP-gated ion channel receptors. Although high glucose increased P2X7 mRNA, neither P2X7 protein nor function was detected in rMC-1 cells. The increased caspase-1 activity stimulated by high glucose, FSK, NECA, or ATP was correlated with increased gene expression of caspase-1 and thioredoxin-interacting-protein (TXNIP). These findings support a novel role for autocrine P1 and P2 purinergic receptors coupled to cAMP signaling cascades and transcriptional induction of caspase-1 in mediating the high-glucose-induced activation of caspase-1 and secretion of IL-1β in a cell culture model of nonhematopoietic retinal Müller cells. PMID:21832250

  5. Retinal VEGF levels correlate with ocular circulation measured by a laser speckle-micro system in an oxygen-induced retinopathy rat model.

    PubMed

    Matsumoto, Tadashi; Saito, Yuta; Itokawa, Takashi; Shiba, Tomoaki; Oba, Mari S; Takahashi, Haruo; Hori, Yuichi

    2017-08-08

    We used a Laser speckle flowgraphy (LSFG)-micro system to examine the relationship between ocular blood flow and retinal vascular endothelial growth factor (VEGF) at retinopathy onset in oxygen-induced ischemic retinopathy (OIR) model rats. Sixteen 50/10 OIR rats were compared with 17 control rats reared in room air. In postnatal day 14 (P14) and P18 rats, we measured and analyzed the left eye's mean blur rate (MBR) by setting a rubber band on the optic nerve head center, using the LSFG-Micro. At P18, the rats were sacrificed and their left-eye retinas were fixed, flat-mounted and stained with adenosine diphosphatase (ADPase). The right-eye retinas were homogenized; the lysate was centrifuged for an enzyme-linked immunosorbent assay (ELISA). The avascular area was measured as the percentage (%AVA) of the total retinal area. Retinal VEGF was measured by an ELISA. The examination's reproducibility was good. Our multivariate linear mixed model analysis revealed significantly high MBRs in the OIR rats (p = 0.0017). In the P18 OIR rats, significant correlations were seen between the MBR and %AVA (r = 0.80, p = 0.0002) and between the MBR and VEGF (r = 0.76, p = 0.0006). The LSFG-Micro provided reproducible blood flow measurements in neonatal rats. Because of the vitreous blood vessels, measurement of only the retinal vessels was not possible. However, the MBR was higher in the OIR rats than in the control rats, and the MBR and %AVA were correlated, as were the MBR and retinal VEGF. The MBR may thus serve as an indicator of OIR severity.

  6. Modulation of Type-1 and Type-2 Cannabinoid Receptors by Saffron in a Rat Model of Retinal Neurodegeneration

    PubMed Central

    Maccarone, Rita; Rapino, Cinzia; Zerti, Darin; di Tommaso, Monia; Battista, Natalia; Di Marco, Stefano; Bisti, Silvia; Maccarrone, Mauro

    2016-01-01

    Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection. PMID:27861558

  7. Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A-dependent eNOS inactivation.

    PubMed

    García, Celina; Aranda, Jorge; Arnold, Edith; Thébault, Stéphanie; Macotela, Yazmín; López-Casillas, Fernando; Mendoza, Valentín; Quiroz-Mercado, Hugo; Hernández-Montiel, Hebert Luis; Lin, Sue-Hwa; de la Escalera, Gonzalo Martínez; Clapp, Carmen

    2008-06-01

    Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

  8. Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A–dependent eNOS inactivation

    PubMed Central

    García, Celina; Aranda, Jorge; Arnold, Edith; Thébault, Stéphanie; Macotela, Yazmín; López-Casillas, Fernando; Mendoza, Valentín; Quiroz-Mercado, Hugo; Hernández-Montiel, Hebert Luis; Lin, Sue-Hwa; de la Escalera, Gonzalo Martínez; Clapp, Carmen

    2008-01-01

    Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies. PMID:18497878

  9. Damage thresholds for cultured retinal pigment epithelial cells exposed to lasers at 532 nm and 458 nm.

    PubMed

    Denton, Michael L; Foltz, Michael S; Schuster, Kurt J; Estlack, Larry E; Thomas, Robert J

    2007-01-01

    The determination of safe exposure levels for lasers has come from damage assessment experiments in live animals, which typically involve correlating visually identifiable damage with laser dosimetry. Studying basic mechanisms of laser damage in animal retinal systems often requires tissue sampling (animal sacrifice), making justification and animal availability problematic. We determined laser damage thresholds in cultured monolayers of a human retinal pigment epithelial (RPE) cell line. By varying exposure duration and laser wavelength, we identified conditions leading to damage by presumed photochemical or thermal mechanisms. A comparison with literature values for ocular damage thresholds validates the in vitro model. The in vitro system described will facilitate molecular and cellular approaches for understanding laser-tissue interaction.

  10. Human pericyte-endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment.

    PubMed

    Tarallo, Sonia; Beltramo, Elena; Berrone, Elena; Porta, Massimo

    2012-12-01

    Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.

  11. Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration.

    PubMed

    Raz-Prag, Dorit; Grimes, William N; Fariss, Robert N; Vijayasarathy, Camasamudram; Campos, Maria M; Bush, Ronald A; Diamond, Jeffrey S; Sieving, Paul A

    2010-07-13

    Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.

  12. Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration

    PubMed Central

    Raz-Prag, Dorit; Grimes, William N.; Fariss, Robert N.; Vijayasarathy, Camasamudram; Campos, Maria M.; Bush, Ronald A.; Diamond, Jeffrey S.; Sieving, Paul A.

    2010-01-01

    Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K+) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research. PMID:20616020

  13. Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

    PubMed Central

    Froger, Nicolas; Cadetti, Lucia; Lorach, Henri; Martins, Joao; Bemelmans, Alexis-Pierre; Dubus, Elisabeth; Degardin, Julie; Pain, Dorothée; Forster, Valérie; Chicaud, Laurent; Ivkovic, Ivana; Simonutti, Manuel; Fouquet, Stéphane; Jammoul, Firas; Léveillard, Thierry; Benosman, Ryad; Sahel, José-Alain; Picaud, Serge

    2012-01-01

    Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases. PMID:23115615

  14. Taurine provides neuroprotection against retinal ganglion cell degeneration.

    PubMed

    Froger, Nicolas; Cadetti, Lucia; Lorach, Henri; Martins, Joao; Bemelmans, Alexis-Pierre; Dubus, Elisabeth; Degardin, Julie; Pain, Dorothée; Forster, Valérie; Chicaud, Laurent; Ivkovic, Ivana; Simonutti, Manuel; Fouquet, Stéphane; Jammoul, Firas; Léveillard, Thierry; Benosman, Ryad; Sahel, José-Alain; Picaud, Serge

    2012-01-01

    Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.

  15. Antioxidant-Rich Extract from Plantaginis Semen Ameliorates Diabetic Retinal Injury in a Streptozotocin-Induced Diabetic Rat Model

    PubMed Central

    Tzeng, Thing-Fong; Liu, Wayne Young; Liou, Shorong-Shii; Hong, Tang-Yao; Liu, I-Min

    2016-01-01

    Plantaginis semen, the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd. (Plantaginaceae), has been traditionally used to treat blurred vision in Asia. The aim of this work was to investigate the effect of plantaginis semen ethanol extract (PSEE) on the amelioration of diabetic retinopathy (DR) in streptozotocin (STZ)-diabetic rats. PSEE has abundant polyphenols with strong antioxidant activity. PSEE (100, 200 or 300 mg/kg) was oral administrated to the diabetic rats once daily consecutively for 8 weeks. Oral administration of PSEE resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and retinal vascular permeability and leukostasis in diabetic rats. In addition, PSEE administration increased the activities of superoxidase dismutase (SOD) and catalase (CAT), and glutathione peroxidase (GSH) level in diabetic retinae. PSEE treatment inhibited the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and the phosphorylation of Akt without altering the Akt protein expression in diabetic retinae. PSEE not only down-regulated the gene expression of hypoxia-inducible factor-1α (TNF-α) and interleukin-1β (IL-1β), but also reduced ICAM-1 and VCAM-1 expression in diabetic retinae. Moreover, PSEE reduced the nuclear factor-κB (NF-κB) activation and corrected imbalance between histone deacetylases (HDAC) and histone acetyltransferases (HAT) activities in diabetic retinae. In conclusion, phenolic antioxidants extract from plantaginis semen has potential benefits in the prevention and/or progression of DR. PMID:27649243

  16. Effects of curcumin on interleukin-23 and interleukin-17 expression in rat retina after retinal ischemia-reperfusion injury.

    PubMed

    Zhang, Hai-Jiang; Xing, Yi-Qiao; Jin, Wei; Li, Dai; Wu, Kaili; Lu, Yi

    2015-01-01

    This study aimed to investigate the effect of curcumin on the retinal structure and the expressions of interleukin-23 (IL-23) and IL-17 in the rat retina after retinal ischemia-reperfusion injury (RIRI). 150 Sprague-Dawley rats were randomly divided into RIRI group (MG), low-dose curcumin group (LDCG) and high-dose curcumin group (HDCG), (n = 50 per group). RIRI was generated by anterior chamber perfusion of normal saline to the right eye. The left eye served as a normal control group (NCG). Rats in LDCG and HDCG received an intraperitoneal injection of 20 mg/kg/d and 100 mg/kg/d curcumin respectively, at 30 min before RIRI and once daily after RIRI. The morphological changes in HDCG group were improved as compared to MG and LDCG groups. Immunohistochemistry showed that IL-23 and IL-17 were mainly expressed in the ganglion cell layer and the inner nuclear layer of the retina. Low IL-23 and IL-17 expressions were observed in NCG, but increased significantly in MG and LDCG groups. Western blot assay and ELISA also showed that IL-23 and IL-17 expressions increased significantly after RIRI (vs. NCG, P<0.01). Moreover, the IL-23 expression reached a peak at 24 h, whereas IL-17 expression peaked at 72 h after RIRI. Curcumin reduced IL-23 and IL-17 expressions significantly in a dose-dependent manner (vs. MG, P<0.01). The IL-23 and IL-17 expressions increase after RIRI and curcumin significantly reduces retinal IL-23 and IL-17 expressions in a dose-dependent manner and is able to prevent the RIRI induced damage to the retina.

  17. Morphological diversity of displaced retinal ganglion cells in the rat: a lucifer yellow study.

    PubMed

    Buhl, E H; Dann, J F

    1988-03-08

    Displaced retinal ganglion cells (DRGCs) were retrogradely labelled by injections of the fluorescent dye Fast Blue into the superior colliculi of pigmented rats. Following fixation these cells were intracellularly injected with Lucifer Yellow to determine their dendritic morphology and distribution. Graphic reconstruction of Lucifer Yellow-filled prelabelled neurones revealed a heterogeneous population of DRGCs. Their stratification within the inner plexiform layer was diverse and cells were classified according to their dendritic morphology. The present sample consists largely of unistratifying neurones, the dendrites of which arborized within a narrow sublamina of the inner plexiform layer. They were characterized by a centrally located soma and densely branched dendritic network with little overlap within the branching pattern. In contrast, bistratifying DRGCs possessed a loose and sparsely branched dendritic structure, while diffusely stratifying neurones contained a high degree of dendritic crossing, culminating in a complex network, in which the soma position was biased toward the periphery. One type of DRGC bore a striking resemblance to type 1 neurones (Perry, 1979; Proc. R. Soc. Lond. [Biol.] 204:363-375) in the ganglion cell layer. They were characterized by a large soma (15.5 micron +/- 2.2 micron s.d.) and a dendritic field diameter averaging 288 micron (s.d. +/- 62 micron) and were on average larger than the rest of the displaced population but smaller than type 1 cells in the ganglion cell layer. Since the stratification patterns of the displaced and nondisplaced type 1 neurones were indistinguishable, it is reasonable to assume that the Lucifer Yellow-filled cells in the present study represent the displaced counterpart of regular type 1 ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Early Gene Expression Changes in the Retinal Ganglion Cell Layer of a Rat Glaucoma Model

    PubMed Central

    Guo, Ying; Johnson, Elaine C.; Cepurna, William O.; Dyck, Jennifer A.; Doser, Tom

    2011-01-01

    Purpose. To identify patterns of early gene expression changes in the retinal ganglion cell layer (GCL) of a rodent model of chronic glaucoma. Methods. Prolonged elevation of intraocular pressure (IOP) was produced in rats by episcleral vein injection of hypertonic saline (N = 30). GCLs isolated by laser capture microdissection were grouped by grading of the nerve injury (<25% axon degeneration for early injury; >25% for advanced injury). Gene expression was determined by cDNA microarray of independent GCL RNA samples. Quantitative PCR (qPCR) was used to further examine the expression of selected genes. Results. By array analysis, 533 GCL genes (225 up, 308 down) were significantly regulated in early injury. Compared to only one major upregulated gene class of metabolism regulation, more were downregulated, including mitochondria, ribosome, proteasome, energy pathways, protein synthesis, protein folding, and synaptic transmission. qPCR confirmed an early upregulation of Atf3. With advanced injury, 1790 GCL genes were significantly regulated (997 up, 793 down). Altered gene categories included upregulated protein synthesis, immune response, and cell apoptosis and downregulated dendrite morphogenesis and axon extension. Of all the early changed genes, 50% were not present in advanced injury. These uniquely affected genes were mainly associated with upregulated transcription regulation and downregulated protein synthesis. Conclusions. Early GCL gene responses to pressure-induced injury are characterized by an upregulation of Atf3 and extensive downregulation in genes associated with cellular metabolism and neuronal functions. Most likely, these changes represent those specific to RGCs and are thus potentially important for enhancing RGC survival in glaucoma. PMID:21051717

  19. Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

    PubMed Central

    Vigneswara, Vasanthy; Berry, Martin; Logan, Ann; Ahmed, Zubair

    2012-01-01

    Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL+ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis. PMID:23285297

  20. Rearrangement of the cone mosaic in the retina of the rat model of retinitis pigmentosa.

    PubMed

    Ji, Yerina; Zhu, Colleen L; Grzywacz, Norberto M; Lee, Eun-Jin

    2012-03-01

    In retinitis pigmentosa (RP), the death of cones normally follows some time after the degeneration of rods. Recently, surviving cones in RP have been studied and reported in detail. These cones undergo extensive remodeling in their morphology. Here we report an extension of the remodeling study to consider possible modifications of spatial-distribution patterns. For this purpose we used S334ter-line-3 transgenic rats, a transgenic model developed to express a rhodopsin mutation causing RP. In this study, retinas were collected at postnatal (P) days P5-30, 90, 180, and P600. We then immunostained the retinas to examine the morphology and distribution of cones and to quantify the total cone numbers. Our results indicate that cones undergo extensive changes in their spatial distribution to give rise to a mosaic comprising an orderly array of rings. These rings first begin to appear at P15 at random regions of the retina and become ubiquitous throughout the entire tissue by P90. Such distribution pattern loses its clarity by P180 and mostly disappears at P600, at which time the cones are almost all dead. In contrast, the numbers of cones in RP and normal conditions do not show significant differences at stages as late as P180. Therefore, rings do not form by cell death at their centers, but by cone migration. We discuss its possible mechanisms and suggest a role for hot spots of rod death and the remodeling of Müller cell process into zones of low density of photoreceptors. Copyright © 2011 Wiley Periodicals, Inc.

  1. Culture Model of Rat Portal Myofibroblasts

    PubMed Central

    El Mourabit, Haquima; Loeuillard, Emilien; Lemoinne, Sara; Cadoret, Axelle; Housset, Chantal

    2016-01-01

    Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4–5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in

  2. Culture Model of Rat Portal Myofibroblasts.

    PubMed

    El Mourabit, Haquima; Loeuillard, Emilien; Lemoinne, Sara; Cadoret, Axelle; Housset, Chantal

    2016-01-01

    Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4-5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in

  3. Protective effect of Aster tataricus extract on retinal damage on the virtue of its antioxidant and anti-inflammatory effect in diabetic rat.

    PubMed

    Du, Hao; Zhang, Meng; Yao, Kejun; Hu, Zhitao

    2017-03-02

    Effect of Aster tataricus (AT) was estimated on the retinal injury in diabetic rats by its antioxidant and anti-inflammatory activity. Streptozotocin (STZ) was used to induce diabetes at a dose of 60mg/kg, i.p. and blood glucose was estimated to confirm the diabetic rats. All the animals were separated in to 5 different groups (n=10) such as control, diabetic retinopathy (DR) receives saline solution, and AT treated group receives AT (100, 200 and 400mg/kg) for the duration of 8 week. After treatment protocol period blood glucose and HbA1c% was estimated in the blood sample of diabetic rats. Retinal tissue was isolated for the fundus photography and retinal vessel diameter, retinal vascular permeability and leukocytosis were estimated. Moreover in the retinal tissue homogenate oxidative stress parameters such as superoxide dismutase (SOD), glutathione peroxidase (GSH) and catalase (CAT) and concentration of cytokines (TNFα, IL10) was estimated. Result of the study suggested that root extract of AT contain rich amount of polyphenol in it which significantly reduces the body weight and concentration of glucose in blood in diabetic rats. Fundus photography suggested that AT extract attenuates the structure and functional abnormalities that develops due to diabetes. Retinal leukocytosis and vascular permeability was significantly decreases in AT treated group than DR group. There was significant increase in the activity of GSH, CAT and SOD in AT treated group than DR group. Moreover AT also attenuates the altered concentration of TNFα, IL10 and NF-κB in the retina of STZ induced diabetic rat. Thus present study concludes that root extract of AT effectively manages the diabetic retinopathy by controlling the blood glucose and also by attenuating the altered oxidative stresss and inflammatory mediators such as TNFα, IL10 and NF-κB in the retina of STZ induced diabetic rat.

  4. Novel Transgenic Mouse Models Develop Retinal Changes Associated with Early Diabetic Retinopathy Similar to Those Observed in Rats with Diabetes Mellitus

    PubMed Central

    Guo, Changmei; Zhang, Zifeng; Zhang, Peng; Makita, Jun; Kawada, Hiroyoshi; Blessing, Karen; Kador, Peter F.

    2014-01-01

    Retinal capillary pericyte degeneration has been linked to aldose reductase (AR) activity in diabetic retinopathy (DR). Since the development of DR in mice and rats has been reported to differ and that this may be linked to differences in retinal sorbitol levels, we have established new murine models of early onset diabetes mellitus as tools for investigating the role of AR in DR. Transgenic diabetic mouse models were developed by crossbreeding diabetic C57BL/6-Ins2Akita/J (AK) with transgenic C57BL mice expressing green fluorescent protein (GFP), human aldose reductase (hAR) or both in vascular tissues containing smooth muscle actin-α (SMAA). Changes in retinal sorbitol levels were determined by HPLC while changes of growth factors and signaling were investigated by Western Blots. Retinal vascular changes were quantitatively analyzed on elastase-digestion flat mounts. Results show that sorbitol levels were higher in neural retinas of diabetic AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors VEGF, IGF-1, bFGF and TGFβ, as well as signaling changes in P-Akt, P-SAPK/JNK, and P-44/42 MAPK. Increased loss of nuclei per capillary length and a significant increase in the percentage of acellular capillaries presented in 18 week old AK-SMAA-GFP-hAR mice. These changes are similar to those observed in streptozotocin-induced diabetic rats. Retinal changes in both mice and rats were prevented by inhibition of AR. These studies confirm that the increased expression of AR in mice results in the development of retinal changes associated with the early stages of DR that are similar to those observed in rats. PMID:24370601

  5. Visual evoked potential in RCS rats with Okayama University-type retinal prosthesis (OUReP™) implantation.

    PubMed

    Alamusi; Matsuo, Toshihiko; Hosoya, Osamu; Uchida, Tetsuya

    2017-02-08

    Photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis or OUReP™, generates light-evoked surface electric potentials and stimulates neurons. The dye-coupled films or plain films were implanted subretinally in both eyes of 10 Royal College of Surgeons rats with hereditary retinal dystrophy at the age of 6 weeks. Visual evoked potentials in response to monocular flashing light stimuli were recorded from cranially-fixed electrodes, 4 weeks and 8 weeks after the implantation. After the recording, subretinal film implantation was confirmed histologically in 7 eyes with dye-coupled films and 7 eyes with plain films. The recordings from these 7 eyes in each group were used for statistical analysis. The amplitudes of visual evoked potentials in the consecutive time points from 125 to 250 ms after flash were significantly larger in the 7 eyes with dye-coupled film implantation, compared to the 7 eyes with plain film implantation at 8 weeks after the implantation (P < 0.05, repeated-measure ANOVA). The photoelectric dye-coupled polyethylene film, as retinal prosthesis, gave rise to visual evoked potential in response to flashing light.

  6. Protective effects of PF-4708671 against N-methyl-d-aspartic acid-induced retinal damage in rats.

    PubMed

    Hayashi, Ikumi; Aoki, Yuto; Ushikubo, Hiroko; Asano, Daiki; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2016-12-01

    We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-d-aspartic acid (NMDA)-induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF-4708671, an inhibitor of S6 kinase, protects against NMDA-induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45-positive leukocytes and Iba1-positive microglia. Surprisingly, simultaneous injection of PF-4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF-4708671 and rapamycin likely protect against NMDA-induced retinal damage via distinct pathways. The neuroprotective effect of PF-4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF-4708671 is reported to be a S6 kinase inhibitor.

  7. Co-Expression of Two Subtypes of Melatonin Receptor on Rat M1-Type Intrinsically Photosensitive Retinal Ganglion Cells

    PubMed Central

    Sheng, Wen-Long; Chen, Wei-Yi; Yang, Xiong-Li; Zhong, Yong-Mei; Weng, Shi-Jun

    2015-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions. PMID:25714375

  8. Visually evoked changes in the rat retinal blood flow measured with Doppler optical coherence tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Tan, Bingyao; Mason, Erik; MacLellan, Ben; Bizheva, Kostadinka

    2017-02-01

    Visually evoked changes of retinal blood flow can serve as an important research tool to investigate eye disease such as glaucoma and diabetic retinopathy. In this study we used a combined, research-grade, high-resolution Doppler OCT+ERG system to study changes in the retinal blood flow (RBF) and retinal neuronal activity in response to visual stimuli of different intensities, durations and type (flicker vs single flash). Specifically, we used white light stimuli of 10 ms and 200 ms single flash, 1s and 2s for flickers stimuli of 20% duty cycle. The study was conducted in-vivo in pigmented rats. Both single flash (SF) and flicker stimuli caused increase in the RBF. The 10 ms SF stimulus did not generate any consistent measurable response, while the 200 ms SF of the same intensity generated 4% change in the RBF peaking at 1.5 s after the stimulus onset. Single flash stimuli introduced 2x smaller change in RBF and 30% earlier RBF peak response compared to flicker stimuli of the same intensity and duration. Doubling the intensity of SF or flicker stimuli increased the RBF peak magnitude by 1.5x. Shortening the flicker stimulus duration by 2x increased the RBF recovery rate by 2x, however, had no effect on the rate of RBF change from baseline to peak.

  9. Involvement of Fra-1 in Retinal Ganglion Cell Apoptosis in Rat Light-Induced Retina Damage Model.

    PubMed

    Liu, Xiaojuan; Yang, Xiaowei; Zhu, Rongrong; Dai, Ming; Zhu, Manhui; Shen, Yuntian; Fang, Hongda; Sang, Aimin; Chen, Hui

    2017-01-01

    Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

  10. Protection of cone photoreceptor M-opsin degradation with 9-cis-β-carotene-rich alga Dunaliella bardawil in Rpe65(-/-) mouse retinal explant culture.

    PubMed

    Ozaki, Taku; Nakazawa, Mitsuru; Kudo, Takashi; Hirano, Satoshi; Suzuki, Kaori; Ishiguro, Sei-ichi

    2014-12-01

    RPE65, a retinal pigment epithelium-specific 65-kDa protein, plays a critical role in the visual cycle of the eye. Rpe65(-/-) mice develop vision loss due to a lack of 11-cis-retinal, degradation of M-opsin and mislocalization of S-opsin. Several studies have suggested that 9-cis-β-carotene, a precursor of 9-cis-retinal and all-trans-retinal, could have therapeutic applications in vision loss. We therefore examined whether Dunaliella bardawil, a 9-cis-β-carotene-rich alga, protects against the degradation of M-opsin using Rpe65(-/-) mouse retinal explant cultures. The eyes of three-week-old Rpe65(-/-) and C57BL/6 J mice were enucleated, and the corneas were removed. The eyecups were incubated with culture medium in the absence or presence of D. bardawil for 6 h to 4 days. Localizations of M-opsin proteins in the retina were observed immunohistochemically. Expression levels of M-opsin, S-opsin and rhodopsin proteins were evaluated by Western blot analysis. In C57BL/6 J mouse retina, no change was observed in localization and expression levels of M-opsin in the explant culture system. In Rpe65(-/-) mouse retina, the amount of M-opsin protein was decreased in the photoreceptor outer segment after 6 h to 4 days of culture. However, the presence of D. bardawil significantly ameliorated this decrease. In contrast, expression levels of S-opsin and rhodopsin were unchanged in the presence of the explant culture. These results demonstrate that D. bardawil treatment protects against M-opsin degradation in Rpe65(-/-) mouse retina and suggest that D. bardawil has therapeutic potential for retinal degeneration caused by Rpe65 gene mutation, such as Leber congenital amaurosis and retinitis pigmentosa.

  11. Quantitative retinal and choroidal blood flow during light, dark adaptation and flicker light stimulation in rats using fluorescent microspheres.

    PubMed

    Shih, Yen-Yu I; Wang, Lin; De La Garza, Bryan H; Li, Guang; Cull, Grant; Kiel, Jeffery W; Duong, Timothy Q

    2013-02-01

    The present study aimed to quantify retinal and choroidal blood flow (BF) during light, dark adaptation and flicker light stimulation using the microsphere technique. Adult male Sprague-Dawley rats were anesthetized with isoflurane. Eyes were dark (Group I, n = 8), light (Group II, n = 8) adapted or stimulated with 10 Hz flicker light (Group III, n = 10). Retinal and choroidal BF were measured by a previously established method, using a mixture of 8 µm yellow-green and 10 µm red fluorescent microspheres. The microspheres were counted ex vivo in the dissected retina and choroid and in the reference arterial blood under a fluorescent microscope. The choroidal BF was 64.8 ± 29 µl/min (mean ± SD) during dark adaptation, not significantly different from that during light adaptation (66.0 ± 17.8 µl/min). The retinal BF was 13.5 ± 3.2 µl/min during 10 Hz flickering light stimulation, significantly higher than that during dark adaptation in the control fellow eyes (9.9 ± 2.9 µl/min). The choroidal BF values were not statistically different between flicker stimulation and dark adaptation. Retinal BF was 11.6 ± 2.9 µl/min during light adaptation. Dark adaptation did not increase retinal BF (Group I, 8.2 ± 2.4 µl/min; Group II, 9.9 ± 2.9 µl/min). These findings argue against a dark-induced or flicker-induced functional hyperemia in the choroid as a result of the demands of the outer retina. Retinal BF was not higher during dark adaptation. Our data support the conclusion that the inner retina has a higher energy demand in flicker conditions relative to dark.

  12. Multi-Modal Proteomic Analysis of Retinal Protein Expression Alterations in a Rat Model of Diabetic Retinopathy

    PubMed Central

    Kutzler, Lydia; Brucklacher, Robert M.; Bronson, Sarah K.; Kimball, Scot R.; Freeman, Willard M.

    2011-01-01

    Background As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. Methodology/Principal Findings A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. Conclusions/Significance These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies. PMID:21249158

  13. Laser speckle imaging of rat retinal blood flow with hybrid temporal and spatial analysis method

    NASA Astrophysics Data System (ADS)

    Cheng, Haiying; Yan, Yumei; Duong, Timothy Q.

    2009-02-01

    Noninvasive monitoring of blood flow in retinal circulation will reveal the progression and treatment of ocular disorders, such as diabetic retinopathy, age-related macular degeneration and glaucoma. A non-invasive and direct BF measurement technique with high spatial-temporal resolution is needed for retinal imaging. Laser speckle imaging (LSI) is such a method. Currently, there are two analysis methods for LSI: spatial statistics LSI (SS-LSI) and temporal statistical LSI (TS-LSI). Comparing these two analysis methods, SS-LSI has higher signal to noise ratio (SNR) and TSLSI is less susceptible to artifacts from stationary speckle. We proposed a hybrid temporal and spatial analysis method (HTS-LSI) to measure the retinal blood flow. Gas challenge experiment was performed and images were analyzed by HTS-LSI. Results showed that HTS-LSI can not only remove the stationary speckle but also increase the SNR. Under 100% O2, retinal BF decreased by 20-30%. This was consistent with the results observed with laser Doppler technique. As retinal blood flow is a critical physiological parameter and its perturbation has been implicated in the early stages of many retinal diseases, HTS-LSI will be an efficient method in early detection of retina diseases.

  14. Connexin43 Mimetic Peptide Improves Retinal Function and Reduces Inflammation in a Light-Damaged Albino Rat Model.

    PubMed

    Guo, Cindy X; Mat Nor, Mohd N; Danesh-Meyer, Helen V; Vessey, Kirstan A; Fletcher, Erica L; O'Carroll, Simon J; Acosta, Monica L; Green, Colin R

    2016-08-01

    Drugs that regulate connexin43 (Cx43) gap junction channels can reduce the spread of injury and improve functional outcomes after nervous system trauma. In the eye, Cx43 expression increases in the choroid following light damage. The aim of this study was to investigate whether Cx43 hemichannel block could preserve retinal function postinjury. Light damage was induced by exposure of adult albino Sprague-Dawley rats to 2700 Lux light for 24 hours. Intravitreal injections of a Cx43 mimetic peptide hemichannel blocker, Peptide5, or sham were administered 2 hours after the onset and at the end of the light damage period. Retinal function was assessed by electroretinogram and inflammatory responses in the choroid and retina were assessed using immunohistochemistry (ionized calcium binding adaptor molecule 1 [Iba-1], leukocyte common antigen [CD45], glial fibrillary acidic protein [GFAP]). Light-damaged rat eyes had (1) reduced neuronal responses in both the rod and cone pathways and (2) marked inflammatory responses in the choroid and retina. Peptide5 significantly preserved function of photoreceptoral and postphotoreceptoral neurons in these animals. This was evident 24 hours after injury and 2 weeks later, as shown by improved mixed a-wave and mixed b-wave amplitudes, isolated rod PII and PIII amplitudes, and cone PII responses when compared with sham-treated controls. Retinal thinning and inflammation were also significantly reduced in Peptide5-treated eyes when compared with sham-treated controls. Blocking Cx43 hemichannels after light damage can significantly improve functional outcomes of neurons in both the rod and cone photo-transduction pathways in the light-damaged animal model, likely by reducing choroid inflammation and suppressing the glial-mediated inflammatory response. These data may have relevance for the treatment of conditions such as diabetic retinopathy and age-related macular degeneration.

  15. Remote Ischemic Preconditioning Protects Retinal Photoreceptors: Evidence From a Rat Model of Light-Induced Photoreceptor Degeneration.

    PubMed

    Brandli, Alice; Johnstone, Daniel M; Stone, Jonathan

    2016-10-01

    To test whether remote ischemic preconditioning (RIP) is protective to photoreceptors, in a light damage model, and to identify mechanisms involved. A pressure cuff was used to induce ischemia (2 × 5 minutes) in one hind limb of 4- to 6-month-old albino Sprague-Dawley rats raised in dim, cyclic light (12 hours 5 lux, 12 hours dark). Immediately following the ischemia, rats were exposed to bright continuous light (1000 lux) for 24 hours. After 7-day survival in dim, cyclic light conditions, retinal function was assessed using the flash electroretinogram (ERG) and retinal structure was examined for photoreceptor survival and death, as well as for stress. Messenger RNA and protein expression of growth factors and brain-derived neurotrophic factor (BDNF) receptors was also assessed at 7-day survival. Bright light exposure reduced the amplitude of the a- and b-waves of the ERG, upregulated the expression of glial fibrillary acidic protein (GFAP) by Müller cells, increased the number of dying (TUNEL+) photoreceptors, and reduced the number of surviving photoreceptors. Remote ischemic preconditioning mitigated all of these bright light-induced effects. Remote ischemic preconditioning-induced protection was associated with increased retinal expression of BDNF and its low-affinity receptor NGFR. The present study provides evidence, for the first time, that RIP protects photoreceptors against bright light-induced photoreceptor degeneration. This observation is consistent with previous reports of RIP-induced protection of the inner retina and of other vital organs. Brain-derived neurotrophic factor may play a role in mediating the RIP-induced neuroprotection through activation of NGFR.

  16. Intracellular Signalling in Retinal Ischemia

    DTIC Science & Technology

    1990-07-01

    36) However, vascularization of the RPE is not known to occur in human diseases of photoreceptor degeneration, such as retinitis pigmentosa ...A.C. (1986) Retinitis pigmentosa and retinal neovascularization. Ophthalmology 91, 1599- 1603. Figure la: Control rat retina, 8 weeks of age, central...TITLE (Include Security Classification) Intracellular Signalling in Retinal Ischemia 12. PERSONAL AUTHOR(S) Burns, Margaret Sue; Bellhorn, Roy William

  17. Glucocorticoid-Induced Leucine Zipper Protects the Retina From Light-Induced Retinal Degeneration by Inducing Bcl-xL in Rats.

    PubMed

    Gu, Ruiping; Tang, Wenyi; Lei, Boya; Ding, Xinyi; Jiang, Cheng; Xu, Gezhi

    2017-07-01

    The aim of the present study was to investigate the neuroprotective effects of glucocorticoid-induced leucine zipper (GILZ) in a light-induced retinal degeneration model and to explore the underlying mechanisms. Intravitreal injection of recombinant GILZ-overexpressing lentivirus (OE-GILZ-rLV) and short hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV) was performed to up- and downregulate retinal GILZ, respectively. Three days after stable transduction, rats were exposed to continuous bright light (5000 lux) for 2 days. Retinal function was assessed by full-field electroretinography (ERG), and the retinal structure was examined for photoreceptor survival and death in rats kept under a 12-hour light:2-hour dark cycle following light exposure. The expression levels of retinal Bcl-xL, caspase-9, and caspase-3 were examined by Western blotting or real-time PCR at 1, 3, 5, and 7 days after light exposure. Exposure to bright light downregulated retinal GILZ in parallel with the downregulation of Bcl-xL and the upregulation of active caspase-3. Overexpression of retinal GILZ attenuated the decrease of Bcl-xL and the activation of caspase-9 and caspase-3 at 1, 3, 5, and 7 days after bright light exposure, respectively. GILZ silencing aggravated the downregulation of Bcl-xL induced by bright light exposure. Bright light exposure reduced the amplitude of ERG, increased the number of apoptotic photoreceptor cells, and decreased retinal thickness; and GILZ overexpression could attenuate all these effects. Overexpression of GILZ by OE-GILZ-rLV transduction protected the retina from light-induced cellular damage by activating antiapoptotic pathways.

  18. The Effect of TIMP-1 on the Cone Mosaic in the Retina of the Rat Model of Retinitis Pigmentosa

    PubMed Central

    Ji, Yerina; Yu, Wan-Qing; Eom, Yun Sung; Bruce, Farouk; Craft, Cheryl Mae; Grzywacz, Norberto M.; Lee, Eun-Jin

    2015-01-01

    Purpose. The array of photoreceptors found in normal retinas provides uniform and regular sampling of the visual space. In contrast, cones in retinas of the S334ter-line-3 rat model for RP migrate to form a mosaic of rings, leaving large holes with few or no photoreceptors. Similar mosaics appear in human patients with other forms of retinal dystrophy. In the current study, we aimed to investigate the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1) on the mosaic of cones in S334ter-line-3 rat retinas. We focused on TIMP-1 because it is one of the regulators of the extracellular matrix important for cellular migration. Methods. Immunohistochemistry was performed to reveal M-opsin cone cells (M-cone) and the results were quantified to test statistically whether or not TIMP-1 restores the mosaics to normal. In particular, the tests focused on the Voronoi and nearest-neighbor distance analyses. Results. Our tests indicated that TIMP-1 led to significant disruption of the M-opsin cone rings in S334ter-line-3 rat retinas and resulted in almost complete homogeneous mosaics. In addition, TIMP-1 induced the M-cone spatial distribution to become closer to random with decreased regularity in S334ter-line-3 rat retinas. Conclusions. These findings confirm that TIMP-1 induced M-cone mosaics in S334ter-line-3 to gain homogeneity without reaching the degree of regularity seen in normal retinal mosaics. Even if TIMP-1 fails to promote regularity, the effects of this drug on homogeneity appear to be so dramatic that TIMP-1 may be a potential therapeutic agent. TIMP-1 improves sampling of the visual field simply by causing homogeneity. PMID:25515575

  19. Effect of Brimonidine on Retinal and Choroidal Neovascularization in a Mouse Model of Retinopathy of Prematurity and Laser-Treated Rats

    PubMed Central

    Kusari, Jyotirmoy; Padillo, Edwin; Zhou, Sheila X.; Bai, Yanyan; Wang, Juanjuan; Song, Zhiming; Zhu, Meili; Le, Yun-Zheng; Gil, Daniel W.

    2011-01-01

    Purpose. To determine whether chronic treatment with brimonidine (BRI) attenuates retinal vascular leakage and neovascularization in neonatal mice after exposure to high oxygen in a mouse model of retinopathy of prematurity (ROP), and choroidal neovascularization (CNV) in rats after laser treatment. Methods. Experimental CNV was induced by laser treatment in Brown Norway (BN) rats. BRI or vehicle (VEH) was administered by osmotic minipumps, and CNV formation was measured 11 days after laser treatment. Oxygen-induced retinopathy was generated in neonatal mice by exposure to 75% oxygen from postnatal day (P)7 to P12. BRI or VEH was administered by gavage, and vitreoretinal vascular endothelial growth factor (VEGF) concentrations and retinal vascular leakage, neovascularization, and vaso-obliteration were measured on P17. Experimental CNV was induced in rabbits by subretinal lipopolysaccharide/fibroblast growth factor-2 injection. Results. Systemic BRI treatment significantly attenuated laser-induced CNV formation in BN rats when initiated 3 days before or within 1 hour after laser treatment. BRI treatment initiated during exposure to high oxygen significantly attenuated vitreoretinal VEGF concentrations, retinal vascular leakage, and retinal neovascularization in P17 mice subjected to oxygen-induced retinopathy. Intravitreal treatment with BRI had no effect on CNV formation in a rabbit model of nonischemic angiogenesis. Conclusions. BRI treatment significantly attenuated vitreoretinal VEGF concentrations, retinal vascular leakage, and retinal and choroidal neovascularization in animal models of ROP and CNV. BRI may inhibit underlying event(s) of ischemia responsible for upregulation of vitreoretinal VEGF and thus reduce vascular leakage and retinal-choroidal neovascularization. PMID:21482645

  20. Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

    PubMed

    Voisin, Pierre; Bernard, Marianne

    2009-07-01

    The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

  1. Light-Emitting Diodes and Cool White Fluorescent Light Similarly Suppress Pineal Gland Melatonin and Maintain Retinal Function and Morphology in the Rat. Part 1

    NASA Technical Reports Server (NTRS)

    Holley, Daniel C.; Heeke, D.; Mele, G.

    1999-01-01

    Currently, the light sources most commonly used in animal habitat lighting are cool white fluorescent or incandescent lamps. We evaluated a novel light-emitting diode (LED) light source for use in animal habitat lighting by comparing its effectiveness to cool white fluorescent light (CWF) in suppressing pineal gland melatonin and maintaining normal retinal physiology and morphology in the rat. Results of pineal melatonin suppression experiments showed equal suppression of pineal melatonin concentrations for LED light and CWF light at five different light illuminances (100, 40, 10, 1 and 0.1 lux). There were no significant differences in melatonin suppression between LED and CWF light when compared to unexposed controls. Retinal physiology was evaluated using electroretinography. Results show no differences in a-wave implicit times and amplitudes or b-wave implicit times and amplitudes between 100-lux LED-exposed rats and 100-lux CWF-exposed rats. Results of retinal histology assessment show no differences in retinal thickness rod outer segment length and number of rod nuclei between rats exposed to 100-lux LED and 100-lux CWF for days. Furthermore, the retinal pigmented epithelium and rod outer segments of all eyes observed were in good condition and of normal thickness. This study indicates that LED light does not cause retinal damage and can suppress pineal melatonin at similar intensities as a conventional CWF light source. These data suggest that LED light sources may be suitable replacements for conventional light sources used in the lighting of rodent vivariums while providing many mechanical and economical advantages.

  2. Bioengineered teeth from cultured rat tooth bud cells.

    PubMed

    Duailibi, M T; Duailibi, S E; Young, C S; Bartlett, J D; Vacanti, J P; Yelick, P C

    2004-07-01

    The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

  3. In-vivo PET imaging of implanted human retinal pigment epithelium cells in a Parkinson's disease rat model.

    PubMed

    Wang, Ruimin; Zhang, Jinming; Guo, Zhe; Shen, Li; Shang, Aijia; Chen, Yingmao; Yao, Shulin; He, Tingting; Yin, Dayi; Tian, Jiahe

    2008-05-01

    Researchers find that monitoring the differentiation of implanted cells in vivo is difficult. This study was designed to show that it is possible to track the efficacy of transplanted human retinal pigment epithelial cells (RPE cells) in a rat model of Parkinson's disease by using positron emission tomography (PET). RPE cells or normal saline were injected into striatum of the injured side of the rat model in treated and control groups, respectively. PET imaging of both groups was undertaken before transplantation and at intervals afterwards, using C-raclopride and C-beta-CFT as the markers. Observation of the rats' behaviour and immunofluorescence confocal microscopy were also used to prove the PET results. PET studies showed increased accumulation of C-raclopride and decreased C-beta-CFT in the injured side of striatum in both groups. C-raclopride decreased along with a concomitant increase of C-beta-CFT after transplantation in the treated group. The changes shown by the PET studies paralleled the behavioural states and confocal microscopy observations in the treated animals. These results suggest that even a clinical PET scanner could, to a certain extent, provide some information on the existence and in-vivo differentiation of RPE cells in a rat model of Parkinson's disease.

  4. Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma.

    PubMed

    Tsai, James C; Wu, Li; Worgul, Basil; Forbes, Max; Cao, Jingtai

    2005-11-01

    The aim of this pilot study was to evaluate the potential neuroprotective effect of an intravitreal injection of erythropoietin (EPO) on retinal ganglion cell (RGC) preservation in an episcleral vessel cautery-induced rat model of glaucoma. The animals were randomly assigned into an unoperated control group (n = 11) and three experimental groups: episcleral vessel cautery only (EVC: n = 4), episcleral vessel cautery with intravitreal normal saline injection (EVC-NS; n = 5), and episcleral vessel cautery with intravitreal EPO treatment (EVC-EPO; n = 9). The intravitreal injections were limited to 5 mul containing either normal saline alone or 200 ng of EPO in normal saline administered immediately after the cautery procedure. RGCs were labeled retrogradely by FluoroGold neuron tracer 5 to 7 days prior to the collection of eyes at day 21 and counted in whole flat-mounted retinas with fluorescence microscopy. Compared to the RGC counts in retinal specimens from unoperated control rats (12,619 +/- 310), the corresponding RGC counts were significantly decreased in both the EVC (9116 +/- 273; p < 0.005) and EVC-NS (9489 +/- 293; p < 0.005) groups but not significantly decreased in the EVC-EPO (11,212 +/- 414; p = 0.051) treated retinas. A single intravitreal 200 ng dose of EPO appears to have a protective effect on RGC viability in an in vivo rat model of glaucoma. Further experimental studies are needed to confirm these preliminary results and to optimize the appropriate dose and frequency of EPO delivery in animal models of glaucoma.

  5. Application of microdialysis to evaluate the efflux transport of estradiol 17-beta glucuronide across the rat blood-retinal barrier.

    PubMed

    Katayama, Kazunori; Ohshima, Yuki; Tomi, Masatoshi; Hosoya, Ken-ichi

    2006-09-30

    The purpose of the present study was to evaluate vitreous humor/retina-to-blood efflux transport in rats and determine the efflux transport of estradiol 17-beta glucuronide (E17betaG) across the blood-retinal barrier (BRB) by the use of microdialysis. [(3)H]E17betaG and [(14)C]D-mannitol, which were used as a model compound for amphipathic organic anions and a bulk flow marker, respectively, were injected into the vitreous humor of rat eye, and a microdialysis probe was placed in the vitreous humor. [(3)H]E17betaG and [(14)C]D-mannitol were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]E17betaG during the terminal phase was 1.9-fold greater than that of [(14)C]D-mannitol and reduced the level of [(14)C]D-mannitol in the retinal presence of 0.3 mM E17betaG, suggesting that [(3)H]E17betaG is transported via a carrier-mediated efflux transport process across the BRB. The efflux transport of [(3)H]E17betaG was significantly inhibited by organic anions, such as probenecid, sulfobromophthalein, digoxin, and dehydroepiandrosterone sulfate, whereas it was not inhibited by p-aminohippuric acid. In conclusion, the efflux transport of [(3)H]E17betaG across the rat BRB was evaluated by microdialysis and its inhibition by organic anions suggests organic anion transporting polypeptide 1a4-mediated E17betaG efflux transport at the BRB.

  6. Nitric oxide regulates cell survival in purified cultures of avian retinal neurons: involvement of multiple transduction pathways.

    PubMed

    Mejía-García, T A; Paes-de-Carvalho, R

    2007-01-01

    Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S-nitroso-acetyl-d-l-penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5'-phosphate (8Br-cGMP) (GMP) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the

  7. Functional Characterization of Carrier-Mediated Transport of Pravastatin across the Blood-Retinal Barrier in Rats.

    PubMed

    Fujii, Shinobu; Setoguchi, Chikako; Kawazu, Kouichi; Hosoya, Ken-ichi

    2015-12-01

    Systemically administered pravastatin effectively treats diabetic retinopathy without central nervous system side effects. The efflux transport mechanism of pravastatin from the brain has already been clarified. In this study, the influx of pravastatin across the blood-retinal and blood-brain barriers (BRB and BBB) and the efflux of pravastatin from the retina were investigated using rats. Pravastatin influx (blood-to-tissues) was assessed using the retinal and brain uptake index (RUI and BUI) methods, and microdialysis was performed to investigate the efflux (retina-to-blood) transport of pravastatin. The RUI and BUI values for [(3)H]pravastatin were lower than those expected based on its lipophilicity, suggesting that the influx transport across the BRB and BBB was less than the reverse-direction transport. The RUI and BUI values for [(3)H]pravastatin were significantly decreased by pravastatin, digoxin, and probenecid, indicating that pravastatin undergoes carrier-mediated influx transport in the blood-to-tissues direction across the BRB and BBB. After intravitreal injection, [(3)H]pravastatin and the bulk flow marker [(14)C]d-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [(3)H]pravastatin during the terminal phase was 1.66-fold greater than that of [(14)C]d-mannitol. Efflux transport was reduced in the retinal presence of pravastatin, digoxin, and benzylpenicillin, suggesting that pravastatin is transported via efflux transporters. In conclusion, pravastatin is transported across the BRB via uptake and efflux transporters in both the blood-to-retina and retina-to-blood directions, and the retina-to-blood transporters are dominant, based on the lower values of the RUI compared with the values expected from the lipophilicity.

  8. ‘Slow activity transients’ in infant rat visual cortex: a spreading synchronous oscillation patterned by retinal waves

    PubMed Central

    Colonnese, Matthew T.; Khazipov, Rustem

    2010-01-01

    A primary feature of the preterm infant electroencephalogram is the presence of large infra-slow potentials containing rapid oscillations called Slow Activity Transients (SATs). Such activity has not been described in animal models, and their generative mechanisms are unknown. Here we use direct-current and multi-site extracellular, as well as whole-cell, recording in vivo to demonstrate the existence of regularly repeating SATs in the visual cortex of infant rats before eye-opening. Present only in absence of anesthesia, SATs at post-natal day 10-11 were identifiable as a separate group of long-duration (∼10s) events that consisted of large (>1 mV) negative local-field potentials produced by the summation of multiple bursts of rapid oscillatory activity (15-30 Hz). SATs synchronized the vast majority of neuronal activity (87%) in the visual cortex before eye-opening. Enucleation eliminated SATs, and their duration, inter-event interval and sub-burst structure matched those of phase III retinal waves recorded in vitro. Retinal waves, however, lacked rapid oscillations, suggesting they arise centrally. Multi-electrode recordings showed that SATs spread horizontally in cortex and synchronize activity at co-active locales via the rapid oscillations. SATs were clearly different from ongoing cortical activity, which was observable as a separate class of short bursts from P9. Together our data suggest that in vivo, early cortical activity is largely determined by peripheral inputs--retinal waves in visual cortex--which provide excitatory input, and by thalamocortical circuitry, which transforms this input to beta oscillations. We propose that the synchronous oscillations of SATs participate in the formation of visual circuitry. PMID:20335468

  9. The contribution of inner and outer retinal photoreceptors to infra-slow oscillations in the rat olivary pretectal nucleus.

    PubMed

    Orlowska-Feuer, Patrycja; Allen, Annette E; Storchi, Riccardo; Szkudlarek, Hanna J; Lewandowski, Marian H

    2016-03-01

    A subpopulation of olivary pretectal nucleus (OPN) neurons discharges action potentials in an oscillatory manner, with a period of approximately two minutes. This 'infra-slow' oscillatory activity depends on synaptic excitation originating in the retina. Signals from rod-cone photoreceptors reach the OPN via the axons of either classic retinal ganglion cells or intrinsically photosensitive retinal ganglion cells (ipRGCs), which use melanopsin for photon capturing. Although both cell types convey light information, their physiological functions differ considerably. The aim of the present study was to disentangle how rod-cone and melanopsin photoresponses contribute to generation of oscillatory activity. Pharmacological manipulations of specific phototransduction cascades were used whilst recording extracellular single-unit activity in the OPN of anaesthetized rats. The results show that under photopic conditions (bright light), ipRGCs play a major role in driving infra-slow oscillations, as blocking melanopsin phototransmission abolishes or transiently disturbs oscillatory firing of the OPN neurons. On the other hand, blocking rod-cone phototransmission does not change firing patterns in photopic conditions. However, under mesopic conditions (moderate light), when melanopsin phototransmission is absent, blocking rod-cone signalling causes disturbances or even the disappearance of oscillations implying that classic photoreceptors are of greater importance under moderate light. Evidence is provided that all photoreceptors are required for the generation of oscillations in the OPN, although their roles in driving the rhythm are determined by the lighting conditions, consistent with their relative sensitivities. The results further suggest that maintained retinal activity is crucial to observe infra-slow oscillatory activity in the OPN. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Quantitative and regional measurement of retinal blood flow in rats using N-isopropyl-p-[14C]-iodoamphetamine ([14C]-IMP).

    PubMed

    Pouliot, Mylène; Deschênes, Micheline C; Hétu, Simon; Chemtob, Sylvain; Lesk, Mark R; Couture, Réjean; Vaucher, Elvire

    2009-12-01

    Quantitative and regional measurement of retinal blood flow in rodents is of prime interest for the investigation of regulatory mechanisms of ocular circulation in physiological and pathological conditions. In this study, a quantitative autoradiographic method using N-isopropyl-p-(14)C-iodoamphetamine ([(14)C]-IMP), a diffusible radioactive tracer, was evaluated for its ability to detect changes in retinal blood perfusion during hypercapnia. Findings were compared to cerebral blood flow values measured simultaneously. Hypercapnia was induced in awaken Wistar rats by inhalation of 5% or 8% CO(2) in medical air for 5 min. [(14)C]-IMP (100 microCi/kg) was injected in the femoral vein over a 30 s period and the rats were sacrificed 2 min later. Blood flow was calculated from whole-mount retinae and 20 microm thick brain sections in discrete regions of interest by quantitative autoradiography or from digested samples of retina and brain by liquid scintillation counting. Retinal blood flow values measured with quantitative and regional autoradiography were higher in the central (108 +/- 20 ml/100 g/min) than in peripheral (84 +/- 15 ml/100 g/min) retina. These values were within the same range as cortical blood flow values (97 +/- 4 ml/100 g/min). The retinal blood flow values obtained on whole-mount retinae were validated by the sampling method. Hypercapnia significantly increased overall blood flow in the retina (24-53%) with a maximal augmentation in the peripheral region and in the brain (22-142%). The changes were stronger in the brain compared to retina (p = 0.016). These results demonstrate that retinal blood flow can be quantified using [(14)C]-IMP and compared with cerebral blood flow. This technique is a powerful tool to study how retinal blood flow is regulated in different regions of the rat retina.

  11. Enhancement of retinal pigment epithelial culture characteristics and subretinal space tolerance of scaffolds with 200 nm fiber topography.

    PubMed

    Liu, Zengping; Yu, Na; Holz, Frank G; Yang, Fang; Stanzel, Boris V

    2014-03-01

    Tissue engineered retinal pigment epithelial (RPE) transplantation is a promising cell-based therapy for age-related macular degeneration. The aim of this work is to develop a supportive scaffold with a favorable topography to aid functional RPE monolayer maintenance while being tolerated underneath the retina. To this end, films and electrospun substrates with fiber diameters ranging from 200 to 1000 nm were made of polyethylene terephthalate or poly(L-lactide-co-ε-caprolactone), and then tested using human fetal RPE cells in vitro and transplanted subretinally in rabbits. The results indicated that RPE on both 200 nm fiber variants showed the highest cell densities, adherent monolayers achieved deeper pigmentation, and more uniform hexagonal tight junctions. Facile subretinal implantation of flat 200 nm fiber membranes was achieved by electrospinning them onto a porous rigid-elastic carrier. Spectral-domain optical coherence tomography showed a reattached, slightly thinned retina overlying the implants over 2 weeks observation. Histology demonstrated native RPE variably migrated onto the nanofibers, and a reactive gliosis with some photoreceptor degeneration. In conclusion, scaffolds with 200 nm fiber topography enhanced RPE culture, showed subretinal biocompatibility, and should thus be considered for future cell-based therapies in blinding retinal diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells

    PubMed Central

    Sohn, Seongsoo; Im, Ji-Eun; Kim, Tae Eun

    2013-01-01

    Purpose To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. Methods Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. Results Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. Conclusions Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease. PMID:23372380

  13. Production of compartmented cultures of rat sympathetic neurons.

    PubMed

    Campenot, Robert B; Lund, Karen; Mok, Sue-Ann

    2009-01-01

    The compartmented culture, in which primary neurons plated in a proximal compartment send their axons under silicone grease barriers and into left and right distal compartments, has enhanced the experimental capabilities of neuronal cultures. Treatments can be applied separately to cell bodies/proximal axons or distal axons, and cell bodies/proximal axons and distal axons can be separately harvested and analyzed. Distal axons can be axotomized, and the neurons can be studied while their axons regenerate. Construction of the culture dishes requires 3 h for 48 cultures, and preparing the neurons also requires 3 h. Compartmented cultures provide enough cellular material for biochemical analyses such as immunoblotting. The uses of compartmented cultures have included studies of neurotrophic factor retrograde signaling, axonal transport, and axonal protein and lipid biosynthesis. Here we focus on sympathetic neurons cultured from neonatal rats and provide protocols for the production and some of the uses of compartmented cultures.

  14. Taurine buffers glutamate homeostasis in retinal cells in vitro under hypoxic conditions.

    PubMed

    Chen, Fang; Mi, Mantian; Zhang, Qianyong; Wei, Na; Chen, Ka; Xu, Hongxia; Yuan, Jialin; Zhou, Yong; Lang, Haibin; Yu, Xiaoping; Wang, Bin; Wang, Jian; Tang, Yong; Chang, Hui

    2010-01-01

    We investigated whether taurine indirectly protects neurons under hypoxia by affecting retinal Müller cells, which are known to play important roles in the regulation of retinal glutamate content. Retinal cells isolated from rats were exposed to hypoxia for 24 h. We evaluated the retinal neuron survival, glutamate content in cultures with and without taurine under hypoxic conditions. The glutamate clearance function correlated with the expression of glutamine synthetase (GS) mRNA and L-glutamate/L-aspartate transporter (GLAST) mRNA. Immunohistochemical staining of glial fibrillary acidic protein (GFAP), vimentin and S-100 protein was performed to examine cytoskeletal changes in retinal Müller cells. Retinal neurons treated with taurine exhibited significantly higher survival rates than those without taurine under hypoxia. Taurine inhibited the upregulation of GFAP and vimentin, and inhibited the downregulation of GLAST, GS and the nuclear-cytoplasmic ratio of S-100 under hypoxia. In addition, taurine inhibited the upregulation of the glutamate content in neurons and retinal Müller cells upon hypoxic exposure. These data suggest that hypoxic damage to cultured retinal cells is decreased by taurine. The neuroprotection by taurine may relate to buffering glutamate homeostasis via modulation of the glutamate clearance by retinal Müller cells. Copyright 2010 S. Karger AG, Basel.

  15. α-Aminoadipic acid protects against retinal disruption through attenuating Müller cell gliosis in a rat model of acute ocular hypertension

    PubMed Central

    Wang, Xiaolei; Su, Jier; Ding, Jingwen; Han, Song; Ma, Wei; Luo, Hong; Hughes, Guy; Meng, Zhaoyang; Yin, Yi; Wang, Yanling; Li, Junfa

    2016-01-01

    Objective Ocular hypertension is an important risk factor for glaucoma. The purpose of this study was to investigate the gliotoxic effects of α-aminoadipic acid (AAA) in a rat model of AOH and its underlying mechanisms. Materials and methods In the rat model of acute ocular hypertension (AOH), intraocular pressure was increased to 110 mmHg for 60 minutes. Animals were divided into four groups: sham operation (Ctrl), AOH, AOH + phosphate-buffered saline (PBS), and AOH + AAA. Cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay, and retinal ganglion cells (RGCs) immunostained with Thy-1 were counted. Müller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein. Tumor necrosis factor-α (TNF-α) was examined using Western blot. Results In the rat model of AOH, cell apoptosis was induced in the ganglion cell layer and the number of RGCs was decreased. Müller cell gliosis in the retinas of rats was induced, and retinal protein levels of TNF-α were increased. Intravitreal treatment of AAA versus PBS control attenuated these retinal abnormalities to show protective effects in the rat model of AOH. Conclusion In the retinas of the rat model of AOH, AAA treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of RGCs, likely through the attenuation of Müller cell gliosis and suppression of TNF-α induction. Our observations suggest that AAA might be a potential therapeutic target in glaucoma. PMID:27799744

  16. α-Aminoadipic acid protects against retinal disruption through attenuating Müller cell gliosis in a rat model of acute ocular hypertension.

    PubMed

    Wang, Xiaolei; Su, Jier; Ding, Jingwen; Han, Song; Ma, Wei; Luo, Hong; Hughes, Guy; Meng, Zhaoyang; Yin, Yi; Wang, Yanling; Li, Junfa

    2016-01-01

    Ocular hypertension is an important risk factor for glaucoma. The purpose of this study was to investigate the gliotoxic effects of α-aminoadipic acid (AAA) in a rat model of AOH and its underlying mechanisms. In the rat model of acute ocular hypertension (AOH), intraocular pressure was increased to 110 mmHg for 60 minutes. Animals were divided into four groups: sham operation (Ctrl), AOH, AOH + phosphate-buffered saline (PBS), and AOH + AAA. Cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay, and retinal ganglion cells (RGCs) immunostained with Thy-1 were counted. Müller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein. Tumor necrosis factor-α (TNF-α) was examined using Western blot. In the rat model of AOH, cell apoptosis was induced in the ganglion cell layer and the number of RGCs was decreased. Müller cell gliosis in the retinas of rats was induced, and retinal protein levels of TNF-α were increased. Intravitreal treatment of AAA versus PBS control attenuated these retinal abnormalities to show protective effects in the rat model of AOH. In the retinas of the rat model of AOH, AAA treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of RGCs, likely through the attenuation of Müller cell gliosis and suppression of TNF-α induction. Our observations suggest that AAA might be a potential therapeutic target in glaucoma.

  17. Effects of increasing numbers of phagocytic inclusions on human retinal pigment epithelial cells in culture: a model for aging.

    PubMed Central

    Boulton, M; Marshall, J

    1986-01-01

    Cultures of human retinal pigment epithelial cells have been challenged with a number of biological (lipofuscin, melanin, and rod outer segments) and non-biological (latex microspheres) particles at a variety of concentrations. The particles were chosen to include examples of both degradable and non-degradable systems. A range of morphological changes were observed by phase contrast microscopy, and these became more atypical with increasing concentration. At the highest concentration cells had ingested so many particles that many had died and others had ruptured. The time course of these changes indicated a relationship between cellular lytic activity and the capacity of the particle to degrade. The potential of this system as a model for studying senescence is discussed. Images PMID:3790481

  18. Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush.

    PubMed

    Barron, K D; Dentinger, M P; Krohel, G; Easton, S K; Mankes, R

    1986-06-01

    Rat retinal ganglion cell layer (GCL) was examined ultrastructurally 1-180 days after intraorbital crushing of one optic nerve. It was confirmed quantitatively that axotomized ganglion cells lost cisternal membranes of the rough endoplasmic reticulum (RER) and showed disintegration of Nissl bodies and ribosomal rosettes 3 days postoperatively. Between 60 and 180 days after neurotomy there was partial reversion of the RER towards normal. At postoperative intervals of 14-60 days, chromatin aggregation became conspicuous and some nuclei were prominently furrowed and contained electron-dense inclusions. Concurrently, profiles of dead ganglion cells were encountered. Mean mitochondrial area increased in axotomized neurons but mitochondrial density declined, while the Golgi apparatus, lamellar specializations of the RER and the size of nuclei did not change significantly. Cytoplasmic atrophy was profound, however. Small nerve cells of the GCL appeared morphologically distinct from ganglion cells and did not undergo appreciable alteration. A decline in neuronal density, approximating 35%, occurred between the third and seventh postoperative day and progressed slowly thereafter. Neuronal density was 32% of normal 180 days postoperatively. A temporary increase in glial density 3-28 days after operation was due to microglial hyperplasia. Müller cell and astrocytic processes hypertrophied, infiltrated nerve fibre bundles, and surrounded and intruded into neuronal somata. Bundles of unmyelinated small axons, invested by astrocytes and basal lamina, were present within the necrotic cavity of the lesioned nerve 28-90 days postoperatively and had cytologic features of regenerative axonal sprouts. We conclude that intraorbital optic nerve crush is followed by a noteworthy degree of regenerative axonal sprouting which occurs and persists against a background of slow but relentless decline in the retinal ganglion cell population. This slow decline follows a rapidly-sustained loss

  19. Improved retinal function in RCS rats after suppressing the over-activation of mGluR5.

    PubMed

    Dai, Jiaman; Fu, Yan; Zeng, Yuxiao; Li, Shiying; Qin Yin, Zheng

    2017-06-14

    Müller cells maintain retinal synaptic homeostasis by taking up glutamate from the synaptic cleft and transporting glutamine back to the neurons. To study the interaction between Müller cells and photoreceptors, we injected either DL-α-aminoadipate or L-methionine sulfoximine-both inhibitors of glutamine synthetase-subretinally in rats. Following injection, the a-wave of the electroretinogram (ERG) was attenuated, and metabotropic glutamate receptor 5 (mGluR5) was activated. Selective antagonism of mGluR5 by 2-methyl-6-(phenylethynyl)-pyridine increased the ERG a-wave amplitude and also increased rhodopsin expression. Conversely, activation of mGluR5 by the agonist, (R,S)-2-chloro-5-hydroxyphenylglycine, decreased both the a-wave amplitude and rhodopsin expression, but upregulated expression of Gq alpha subunit and phospholipase C βIII. Overexpression of mGluR5 reduced the inward-rectifying potassium ion channel (Kir) current and decreased the expression of Kir4.1 and aquaporin-4 (AQP4). Further experiments indicated that mGluR5 formed a macromolecular complex with these two membrane channels. Lastly, increased expression of mGluR5 was found in Royal College of Surgeons rats-a model of retinitis pigmentosa (RP). Inhibition of mGluR5 in this model restored the amplitude of ERG features, and reduced the expression of glial fibrillary acidic protein. These results suggest that mGluR5 may be worth considering as a potential therapeutic target in RP.

  20. Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model.

    PubMed

    Martin, Keith R G; Quigley, Harry A; Zack, Donald J; Levkovitch-Verbin, Hana; Kielczewski, Jennifer; Valenta, Danielle; Baumrind, Lisa; Pease, Mary Ellen; Klein, Ronald L; Hauswirth, William W

    2003-10-01

    To develop a modified adenoassociated viral (AAV) vector capable of efficient transfection of retinal ganglion cells (RGCs) and to test the hypothesis that use of this vector to express brain-derived neurotrophic factor (BDNF) could be protective in experimental glaucoma. Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n = 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n = 30), or AAV-green fluorescent protein (GFP)-WPRE (n = 33). Two weeks later, experimental glaucoma was induced in the injected eye by laser application to the trabecular meshwork. Survival of RGCs was estimated by counting axons in optic nerve cross sections after 4 weeks of glaucoma. Transgene expression was assessed by immunohistochemistry, Western blot analysis, and direct visualization of GFP. The density of GFP-positive cells in retinal wholemounts was 1,828 +/- 299 cells/mm(2) (72,273 +/- 11,814 cells/retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% +/- 27.1% in the saline-treated group (n = 25) and 52.3% +/- 24.2% in the AAV-GFP-WPRE group (n = 30), but only 32.3% +/- 23.0% in the AAV-BDNF-WPRE group (n = 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P = 0.002, t-test) or saline (P = 0.006, t-test). Overexpression of the BDNF gene protects RGC as estimated by axon counts in a rat glaucoma model, further supporting the potential feasibility of neurotrophic therapy as a complement to the lowering of IOP in the treatment of glaucoma.

  1. Alzheimer's and Danish dementia peptides induce cataract and perturb retinal architecture in rats.

    PubMed

    Reddy, G Bhanuprakash; Reddy, P Yadagiri; Surolia, Avadhesha

    2017-03-01

    Familial Danish dementias (FDDs) are autosomal dominant neurodegenerative disorders that are associated with visual defects. In some aspects, FDD is similar to Alzheimer's disease (AD)- the amyloid deposits in FDD and AD are made of short peptides: amyloid β (Aβ) in AD and ADan in FDD. Previously, we demonstrated an interaction between the dementia peptides and α-crystallin leading to lens opacification in organ culture due to impaired chaperone activity of α-crystallin. Herein, we report the in vivo effects of ADan and Aβ on the eye. ADan [reduced (ADan-red) and oxidized (ADan-oxi)] and Aβ (Aβ1-40 and Aβ1-42) were injected intravitreally in rats. The onset of cataract was seen after injection of all the peptides, but the cataract matured by 2 weeks in the case of ADan-red, 5 weeks for ADan-oxi and 6 weeks for Aβ1-40, while Aβ1-42 had minimal effect on cataract progression. The severity of cataract is associated with insolubilization and alterations in crystallins and loss of chaperone activity of α-crystallin. Further, disruption of the architecture of the retina was evident from a loss of rhodopsin, increased gliosis, and the thinning of the retina. These results provide a basis for the dominant heredo-otoophthalmo-encephalopathy (HOOE)/FDD syndrome and indicate that ADan peptides are more potent than Aβpeptides in inflicting visual impairment.

  2. Argon inhalation attenuates retinal apoptosis after ischemia/reperfusion injury in a time- and dose-dependent manner in rats.

    PubMed

    Ulbrich, Felix; Schallner, Nils; Coburn, Mark; Loop, Torsten; Lagrèze, Wolf Alexander; Biermann, Julia; Goebel, Ulrich

    2014-01-01

    Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat's eye. IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA. IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%. Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon's possible role as a therapeutic option.

  3. Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats

    PubMed Central

    Ulbrich, Felix; Schallner, Nils; Coburn, Mark; Loop, Torsten; Lagrèze, Wolf Alexander; Biermann, Julia; Goebel, Ulrich

    2014-01-01

    Purpose Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat's eye. Methods IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA. Results IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%. Conclusion Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon's possible role as a therapeutic option. PMID

  4. Neuropathy optic glaucomatosa induced by systemic hypertension through activation endothelin-1 signaling pathway in central retinal artery in rats

    PubMed Central

    Prayitnaningsih, Seskoati; Sujuti, Hidayat; Effendi, Maksum; Abdullah, Aulia; Anandita, Nanda Wahyu; Yohana, Febriani; Permatasari, Nur; Widodo, Mohamad Aris

    2016-01-01

    AIM To evaluate effect of hypertension on retinal ganglion cell (RGC) apoptosis, intraocular pressure (IOP), and the activation of endothelin-1 (ET-1) signaling pathway in central retinal artery (CRA) in rats. METHODS The experimental study was performed on 20 male Sprague Dawley rats that were divided into control group, and hypertension groups. The hypertension was induced by subcutaneous deoxycorticoacetate (DOCA) 10 mg/kg twice a week and administered 0.9% NaCl solution daily for 2, 6, and 10wk. Blood pressure (BP) was measured using animal BP analyzer. IOP was measured by handheld tonometry. Retinal tissue preparations by paraffin blocks were made after enucleation. The expression of ET-1, eNOS, ET-1 receptor A (ETRA), ET-1 receptor B (ETRB), and phosphorylated myosin light chain kinase (MLCK), and caldesmon (CaD) in CRA and RGC apoptosis were evaluated through immunofluorescent staining method then observed using laser scanning confocal microscopy. RESULTS BP significantly increased in all of the hypertension groups compared to control (P=0.001). Peak IOP elevation (7.78±4.14 mm Hg) and RGC apoptosis (576.15±33.28 Au) occurred on 2wk of hypertension. ET-1 expression (1238.6±55.1 Au) and eNOS expression (2814.2±70.7 Au) were found highest in 2wk of hypertension, although the ratio of ET-1/eNOS decreased since 2wk. ETRA reached peak expression in 10wk of hypertension (1219.4±6.3 Au), while ETRB significantly increased only in 2 weeks group (1069.2±9.6 Au). The highest MLCK expression (1190.09±58.32 Au), CaD (1670.28±18.36 Au) were also found in 2wk of hypertension. CONCLUSION Hypertension effects to activation of ET-1 signaling pathway significantly in CRA, elevation of IOP, and RGC apoptosis. The highest value was achieved at 2wk, which is the development phase of hypertension. PMID:27990358

  5. Characterization of GABA- and glycine-induced currents of solitary rodent retinal ganglion cells in culture.

    PubMed

    Tauck, D L; Frosch, M P; Lipton, S A

    1988-10-01

    Ganglion cells were fluorescently labeled, dissociated from 7- to 11-day-old rodent retinas, and placed in tissue culture. Whole-cell recordings with patch electrodes were obtained from solitary cells lacking processes, which permitted a high-quality space clamp. Both GABA (1-200 microM) and glycine (10-300 microM) produced large increases in membrane conductance in virtually every ganglion cell tested, including ganglion cells from different size classes in both rats and mice. Taurine evoked responses similar to those of glycine, but considerably greater concentrations of taurine (150-300 microM) were necessary to observe any effect. Since 20 microM GABA produced approximately the same response as 100 microM glycine, the effects of these two concentrations were compared under various conditions. When recording with chloride distributed equally across the membrane, the reversal potential of the agonist-induced currents was approximately 0 mV. When the internal chloride was reduced by substitution with aspartate, the reversal potential shifted in a negative direction by about 42 mV, indicating that the current was carried mainly by chloride ions. Strychnine (1-5 microM) completely and reversibly blocked the actions of glycine (100 microM) but not those of GABA (20 microM); however, higher concentrations of strychnine (20 microM) nearly totally inhibited the current elicited by GABA (20 microM). The responses to glycine (100 microM) were not affected by bicuculline methiodide (20 microM) or picrotoxinin (20 microM). In contrast, bicuculline methiodide (10 microM) and picrotoxinin (10 microM) reversibly blocked the current evoked by GABA (20 microM); d-tubocurarine (100 microM) only slightly decreased the response to GABA (20 microM). The antagonists were effective over a wide range of holding potentials (-90 mV to +30 mV). The responses to a steady application of both GABA and glycine decayed in a few seconds when recorded under conditions of both symmetric and

  6. Low-density lipoprotein receptor-related protein-1 (LRP-1) expression in a rat model of oxygen-induced retinal neovascularization.

    PubMed

    Sánchez, María C; Barcelona, Pablo F; Luna, Jose D; Ortiz, Susana G; Juarez, Patricio C; Riera, Clelia M; Chiabrando, Gustavo A

    2006-12-01

    The low-density lipoprotein receptor-related protein-1 (LRP-1) is a high-molecular weight receptor of the LDL receptor gene family. Its ability to bind and internalize both proteinases and proteinase-inhibitor complexes from the extracellular space suggests that it has a major role in modulating uncontrolled retinal cell proliferation. In order to test this assumption, we investigated the expression of LRP-1 and receptor-associated ligands in a rat model of oxygen-induced retinal neovascularization. Wistar albino rats were placed into incubators at birth and exposed to an atmosphere alternating between 50% and 10% of oxygen every 24 h. After 14 days, the animals were allowed to recover in room air and sacrificed at postnatal day 20 (P20). The protein expression of LRP-1 and alpha2-macroglobulin (alpha2M) in the retina from unexposed and hyperoxia-exposed rats was investigated by Western blot. The localization of LRP-1 after neovascularization was assessed by immunohistochemical staining. The activity of metalloproteinases (MMPs) was determined by zymography. Histological analysis was done to quantitate the neovascular response in these animals. Western blot analysis showed that LRP-1 was expressed, along with alpha2M, in the retina of rats with oxygen-induced neovascularization at P20. By immunohistochemical analysis, positive staining for LRP-1 appeared in cells extending from the inner limiting membrane (ILM) to the outer limiting membrane (OLM). The cells of the retina that expressed LRP-1 were identified by immunofluorescence as Müller cells. Zymographic analysis demonstrated increased activity of MMP-2 and MMP-9 under neovascular conditions. This is the first demonstration of the involvement of LRP-1 in retinal neovascularization. In retinas of rats with oxygen-induced neovascularization, the expression of LRP-1 and alpha2M was increased along with an enhanced activity of MMPs, suggesting that LRP-1 expression may play a role in modulating retinal

  7. Red light of the visual spectrum attenuates cell death in culture and retinal ganglion cell death in situ.

    PubMed

    Del Olmo-Aguado, Susana; Núñez-Álvarez, Claudia; Osborne, Neville N

    2016-09-01

    To ascertain whether red light, known to enhance mitochondrial function, can blunt chemical insults to cell cultures and ischaemic insults to the rat retina. Raised intraocular pressure (IOP, 140 mmHg, 60 min) or ischaemia was delivered in complete darkness or in the presence of low intensity red light (16.5 watts/m(2) , 3000 lux, 625-635 nm) to one eye of each rat. Animals were killed at specific times after ischemia and retinas analysis for ganglion cell numbers, the localization of specific antigens or for changes in defined RNAs. RGC-5 cell cultures were also exposed to various chemical insults in the presence or absence of red light. Significant differences were determined by t-test and anova. Elevation of IOP causes changes in the localization of glial fibrillary acid protein (GFAP), calretinin, calbindin, choline acetyltransferase, ganglion cell numbers and an elevation (GFAP, vimentin, HO-1 and mTORC1) or reduction (Thy-1 and Brn3a) of mRNAs in the rat retina. These negative effects to the rat retina caused by ischaemia are reduced by red light. Moreover, chemical insults to cell cultures are blunted by red light. Low, non-toxic levels of red light focussed on the retina for a short period of time are sufficient to attenuate an insult of raised IOP to the rat retina. Since mitochondrial dysfunctions are thought to play a major role in ganglion cell death in glaucoma, we propose the potential use of red light therapy for the treatment of the disease. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  8. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    PubMed Central

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  9. Kv3 channels modulate calcium signals induced by fast firing patterns in the rat retinal ganglion cells.

    PubMed

    Kuznetsov, Kirill I; Grygorov, Oleksii O; Maslov, Vitaly Yu; Veselovsky, Nikolay S; Fedulova, Svetlana A

    2012-11-01

    Expression of non-inactivating Kv3.1/Kv3.2 potassium channels determines fast-spiking phenotype of many types of neurones including retinal ganglion cells (RGCs); furthermore Kv3 channels regulate neurotransmitter release from presynaptic terminals. In the present study we investigated how inhibition of Kv3 channel by low TEA concentrations modifies firing properties and Ca2+ influx in the rat RGCs. Experiments were performed on the whole-mount retinal preparations from 4 to 6 weeks old Wistar rats using simultaneous whole cell patch clamp and intracellular Ca2+ measurements in combination with single-cell RT-PCR. In response to 500-ms depolarization step the RGCs demonstrated fast firing tonic behaviour with a mean frequency of spiking 61±5 Hz (n=28). All of the tonic cells tested (n=9) expressed specific mRNA for either Kv3.1 or Kv3.2 or for both channels. Bath applications of TEA (250 μM, 500 μM and 1 mM) modified firing patterns dose-dependently as follows: firing frequency was decreased, mean action potential (AP) half-width increased and mean amplitude of after hyperpolarization was reduced. The amplitude of the Ca2+ signals induced by the cells firing was linearly dependent on number of APs with a mean slope of 7.3±0.9 nM per one AP (n=8). APs widening by TEA increased the slope of the amplitude vs. AP number plots in a dose-dependent manner: 250 μM of TEA increased the mean slope value to 9.5±1.2 nM/AP, 500 μM to 12.4±2.4 nM/AP and 1 mM to 13.2±2.9 nM/AP (n=6). All these parameters, as well as the cells firing properties, were significantly different from controls and from each other except between 500 μM and 1 mM. This is consistent with the pharmacological properties of Kv3.1/Kv3.2 channels: the TEA IC50 is in the range 150-300 μM with almost complete block at 1 mM. This suggests that Kv3.1/Kv3.2 channels underlie the fast firing of the rat RGCs and provide at a given firing frequency 1.8-fold restriction Ca2+ influx, thus protecting the cells

  10. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

    S. Lin1, L. M. Del Razo1, M. Styblo1, C. Wang2, W. R. Cullen2, and D.J. Thomas3. 1Univ. North Carolina, Chapel Hill, NC; 2Univ. British Columbia, Vancouver, BC, Canada; 3National Health and En...

  11. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

    S. Lin1, L. M. Del Razo1, M. Styblo1, C. Wang2, W. R. Cullen2, and D.J. Thomas3. 1Univ. North Carolina, Chapel Hill, NC; 2Univ. British Columbia, Vancouver, BC, Canada; 3National Health and En...

  12. Rat embryonic palatal shelves respond to TCDD in organ culture

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1990-05-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in (3H)TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves.

  13. Comparison of the in vitro toxicity of indocyanine green to that of trypan blue in human retinal pigment epithelium cell cultures.

    PubMed

    Gale, Jeffrey S; Proulx, Alain A; Gonder, John R; Mao, Alexander J; Hutnik, Cindy M L

    2004-07-01

    To compare the in vitro toxicity of indocyanine green (ICG) to that of trypan blue (TB) in human retinal pigment epithelium cell cultures. The use of ICG and TB in macular hole surgery is discussed. In vitro cell biology experimental study. The ICG dye and TB were applied to ARPE-19, a commercially available human retinal pigment epithelium cell line. Cultures were established and maintained according to supplier protocols. The ICG dye, TB or Hank's balanced salt solution (controls) were then applied to the cells at varying concentrations and over various exposure periods. Fiberoptic light was also applied to cells to assess for the possibility of a potentiating phototoxic effect. Cell viability fractions were determined using a well-studied mitochondrial dehydrogenase assay. The TB was not toxic to the retinal pigment epithelium cell cultures at any concentration or over any period of exposure, whereas ICG dye demonstrated dose-dependent and exposure-dependent toxicity. The ICG dye was found to be toxic to the cells at all tested concentrations between 5.0 mg/ml (stock concentration, 26.1% cell survival) and 0.5 mg/ml (92.8% cell survival) over a 3-minute exposure. No toxicity to TB was seen at the stock concentration of 1.5 mg/mL. Addition of light to the cultures did not significantly alter cell viability with either dye. Long periods of exposure, 2 hours, 24 hours, and 72 hours, to minute concentrations of either dye did not produce significant cell death. Indocyanine green demonstrates more toxicity than TB to human retinal pigment epithelium cell cultures. This is independent of any phototoxic potentiating effect of fiberoptic light or solvent toxicity. A clinically useful concentration of 0.5-mg/ml ICG causes low cytotoxicity at 3 minutes' exposure (cell survival 92.8%) and shows no detectable toxicity at 1-minute exposure (cell survival 102%).

  14. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    SciTech Connect

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  15. Contributions of age-related alterations of the retinal pigment epithelium and of glia to the AMD-like pathology in OXYS rats

    PubMed Central

    Telegina, Darya V.; Kozhevnikova, Oyuna S.; Bayborodin, Sergey I.; Kolosova, Nataliya G.

    2017-01-01

    Age-related macular degeneration (AMD) is a major cause of blindness in developed countries, and the molecular pathogenesis of early events of AMD is poorly understood. It is known that age-related alterations of retinal pigment epithelium (RPE) cells and of glial reactivity are early hallmarks of AMD. Here we evaluated contributions of the age-related alterations of the RPE and of glia to the development of AMD-like retinopathy in OXYS rats. We showed that destructive alterations in RPE cells are a primary change during the development of retinopathy in OXYS rats. Furthermore, a defect of retinal maturation and decreased immune function at the preclinical stage of retinopathy were observed in OXYS rats in addition to the impairment of RPE cell proliferation and of their capacity for division. At the active stage of the disease, the atrophic alterations increased, and reactive gliosis was observed when disease progressed, but immune function stayed weakened. Unexpectedly, we did not observe migration of microglia and macrophages into the photoreceptor layer. These results and the wide spectrum of age-related retinal alterations in humans as well as individual differences in the risk of AMD may be attributed to genetic factors and to differences in the underlying molecular events. PMID:28134357

  16. Effect of retinoic acid on proliferation and polyamine metabolism in cultured bovine retinal pigment epithelial cells.

    PubMed

    Yasunari, T; Yanagihara, N; Komatsu, T; Moriwaki, M; Shiraki, K; Miki, T; Yano, Y; Otani, S

    1999-01-01

    Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.

  17. Low Power Laser Irradiation Stimulates the Proliferation of Adult Human Retinal Pigment Epithelial Cells in Culture

    PubMed Central

    Song, Qing; Uygun, Basak; Banerjee, Ipsita; Nahmias, Yaakov; Zhang, Quan; Berthiaume, François; Latina, Mark; Yarmush, Martin L.

    2015-01-01

    We investigated the effects of low power laser irradiation on the proliferation of retinal pigment epithelial (RPE) cells. Adult human RPE cells were artificially pigmented by preincubation with sepia melanin, and exposed to a single sublethal laser pulse (590 nm, 1 µs, <200 mJ/cm2). DNA synthesis, cell number, and growth factor activity in irradiated RPE cells were subsequently monitored. The effect of sublethal laser irradiation on the “wound” healing response of an RPE monolayer in an in vitro scratch assay was also investigated. Single pulsed laser irradiation increased DNA synthesis in pigmented RPE cells measured 6 h post-treatment. In the scratch assay, laser irradiation increased the rates of cell proliferation and wound closure. Conditioned medium, collected 48 h following laser treatment, increased cell proliferation of unirradiated cells. Irradiation increased RPE cell secretion of platelet-derived growth factor (PDGF)-B chain, and increased mRNA levels of several growth factors and their receptors, including PDGF, transforming growth factor-β1, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, as well as heat shock proteins. This demonstrates, for the first time, that low power single pulsed laser irradiation stimulates the proliferation of RPE cells, and upregulates growth factors that are mitogenic for RPE cells. PMID:26740823

  18. Hearing impairment in the P23H-1 retinal degeneration rat model

    PubMed Central

    Sotoca, Jorge V.; Alvarado, Juan C.; Fuentes-Santamaría, Verónica; Martinez-Galan, Juan R.; Caminos, Elena

    2014-01-01

    The transgenic P23H line 1 (P23H-1) rat expresses a variant of rhodopsin with a mutation that leads to loss of visual function. This rat strain is an experimental model usually employed to study photoreceptor degeneration. Although the mutated protein should not interfere with other sensory functions, observing severe loss of auditory reflexes in response to natural sounds led us to study auditory brain response (ABR) recording. Animals were separated into different hearing levels following the response to natural stimuli (hand clapping and kissing sounds). Of all the analyzed animals, 25.9% presented auditory loss before 50 days of age (P50) and 45% were totally deaf by P200. ABR recordings showed that all the rats had a higher hearing threshold than the control Sprague-Dawley (SD) rats, which was also higher than any other rat strains. The integrity of the central and peripheral auditory pathway was analyzed by histology and immunocytochemistry. In the cochlear nucleus (CN), statistical differences were found between SD and P23H-1 rats in VGluT1 distribution, but none were found when labeling all the CN synapses with anti-Syntaxin. This finding suggests anatomical and/or molecular abnormalities in the auditory downstream pathway. The inner ear of the hypoacusic P23H-1 rats showed several anatomical defects, including loss and disruption of hair cells and spiral ganglion neurons. All these results can explain, at least in part, how hearing impairment can occur in a high percentage of P23H-1 rats. P23H-1 rats may be considered an experimental model with visual and auditory dysfunctions in future research. PMID:25278831

  19. Retinal gap junctions are involved in rhythmogenesis of neuronal activity at remote locations - Study on infra-slow oscillations in the rat olivary pretectal nucleus.

    PubMed

    Orlowska-Feuer, Patrycja; Jeczmien-Lazur, Jagoda S; Szkudlarek, Hanna J; Lewandowski, Marian H

    2016-12-17

    A subpopulation of olivary pretectal nucleus (OPN) neurons fire action potentials in a rhythmic manner with an eruption of activity occurring approximately every two minutes. These infra-slow oscillations depend critically on functional retinal input and are subject to modulation by light. Interestingly, the activity of photoreceptors is necessary for the emergence of the rhythm and while classic photoreceptors (rods and cones) are necessary in darkness and dim light, melanopsin photoreceptors are indispensable in bright light. Using pharmacological and electrophysiological approaches in vivo, we show that also blocking retinal gap junctions (GJs), which are expressed by multitude of retinal cells, leads to the disruption of oscillatory activity in the rat OPN. Intravitreal injection of carbenoxolone (CBX) quenched oscillations in a concentration-dependent manner with 1mM being ineffective, 5mM showing partial and 20mM showing complete effectiveness in disrupting oscillations. Moreover, the most effective CBX concentration depressed cone-mediated light-induced responses of oscillatory neurons suggesting that CBX is also acting on targets other than GJs. In contrast, intravitreal injection of meclofenamic acid (MFA, 20mM) led to disruption of the rhythm but did not interfere with cone-mediated light-induced responses of oscillatory neurons, implying that MFA is more specific toward GJs than CBX, as suggested before. We conclude that electrical coupling between various types of retinal cells and resultant synchronous firing of retinal ganglion cells is necessary for the generation of infra-slow oscillations in the rat OPN. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Brain-derived neurotrophic factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by activation of basic fibroblast growth factor signaling.

    PubMed

    Berk, B-A; Vogler, S; Pannicke, T; Kuhrt, H; Garcia, T B; Wiedemann, P; Reichenbach, A; Seeger, J; Bringmann, A

    2015-06-04

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Intravitreal administration of neurotrophins such as brain-derived neurotrophic factor (BDNF) is known to promote survival of retinal neurons. Here, we show that exogenous BDNF inhibits the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices or freshly isolated cells with a hypoosmotic solution containing barium ions. BDNF also inhibited the osmotic swelling of bipolar cell somata in retinal slices, but failed to inhibit the osmotic soma swelling of freshly isolated bipolar cells. The inhibitory effect of BDNF on Müller cell swelling was mediated by activation of tropomyosin-related kinase B (TrkB) and transactivation of fibroblast growth factor receptors. Exogenous basic fibroblast growth factor (bFGF) fully inhibited the osmotic swelling of Müller cell somata while it partially inhibited the osmotic swelling of bipolar cell somata. Isolated Müller cells displayed immunoreactivity of truncated TrkB, but not full-length TrkB. Isolated rod bipolar cells displayed immunoreactivities of both TrkB isoforms. Data suggest that the neuroprotective effect of exogenous BDNF in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. While BDNF directly acts on Müller cells by activation of TrkB, BDNF indirectly acts on bipolar cells by inducing glial release of factors like bFGF that inhibit bipolar cell swelling.

  1. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

    1997-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

  2. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

    1997-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

  3. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

    1994-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

  4. Locational heterogeneity of maturation by changes in migratory behaviors of human retinal pigment epithelial cells in culture.

    PubMed

    Sonoi, Rie; Kim, Mee-Hae; Kino-oka, Masahiro

    2015-01-01

    To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 μm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 μm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 μg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 μm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Involvement of microRNA-181a and Bim in a rat model of retinal ischemia-reperfusion injury.

    PubMed

    He, Yu; Liu, Jin-Nan; Zhang, Jun-Jun; Fan, Wei

    2016-01-01

    To investigate the changes in the expression of microRNA-181a (miR-181a) and Bim in a rat model of retinal ischemia-reperfusion (RIR), to explore their target relationship in RIR and their involvement in regulating apoptosis of retinal ganglion cells (RGCs). Target gene prediction for miR-181a was performed with the aid of bioinformatics and Bim was identified as a potential target gene of miR-181a. A rat model of RIR was created by increasing the intraocular pressure. RGCs in the flatmounted retinas were labeled with Brn3, a marker for alive RGCs, by immunofluorescent staining. The changes in the number of RGCs after RIR were recorded. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression level of miR-181a in the retina. Bim/Brn3 double immunofluorescence was used to detect the localization of Bim. The expression of Bim in the retina was determined with the aids of Western blot and qRT-PCR. Compared with the negative control group, the density of RGCs was significantly lower in the ischemia/reperfusion (I/R)-24h and I/R-72h groups (P<0.001). The expression level of miR-181a started to decrease at 0h after RIR, and further decreased at 24h and 72h compared with the negative control group (P<0.001). Bim was significantly upregulated at 12h after RIR (P<0.05) and reached peak at 24, 72h compared with the negative control group (P<0.01). Pearson correlation analysis showed that the expression level of Bim was negatively correlated with the expression level of miR-181a and the density of RGCs. Bim may be a potential target gene of miR-181a. Both miR-181a and Bim are involved in RGCs death in RIR. RIR may promote RGCs apoptosis in the retina via downregulation of miR-181a and its inhibition on Bim expression.

  6. Increased phase synchronization of spontaneous calcium oscillations in epileptic human versus normal rat astrocyte cultures

    NASA Astrophysics Data System (ADS)

    Balázsi, Gábor; Cornell-Bell, Ann H.; Moss, Frank

    2003-06-01

    Stochastic synchronization analysis is applied to intracellular calcium oscillations in astrocyte cultures prepared from epileptic human temporal lobe. The same methods are applied to astrocyte cultures prepared from normal rat hippocampus. Our results indicate that phase-repulsive coupling in epileptic human astrocyte cultures is stronger, leading to an increased synchronization in epileptic human compared to normal rat astrocyte cultures.

  7. THE ORGANOPHOSPHOROUS INSECTICIDE FENTHION DOES NOT AFFECT PHAGOCYTOSIS OF ROD OUTER SEGMENTS BY RETINAL PIGMENT EPITHELIUM CELLS IN CULTURE.

    EPA Science Inventory

    :
    Exposure to the organophosphorous insecticide fenthion has been associated with retinal degeneration in occupational studies. It has also been associated with pigmentary changes of the retina. Because retinal degeneration and pigmentary changes may be due to dysfunction of t...

  8. THE ORGANOPHOSPHOROUS INSECTICIDE FENTHION DOES NOT AFFECT PHAGOCYTOSIS OF ROD OUTER SEGMENTS BY RETINAL PIGMENT EPITHELIUM CELLS IN CULTURE.

    EPA Science Inventory

    :
    Exposure to the organophosphorous insecticide fenthion has been associated with retinal degeneration in occupational studies. It has also been associated with pigmentary changes of the retina. Because retinal degeneration and pigmentary changes may be due to dysfunction of t...

  9. Quantification of rat retinal growth and vascular population changes after single and split doses of proton irradiation: translational study using stereology methods

    NASA Technical Reports Server (NTRS)

    Mao, Xiao W.; Archambeau, John O.; Kubinova, Lucie; Boyle, Soames; Petersen, Georgia; Grove, Roger; Nelson, G. A. (Principal Investigator)

    2003-01-01

    This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (P<0.01) after 28 Gy given in single and split doses and after 20 Gy given as a split dose (P<0.05). Total vessel length in microvessel was significantly shortened at 20 and 28 Gy compared to that of controls (P<0.05). No evident dose recovery was observed in the endothelial populations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

  10. Sigma-1 receptor activation inhibits osmotic swelling of rat retinal glial (Müller) cells by transactivation of glutamatergic and purinergic receptors.

    PubMed

    Vogler, Stefanie; Winters, Helge; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-01-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Müller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Müller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells.

  11. Quantification of rat retinal growth and vascular population changes after single and split doses of proton irradiation: translational study using stereology methods

    NASA Technical Reports Server (NTRS)

    Mao, Xiao W.; Archambeau, John O.; Kubinova, Lucie; Boyle, Soames; Petersen, Georgia; Grove, Roger; Nelson, G. A. (Principal Investigator)

    2003-01-01

    This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (P<0.01) after 28 Gy given in single and split doses and after 20 Gy given as a split dose (P<0.05). Total vessel length in microvessel was significantly shortened at 20 and 28 Gy compared to that of controls (P<0.05). No evident dose recovery was observed in the endothelial populations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

  12. Neuroprotective effects of citicoline in in vitro models of retinal neurodegeneration.

    PubMed

    Matteucci, Andrea; Varano, Monica; Gaddini, Lucia; Mallozzi, Cinzia; Villa, Marika; Pricci, Flavia; Malchiodi-Albedi, Fiorella

    2014-04-14

    In recent years, citicoline has been the object of remarkable interest as a possible neuroprotectant. The aim of this study was to investigate if citicoline affected cell survival in primary retinal cultures and if it exerted neuroprotective activity in conditions modeling retinal neurodegeneration. Primary retinal cultures, obtained from rat embryos, were first treated with increasing concentrations of citicoline (up to 1000 µM) and analyzed in terms of apoptosis and caspase activation and characterized by immunocytochemistry to identify neuronal and glial cells. Subsequently, excitotoxic concentration of glutamate or High Glucose-containing cell culture medium (HG) was administered as well-known conditions modeling neurodegeneration. Glutamate or HG treatments were performed in the presence or not of citicoline. Neuronal degeneration was evaluated in terms of apoptosis and loss of synapses. The results showed that citicoline did not cause any damage to the retinal neuroglial population up to 1000 µM. At the concentration of 100 µM, it was able to counteract neuronal cell damage both in glutamate- and HG-treated retinal cultures by decreasing proapoptotic effects and contrasting synapse loss. These data confirm that citicoline can efficiently exert a neuroprotective activity. In addition, the results suggest that primary retinal cultures, under conditions inducing neurodegeneration, may represent a useful system to investigate citicoline neuroprotective mechanisms.

  13. Transport of 3-hydroxybutyrate by cultured rat brain astrocytes

    SciTech Connect

    McKenna, M.C.; Tildon, J.T.; Stevenson, J.H.; Couto, R.; Caprio, F.J. )

    1990-02-26

    Studies by a number of investigators have shown that 3-hydroxybutyrate is a preferred energy substrate for brain during early development. Since recent studies by the authors group suggest that the utilization of oxidizable substrates by brain may be regulated in part by transport across the plasma membrane, the authors investigated the transport of ({sup 3}H) D- and L-3-hydroxybutyrate and 3-hydroxy-(3-{sup 14}C) butyrate by primary cultures of rat brain astrocytes. The data is consistent with the hypothesis that 3-hydroxybutyrate is taken up into cultured rat brain astrocytes by both diffusion and a carrier mediated transport system, and further support the concept that transport at the cellular level contributes to the regulation of substrate utilization by brain cells.

  14. Triptolide upregulates NGF synthesis in rat astrocyte cultures.

    PubMed

    Xue, Bing; Jiao, Jian; Zhang, Lei; Li, Kai-Rong; Gong, Yun-Tao; Xie, Jun-Xia; Wang, Xiao-Min

    2007-07-01

    Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by D: -amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson's disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes.

  15. Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos.

    PubMed

    Usami, Makoto; Nakajima, Mikio; Mitsunaga, Katsuyoshi; Miyajima, Atsuko; Sunouchi, Momoko; Doi, Osamu

    2009-12-01

    Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

  16. Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons.

    PubMed

    Cui, Qi; Yip, Henry K; Zhao, Robert C H; So, Kwok-Fai; Harvey, Alan R

    2003-01-01

    In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

  17. Protective Effects of Panax notoginseng Saponins against High Glucose-Induced Oxidative Injury in Rat Retinal Capillary Endothelial Cells

    PubMed Central

    Fan, Yue; Qiao, Yuan; Huang, Jianmei

    2016-01-01

    Diabetic retinopathy, a leading cause of visual loss and blindness, is characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for diabetic retinopathy and is associated with increased oxidative stress in the retina. In this study, we investigated the potential protective effects of Panax notoginseng Saponins (PNS) in retinal capillary endothelial cells (RCECs) exposed to high glucose conditions. We found a pronounced increase in cell viability in rat RCECs incubated with both PNS and high glucose (30 mM) for 48 h or 72 h. The increased viability was accompanied by reduced intracellular hydrogen peroxide (H2O2) and superoxide (O2−), decreased mitochondrial reactive oxygen species (ROS), and lowered malondialdehyde (MDA) levels. PNS also increased the activities of total superoxide dismutase (SOD), MnSOD, catalase (CAT), and glutathione peroxidase (GSH-PX). The glutathione (GSH) content also increased after PNS treatment. Furthermore, PNS reduced NADPH oxidase 4 (Nox4) expression. These results indicate that PNS exerts a protective effect against high glucose-induced injury in RCECs, which may be partially attributed to its antioxidative function. PMID:27019662

  18. Effect of Microcystin-LR on Cultured Rat Endothelial Cells

    DTIC Science & Technology

    1990-02-26

    protection, silymarin, and dithioerythritol 19. ABSTRACT (Continue on reverse if necessary and identify by block number) Primary cultureLs of adult rat...hepatotoxicity is not known, Microcystin-LR effects do not appear to be mediated by the inhibition of macromolecular biosynthesis; i.e. protein, RNA or DNA...hepatocytes treated with a cytotoxic dose (4 wM) of microcystin-LR release factors ( mediators ) tlaL could induce changes, or cytotoxicity in endothelial 3

  19. Oncostatin M protects rod and cone photoreceptors and promotes regeneration of cone outer segment in a rat model of retinal degeneration.

    PubMed

    Xia, Xin; Li, Yiwen; Huang, Deqiang; Wang, Zhengying; Luo, Lingyu; Song, Ying; Zhao, Lian; Wen, Rong

    2011-03-30

    Retinitis pigmentosa (RP) is a group of photoreceptor degenerative disorders that lead to loss of vision. Typically, rod photoreceptors degenerate first, resulting in loss of night and peripheral vision. Secondary cone degeneration eventually affects central vision, leading to total blindness. Previous studies have shown that photoreceptors could be protected from degeneration by exogenous neurotrophic factors, including ciliary neurotrophic factor (CNTF), a member of the IL-6 family of cytokines. Using a transgenic rat model of retinal degeneration (the S334-ter rat), we investigated the effects of Oncostatin M (OSM), another member of the IL-6 family of cytokines, on photoreceptor protection. We found that exogenous OSM protects both rod and cone photoreceptors. In addition, OSM promotes regeneration of cone outer segments in early stages of cone degeneration. Further investigation showed that OSM treatment induces STAT3 phosphorylation in Müller cells but not in photoreceptors, suggesting that OSM not directly acts on photoreceptors and that the protective effects of OSM on photoreceptors are mediated by Müller cells. These findings support the therapeutic strategy using members of IL-6 family of cytokines for retinal degenerative disorders. They also provide evidence that activation of the STAT3 pathway in Müller cells promotes photoreceptor survival. Our work highlights the importance of Müller cell-photoreceptor interaction in the retina, which may serve as a model of glia-neuron interaction in general.

  20. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery.

    PubMed

    Weber, Michel; Rabinowitz, Joseph; Provost, Nathalie; Conrath, Hervé; Folliot, Sébastien; Briot, Delphine; Chérel, Yan; Chenuaud, Pierre; Samulski, Jude; Moullier, Philippe; Rolling, Fabienne

    2003-06-01

    We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

  1. Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells.

    PubMed

    Szurman, Peter; Kaczmarek, Radoslaw; Spitzer, Martin S; Jaissle, Gesine B; Decker, Patrice; Grisanti, Salvatore; Henke-Fahle, Sigrid; Aisenbrey, Sabine; Bartz-Schmidt, Karl U

    2006-09-01

    The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01-1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025%-0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically

  2. Evaluation of the effect of elevated intraocular pressure and reduced ocular perfusion pressure on retinal capillary bed filling and total retinal blood flow in rats by OMAG/OCT.

    PubMed

    Zhi, Zhongwei; Cepurna, William; Johnson, Elaine; Jayaram, Hari; Morrison, John; Wang, Ruikang K

    2015-09-01

    To determine if retinal capillary filling is preserved in the face of acutely elevated intraocular pressure (IOP) in anesthetized rats, despite a reduction in total retinal blood flow (RBF), using optical microangiography/optical coherence tomography (OMAG/OCT). OMAG provided the capability of depth-resolved imaging of the retinal microvasculature down to the capillary level. Doppler OCT was applied to measure the total RBF using an enface integration approach. The microvascular pattern, capillary density, and total RBF were monitored in vivo as the IOP was increased from 10 to 100mmHg in 10mmHg intervals and returned back to 10mmHg. In animals with mean arterial pressure (MAP) of 102±4mmHg (n=10), when IOP was increased from 0 to 100mmHg, the capillary density remained at or above 80% of baseline for the IOP up to 60mmHg [or ocular perfusion pressure (OPP) at 40mmHg]. This was then decreased, achieving 60% of baseline at IOP 70mmHg and OPP of 30mmHg. Total RBF was unaffected by moderate increases in IOP up to 30mmHg, beyond which total RBF decreased linearly, reaching 50% of baseline at IOP 60mmHg and OPP 40mmHg. Both capillary density and total RBF were totally extinguished at 100mmHg, but fully recovered when IOP returned to baseline. By comparison, a separate group of animals with lower MAP (mean=75±6mmHg, n=7) demonstrated comparable decreases in both capillary filling and total RBF at IOPs that were 20mmHg lower than in the initial group. Both were totally extinguished at 80mmHg, but fully recovered when IOP returned to baseline. Relationships of both parameters to OPP were unchanged. Retinal capillary filling and total RBF responses to IOP elevation can be monitored non-invasively by OMAG/OCT and both are influenced by OPP. Retinal capillary filling was relatively preserved down to a perfusion pressure of 40mmHg, despite a linear reduction in total RBF. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Apigenin prevents TNF-α induced apoptosis of primary rat retinal ganglion cells.

    PubMed

    Fu, M-S; Zhu, B-J; Luo, D-W

    2014-11-25

    TNF-α has recently been identified to be a mediator of retinal ganglion cell (RGC) death, while glial cells are relatively protected against this death stimulus. Exposure of RGCs to TNF-α is thought to contribute to RGC apoptosis. Apigenin is a flavone with powerful anti-inflammatory properties that exists naturally in various plants and Chinese medicine. In our study, MTT assays showed that apigenin significantly inhibited the decrease of RGC viability induced by TNF-α in a dose-dependent manner. Pretreatment with apigenin prevented TNF-α-induced apoptosis in a dose-dependent manner as shown by flow cytometry. The production of ATP and the total oxygen uptake were also promoted after apigenin administration. TNF-α stimulation led to a significant reduction of bcl-2 and enhancement of bax, which was reversed by apigenin treatment. Apigenin treatment also alleviated the increased caspase-3 activity induced by TNF-α. Moreover, luciferase reporter assay indicated that apigenin dose-dependently decreased NF-κB activation induced by TNF-α, but had no significant effect on activation of AP-1. Collectively, these data demonstrated that apigenin alleviated TNF-α-induced apoptosis through inhibition of caspase-dependent apoptotic pathway and activation of nuclear factor-kappaB. Therefore, apigenin may be developed as an anti-apoptotic drug to treat retinopathy.

  4. Ginsenoside Rb1 protects rat retinal ganglion cells against hypoxia and oxidative stress.

    PubMed

    Liu, Zhaochun; Chen, Juying; Huang, Wendong; Zeng, Zhi; Yang, Yongfei; Zhu, Banghao

    2013-11-01

    The current study was designed to investigate the effect of ginsenoside Rb1 (Rb1) on apoptosis induced by hypoxia and oxidative stress in a retinal ganglion cell line (RGC-5). The underlying mechanism was also investigated. RGC-5 cells were pretreated with 10 µmol/l Rb1 for 24 h and exposed to 400 µmol/l cobalt chloride (CoCl2) for 48 h or 600 µmol/l H2O2 for 24 h. The percentage of cells actively undergoing apoptosis was determined by flow cytometry with Annexin V/propidium iodide (PI) double staining. The expression of caspases was determined using western blot analysis. CoCl2 and H2O2 treatments significantly increased the apoptotic percentages to 24.5 and 21.63%, respectively. Pretreatment of Rb1 reduced the total apoptotic percentages to 15.12 and 12.03%, respectively. The expression of cleaved caspase-3, -9 and -8 was increased in the CoCl2-treated group, however, caspase-3 was not increased in the H2O2-treated group. Pretreatment of Rb1 reduced the expression of cleaved caspase-3 and -9 in the CoCl2-treated group, but reduced only cleaved caspase-9 in the H2O2-treated group. These results suggest that Rb1 may prevent RGC-5 cells from apoptosis against hypoxia and oxidative stress via the mitochondrial pathway.

  5. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-03-04

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

  6. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  7. Retinitis pigmentosa

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/001029.htm Retinitis pigmentosa To use the sharing features on this page, please enable JavaScript. Retinitis pigmentosa is an eye disease in which there is ...

  8. Retinitis Pigmentosa.

    ERIC Educational Resources Information Center

    Carr, Ronald E.

    1979-01-01

    The author describes the etiology of retinitis pigmentosa, a visual dysfunction which results from progressive loss of the retinal photoreceptors. Sections address signs and symptoms, ancillary findings, heredity, clinical diagnosis, therapy, and research. (SBH)

  9. Retinitis Pigmentosa.

    ERIC Educational Resources Information Center

    Carr, Ronald E.

    1979-01-01

    The author describes the etiology of retinitis pigmentosa, a visual dysfunction which results from progressive loss of the retinal photoreceptors. Sections address signs and symptoms, ancillary findings, heredity, clinical diagnosis, therapy, and research. (SBH)

  10. Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

    PubMed

    Jacques, Flavia Jesus; Silva, Thayane Martins; da Silva, Flavia Emenegilda; Ornelas, Isis Moraes; Ventura, Ana Lucia Marques

    2017-03-24

    Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [(3)H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY241385 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [(3)H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [(3)H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca(2+)]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca

  11. Proteomic Study of Retinal Proteins Associated with Transcorneal Electric Stimulation in Rats

    PubMed Central

    Kanamoto, Takashi; Souchelnytskyi, Nazariy; Kurimoto, Takuji; Ikeda, Yasuhiro; Sakaue, Hiroaki; Munemasa, Yasunari; Kiuchi, Yoshiaki

    2015-01-01

    Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina. PMID:25821588

  12. Proteomic study of retinal proteins associated with transcorneal electric stimulation in rats.

    PubMed

    Kanamoto, Takashi; Souchelnytskyi, Nazariy; Kurimoto, Takuji; Ikeda, Yasuhiro; Sakaue, Hiroaki; Munemasa, Yasunari; Kiuchi, Yoshiaki

    2015-01-01

    Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina.

  13. Loofa sponge as a scaffold for culture of rat hepatocytes.

    PubMed

    Chen, Jyh-Ping; Lin, Tsung-Cheng

    2005-01-01

    The dried fruit from Luffa cylindrica (loofa sponge, LS), which represents a new chitinous source material, was used as a 3-D scaffold for the culture of rat hepatocytes. With the macroporous structure and large pore size (ca. 800 microm) of LS, cell loading to the scaffold should be carried out by dynamic seeding with continuous shaking throughout the seeding period. Hepatocytes attach well to the surface of loofa fibers after seeding and maintain their round shapes. The initial ammonia removal and urea-N synthesis rates of hepatocytes immobilized within LS slightly decreased with increasing cell densities, but their metabolic activities were comparable to or better than those in monolayer culture on tissue culture polystyrene control surfaces. Both urea-N synthesis and albumin secretion rates could be maintained up to 7 days for cells immobilized within LS and spheroid-like cell aggregates could be found after the second day.

  14. RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride.

    PubMed

    del Olmo-Aguado, Susana; Núñez-Álvarez, Claudia; Ji, Dan; Manso, Alberto García; Osborne, Neville N

    2013-09-01

    RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl2) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl2 increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl2 was blunted. Rapamycin nullified the CoCl2-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10μM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.

  15. Glaucoma, capillaries and pericytes. 4. Beta-adrenergic activation of cultured retinal pericytes.

    PubMed

    Zschauer, A O; Davis, E B; Anderson, D R

    1996-01-01

    To characterize the relaxation of pericytes induced by beta-adrenergic stimulation, changes in the contractile tone of pericytes were quantified as a change in the wrinkling of an elastic silicone surface on which they were cultured. Isoproterenol produced relaxation of pericytes in a dose-dependent manner over a range of 5 nM to 1 microM. Low concentrations of the nonselective beta-blockers propanolol and timolol blocked the relaxation produced by isoproterenol. The specific beta 2-adrenergic component of isoproterenol-induced relaxation was shown by blockage with bromoacetyl alprenolol menthane. In contrast, atenolol and betaxolol, as relatively selective beta 1-adrenergic blockers, had no effect on the isoproterenol-induced relaxation.

  16. Tamoxifen Toxicity in Cultured Retinal Pigment Epithelial Cells Is Mediated by Concurrent Regulated Cell Death Mechanisms

    PubMed Central

    Kim, Leo A.; Amarnani, Dhanesh; Gnanaguru, Gopalan; Tseng, Wen Allen; Vavvas, Demetrios G.; D'Amore, Patricia A.

    2014-01-01

    Purpose. To evaluate the mechanism of tamoxifen-induced cell death in human cultured RPE cells, and to investigate concurrent cell death mechanisms including pyroptosis, apoptosis, and necroptosis. Methods. Human RPE cells were cultured until confluence and treated with tamoxifen; cell death was measured by detecting LDH release. Tamoxifen-induced cell death was further confirmed by 7-aminoactinomycin D (7-AAD) and annexin V staining. Lysosomal destabilization was assessed using lysosomal-associated membrane protein-1 (LAMP-1) and acridine orange staining. The roles of lysosomal enzymes cathepsin B and L were examined by blocking their activity. Caspase activity was evaluated by caspase-1, -3, -8, and -9 specific inhibition. Cells were primed with IL-1α and treated with tamoxifen; mature IL-1β production was quantified via ELISA. Caspase activity was verified with the fluorochrome-labeled inhibitor of caspases (FLICA) probe specific for each caspase. Regulated cell necrosis or necroptosis was examined with 7-AAD and inhibition of receptor-interacting protein 1 (RIP1) kinase using necrostatin-1 (Nec-1). Results. Cell death occurred within 2 hours of tamoxifen treatment of confluent RPE cells and was accompanied by lysosomal membrane permeabilization. Blockade of cathepsin B and L activity led to a significant decrease in cell death, indicating that lysosomal destabilization and cathepsin release occur prior to regulated cell death. Tamoxifen-induced toxicity was shown to occur through both caspase-dependent and caspase-independent cell death pathways. Treatment of RPE cells with caspase inhibitors and Nec-1 resulted in a near complete rescue from cell death. Conclusions. Tamoxifen-induced cell death occurs through concurrent regulated cell death mechanisms. Simultaneous inhibition of caspase-dependent and caspase-independent cell death pathways is required to protect cells from tamoxifen. Inhibition of upstream activators, such as the cathepsins, may represent a

  17. Transformation of adult rat cardiac myocytes in primary culture.

    PubMed

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  18. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  19. The neurotoxic effect of monosodium glutamate (MSG) on the retinal ganglion cells of the albino rat.

    PubMed

    van Rijn, C M; Marani, E; Rietveld, W J

    1986-07-01

    Monosodium glutamate (MSG) administered postnatally to the albino rat causes extensive destruction of the retina. This MSG effect does not result in complete blindness. Ganglion cells surviving the MSG treatment are healthy and functional. Using retrogradely transported HRP and Nissl staining in whole mounted retinas, it was found that the ganglion cells left after MSG treatment are not smaller than those in controls, that these cells do not belong to one cell size group, and that no cells size group is selectively missed. The results explain why photic entrainment of MSG treated animals is still possible.

  20. The novel induction of retinal ganglion cell apoptosis in porcine organ culture by NMDA - an opportunity for the replacement of animals in experiments.

    PubMed

    Kuehn, Sandra; Hurst, Jose; Jashari, Adelina; Ahrens, Kathrin; Tsai, Teresa; Wunderlich, Ilan M; Dick, H Burkhard; Joachim, Stephanie C; Schnichels, Sven

    2016-12-01

    Some of the advantages of retina organ culture models include their efficient and easy handling and the ability to standardise relevant parameters. Additionally, when porcine eyes are obtained from the food industry, no animals are killed solely for research purposes. To induce retinal degeneration, a commonly used toxic substance, N-methyl-D-aspartate (NMDA), was applied to the cultures. To this end, organotypic cultures of porcine retinas were cultured and treated with different doses of NMDA (0 [control], 50, 100 and 200μM) on day 2 for 48 hours. On day 7, the retinas were cryo-conserved for histological, Western blot and quantitative rt-PCR (qrt-PCR) analyses. NMDA treatment was found to significantly increase retinal ganglion cell (RGC) apoptosis in all the treated groups, without a profound RGC loss. In addition, the intrinsic apoptotic pathway was activated in the 50μM and 100μM NMDA groups, whereas induced nitric oxide synthase (iNOS) expression was increased in the 200μM group. A slight microglial response was detectable, especially in the 100μM group. NMDA treatment induced apoptosis, oxidative stress and a slight microglia activation. All these effects mimic a chronic slow progressive disease that especially affects RGCs, such as glaucoma. A particular advantage of this model is that mediators that can interact in the very early stages of the onset of RGC death, can be easily detected and potential therapies can be tested.

  1. Discovery of a novel class of targeted kinase inhibitors that blocks protein kinase C signaling and ameliorates retinal vascular leakage in a diabetic rat model.

    PubMed

    Grant, Stephan; Tran, Phong; Zhang, Qin; Zou, Aihua; Dinh, Dac; Jensen, Jordan; Zhou, Sue; Kang, Xiaolin; Zachwieja, Joseph; Lippincott, John; Liu, Kevin; Johnson, Sarah Ludlum; Scales, Stephanie; Yin, Chunfeng; Nukui, Seiji; Stoner, Chad; Prasanna, Ganesh; Lafontaine, Jennifer; Wells, Peter; Li, Hui

    2010-02-10

    Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

  2. Retinal detachment.

    PubMed

    Cavallerano, A A

    1992-01-01

    Retinal detachment is a separation of the neurosensory retina from the retinal pigment epithelium. Most retinal detachments are rhegmatogenous, and identification of risk factors and predisposing lesions are important aspects of patient management. Retinal detachment is relatively rare, but can pose a significant threat to vision if there is macular involvement. Prompt diagnosis combined with patient education and appropriate intervention often can avert irrevocable visual impairment. This paper presents an overview of the categories of retinal detachment, discusses the pathogenesis of the various types of detachment, and provides recommendations for primary care of patients with predisposing factors and high-risk characteristics.

  3. The dark phase intraocular pressure elevation and retinal ganglion cell degeneration in a rat model of experimental glaucoma.

    PubMed

    Kwong, Jacky M K; Vo, Nancy; Quan, Ann; Nam, Michael; Kyung, Haksu; Yu, Fei; Piri, Natik; Caprioli, Joseph

    2013-07-01

    Intraocular pressure (IOP) elevation is considered as a major risk factor causing the progression of vision deterioration in glaucoma. Although it is known that the IOP level changes widely throughout the day and night, how the dark or light phase IOP elevation contributes to retinal ganglion cell (RGC) degeneration is still largely unclear. To examine the profile of IOP, modified laser photocoagulation was applied to the trabecular meshwork of Brown Norway rats and both light and dark phase IOPs were monitored approximately 1-2 times a week. The relationship between IOP elevation and RGC degeneration was investigated while RGC body loss was analyzed with Rbpms immunolabeling on retinal wholemount and axonal injury in the optic nerve was semi-quantified. The baseline awake dark and light IOPs were 30.4 ± 2.7 and 20.2 ± 2.1 mmHg respectively. The average dark IOP was increased to 38.2 ± 3.2 mmHg for five weeks after the laser treatment on 270° trabecular meshwork. However, there was no significant loss of RGC body and axonal injury. After laser treatment on 330° trabecular meshwork, the dark and light IOPs were significantly increased to 43.8 ± 4.6 and 23 ± 3.7 mmHg respectively for 5 weeks. The cumulative dark and light IOP elevations were 277 ± 86 and 113 ± 50 mmHg days respectively while the cumulative total (light and dark) IOP elevation was 213 ± 114 mmHg days. After 5 weeks, regional RGC body loss of 29.5 ± 15.5% and moderate axonal injury were observed. Axonal injury and loss of RGC body had a high correlation with the cumulative total IOP elevation (R(2) = 0.60 and 0.65 respectively). There was an association between the cumulative dark IOP elevation and RGC body loss (R(2) = 0.37) and axonal injury (R(2) = 0.51) whereas the associations between neuronal damages and the cumulative light IOP elevation were weak (for RGC body loss, R(2) = 0.01; for axonal injury, R(2) = 0.26). Simple linear regression model

  4. Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways

    PubMed Central

    Fu, Pingping; Wu, Qiang; Hu, Jianyan; Li, Tingting; Gao, Fengjuan

    2016-01-01

    Retinal ganglion cells (RGCs) consume large quantities of energy to convert light information into a neuronal signal, which makes them highly susceptible to hypoxic injury. This study aimed to investigate the potential protection by baclofen, a GABAB receptor agonist of RGCs against hypoxia-induced apoptosis. Cobalt chloride (CoCl2) was applied to mimic hypoxia. Primary rat RGCs were subjected to CoCl2 with or without baclofen treatment, and RNA interference techniques were used to knock down the GABAB2 gene in the primary RGCs. The viability and apoptosis of RGCs were assessed using cell viability and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, Hoechst staining, and flow cytometry. The expression of cleaved caspase-3, bcl-2, bax, Akt, phospho-Akt, protein kinase RNA (PKR)-like ER kinase (PERK), phospho-PERK, eIF2α, phospho-eIF2α, ATF-4 and CCAAT/enhancer-binding protein homologous protein (CHOP) were measured using western blotting. GABAB2 mRNA expression was determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Our study revealed that CoCl2 significantly induced RGC apoptosis and that baclofen reversed these effects. CoCl2-induced reduction of Akt activity was also reversed by baclofen. Baclofen prevented the activation of the PERK pathway and the increase in CHOP expression induced by CoCl2. Knockdown of GABAB2 and the inactivation of the Akt pathway by inhibitors reduced the protective effect of baclofen on CoCl2-treated RGCs. Taken together, these results demonstrate that baclofen protects RGCs from CoCl2-induced apoptosis by increasing Akt activity and by suppressing the PERK pathway and CHOP activation. PMID:27867349

  5. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  6. Characterization of barrier properties and inducible VEGF expression of several types of retinal pigment epithelium in medium-term culture.

    PubMed

    Geisen, Pete; McColm, Janet R; King, Bradley M; Hartnett, M Elizabeth

    2006-09-01

    To investigate and compare the characteristics of four different types of retinal pigment epithelium (RPE) cells cultured for 2 to 5 weeks to provide guidance when choosing RPE cells for experimentation. Human cell lines ARPE-19 (ARPE) and D407, primary RPE cells from C57Bl/6 mouse (mRPE), and primary human fetal RPE (hfRPE) cells were grown in respective media previously reported to be optimal for each cell type. Two methods to obtain hfRPE were used: one isolated outside and transported to our laboratory, and one isolated primarily within our laboratory from donor human fetal eyes. Barrier function was determined by transepithelial electrical resistance (TER) and permeability and structure by localization of Na+,K+-ATPase alpha-1, ZO-1, and actin. VEGF expression, determined by real-time polymerase chain reaction (PCR) for mRNA and ELISA for protein, was determined after exposure to 24 h of 1% oxygen. Madin-Darby canine kidney (MDCK) cells were compared as a non-RPE epithelial cell line. ARPE at passage 15, but not passage 32, maintained steady low TER measurements (up to 30 ohms x cm(2)) despite forming a monolayer with apical Na+,K+-ATPase alpha-1 labeling after 35 days. mRPE developed and maintained a TER of 30 ohms x cm(2) for 2 weeks but did not localize ATPase. hfRPE showed two phenotypes. hfRPE isolated remotely and sent to us appeared more mesenchymal and undifferentiated (hfRPE-U) and had a slow but steady increase in measured TER to approximately 25 ohms x cm(2), whereas hfRPE isolated from donor eyes in our laboratory showed well-differentiated monolayers (hfRPE-D) with TER measurements > 500 ohms x cm(2) within 1 month of culture. TER measurements reflected permeability determined by the measurement of paracellular movement of sodium fluorescein. All human RPE cell types showed expression of VEGF mRNA and protein, and expression was upregulated by hypoxia in hfRPE and D407, but not in ARPE, which had constitutively high expression. ARPE expressed high

  7. Culturing primary rat inner medullary collecting duct cells.

    PubMed

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussmann, Enno; Klussman, Enno

    2013-06-21

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

  8. Culturing Primary Rat Inner Medullary Collecting Duct Cells

    PubMed Central

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussman, Enno

    2013-01-01

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories. PMID:23852264

  9. Purification and culture of adult rat dorsal root ganglia neurons.

    PubMed

    Delree, P; Leprince, P; Schoenen, J; Moonen, G

    1989-06-01

    To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Candesartan Attenuates Diabetic Retinal Vascular Pathology by Restoring Glyoxalase-I Function

    PubMed Central

    Miller, Antonia G.; Tan, Genevieve; Binger, Katrina J.; Pickering, Raelene J.; Thomas, Merlin C.; Nagaraj, Ram H.; Cooper, Mark E.; Wilkinson-Berka, Jennifer L.

    2010-01-01

    OBJECTIVE Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. RESEARCH DESIGN AND METHODS Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. RESULTS In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO•), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO•. Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO•, and restored GLO-I. CONCLUSIONS We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I. PMID:20852029

  11. Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

    PubMed

    Miller, Antonia G; Tan, Genevieve; Binger, Katrina J; Pickering, Raelene J; Thomas, Merlin C; Nagaraj, Ram H; Cooper, Mark E; Wilkinson-Berka, Jennifer L

    2010-12-01

    Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO(•). Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO(•), and restored GLO-I. We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

  12. Safety, efficacy, and quality control of a photoelectric dye-based retinal prosthesis (Okayama University-type retinal prosthesis) as a medical device.

    PubMed

    Matsuo, Toshihiko; Uchida, Tetsuya; Takarabe, Kenichi

    2009-01-01

    Patients with retinitis pigmentosa lose photoreceptor cells as a result of genetic abnormalities and hence become blind. Neurons such as bipolar cells and ganglion cells remain alive even in the retina of these patients, and ganglion cells send axons to the brain as the optic nerve. The basic concept of retinal prostheses is to replace dead photoreceptor cells with artificial devices to stimulate the remaining neurons with electric currents or potentials. Photodiode arrays and digital camera-type electrode arrays are the two main approaches for retinal prostheses to stimulate retinal neurons, but these arrays have the problems of poor biocompatibility, low sensitivity, and low output of electric currents, and hence have a requirement for external electric sources (batteries). To overcome these problems, we are developing photoelectric dye-based retinal prostheses that absorb light and convert photon energy to generate electric potentials. The prototype, using a photoelectric dye-coupled polyethylene film, could induce intracellular calcium elevation in photoreceptor-lacking embryonic retinal tissues and cultured retinal neurons. The subretinal implantation of the prototype in the eyes of Royal College of Surgeons (RCS) rats led to vision recovery as proved by a behavior test. The photoelectric dye that was chosen for the prototype did not exhibit any cytotoxicity. The surface potentials of the photoelectric dye-coupled film showed a rapid on-and-off response to illumination with a threshold for light intensity as measured by a Kelvin probe system. Photoelectric dye-based retinal prostheses are thin and soft, and therefore, a sheet of the film of large size, corresponding to a large visual field, could be inserted into the vitreous and then to the subretinal space through a small opening by rolling up the film. Clinical studies of photoelectric dye-based retinal prostheses in patients with retinitis pigmentosa who lose sight will be planned after the manufacturing

  13. Effects of GABA receptor antagonists on thresholds of P23H rat retinal ganglion cells to electrical stimulation of the retina

    NASA Astrophysics Data System (ADS)

    Jensen, Ralph J.; Rizzo, Joseph F., III

    2011-06-01

    An electronic retinal prosthesis may provide useful vision for patients suffering from retinitis pigmentosa (RP). In animal models of RP, the amount of current needed to activate retinal ganglion cells (RGCs) is higher than in normal, healthy retinas. In this study, we sought to reduce the stimulation thresholds of RGCs in a degenerate rat model (P23H-line 1) by blocking GABA receptor mediated inhibition in the retina. We examined the effects of TPMPA, a GABAC receptor antagonist, and SR95531, a GABAA receptor antagonist, on the electrically evoked responses of RGCs to biphasic current pulses delivered to the subretinal surface through a 400 µm diameter electrode. Both TPMPA and SR95531 reduced the stimulation thresholds of ON-center RGCs on average by 15% and 20% respectively. Co-application of the two GABA receptor antagonists had the greatest effect, on average reducing stimulation thresholds by 32%. In addition, co-application of the two GABA receptor antagonists increased the magnitude of the electrically evoked responses on average three-fold. Neither TPMPA nor SR95531, applied alone or in combination, had consistent effects on the stimulation thresholds of OFF-center RGCs. We suggest that the effects of the GABA receptor antagonists on ON-center RGCs may be attributable to blockage of GABA receptors on the axon terminals of ON bipolar cells.

  14. Chronic CXCL10 Alters Neuronal Properties in Rat Hippocampal Culture

    PubMed Central

    Cho, Jungsook; Nelson, Thomas E.; Bajova, Hilda; Gruol, Donna L.

    2009-01-01

    The chemokine CXCL10 is expressed in the central nervous system (CNS) during neuroinflammatory conditions. Neurons express CXCR3, the receptor for CXCL10, and neuronal function has been shown to be altered by acute exposure to CXCL10. Little is known about the effects of chronic exposure to CXCL10 on neuronal function. Results from our studies show that chronic exposure of cultured rat hippocampal neurons to CXCL10 results in altered levels of protein for GABA and glutamate receptors and altered synaptic network activity. These effects of CXCL10 may contribute to altered CNS function that occurs in some chronic neuroinflammatory conditions. PMID:19167097

  15. Retinal Vasculitis

    PubMed Central

    Rosenbaum, James T.; Sibley, Cailin H.; Lin, Phoebe

    2016-01-01

    Purpose of review Ophthalmologists and rheumatologists frequently miscommunicate in consulting on patients with retinal vasculitis. This report seeks to establish a common understanding of the term, retinal vasculitis, and to review recent papers on this diagnosis. Recent findings 1) The genetic basis of some rare forms of retinal vascular disease have recently been described. Identified genes include CAPN5, TREX1, and TNFAIP3; 2) Behçet’s disease is a systemic illness that is very commonly associated with occlusive retinal vasculitis; 3) retinal imaging including fluorescein angiography and other newer imaging modalities has proven crucial to the identification and characterization of retinal vasculitis and its complications; 4) although monoclonal antibodies to IL-17A or IL-1 beta failed in trials for Behçet’s disease, antibodies to TNF alpha, either infliximab or adalimumab, have demonstrated consistent benefit in managing this disease. Interferon treatment and B cell depletion therapy via rituximab may be beneficial in certain types of retinal vasculitis. Summary Retinal vasculitis is an important entity for rheumatologists to understand. Retinal vasculitis associated with Behçet’s disease responds to monoclonal antibodies that neutralize TNF, but the many other forms of non-infectious retinal vasculitis may require alternate therapeutic management. PMID:26945335

  16. Endotoxin suppresses surfactant synthesis in cultured rat lung cells

    SciTech Connect

    Li, J.J.; Sanders, R.L.; McAdam, K.P.; Gelfand, J.A.; Burke, J.F.

    1989-02-01

    Pulmonary complications secondary to postburn sepsis are a major cause of death in burned patients. Using an in vitro organotypic culture system, we examined the effect of E. coli endotoxin (LPS) on lung cell surfactant synthesis. Our results showed that E. coli endotoxin (1.0, 2.5, 10 micrograms LPS/ml) was capable of suppressing the incorporation of /sup 3/H-choline into de novo synthesized surfactant, lamellar bodies (LB), and common myelin figures (CMF) at 50%, 68%, and 64%, respectively. In a similar study, we were able to show that LPS also inhibited /sup 3/H-palmitate incorporation by cultured lung cells. LPS-induced suppression of surfactant synthesis was reversed by hydrocortisone. Our results suggest that LPS may play a significant role in reducing surfactant synthesis by rat lung cells, and thus contribute to the pathogenesis of sepsis-related respiratory distress syndrome (RDS) in burn injury.

  17. Quantification of rat retinal and choroidal blood plasma kinetics, volume, and flow in vivo using dynamic contrast optical coherence tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Merkle, Conrad W.; Srinivasan, Vivek J.

    2017-02-01

    Blood flow patterns and kinetics in the choriocapillaris are poorly understood owing to a lack of quantitative ophthalmic imaging techniques for studying microvascular flow in the eye. Compared with the proximal retinal vasculature, the more distal choroidal vasculature is relatively more challenging to probe. Magnetic Resonance Imaging and Doppler Ultrasound can assess the retina and choroid, but do not resolve the finer layers or microvasculature. While Optical Coherence Tomography (OCT) angiography produces high-quality choroidal images, attempts at quantification through Doppler-based methods have had mixed success. Here, we use a new technique called Dynamic Contrast OCT (DyC-OCT), which tracks the passage of an intravascular scattering contrast agent, to reveal laminar blood flow patterns in the retina and choroid in vivo. While conceptually similar to fluorescence angiography, DyC-OCT has the substantial benefit of depth resolution, which enables separation of retinal and choroidal microvasculature. The scattering contrast agent enables improved angiography of both macro- and microvasculature in the retina and choroid. Blood plasma transit times are measured in individual vessels, while flow and volume are quantified for each of the microvascular layers. As expected, the choriocapillaris had the highest volume and flow. Blood flow rates were estimated with an average retinal blood flow of 9.1 ± 4.3 μL/min and an average choroidal blood flow of 40 ± 18.3 μL/min in the rat eye. These rates are consistent with previous literature. DyC-OCT affords a new perspective on the poorly understood choriocapillaris blood flow and kinetics and may be useful for studying outer retinal diseases.

  18. Zinc and zinc chelators modify taurine transport in rat retinal cells.

    PubMed

    Márquez, Asarí; Urbina, Mary; Lima, Lucimey

    2014-11-01

    Zinc regulates Na(+)/Cl(-)-dependent transporters, similar to taurine one, such as those for dopamine, serotonin and norepinephrine. This study examined the ex vivo effect of zinc (ZnSO4), N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino (TPEN) and diethylenetriaminepenta-acetic acid (DTPA), intracellular and extracellular zinc chelators, respectively, on rat retina [(3)H]taurine transport. Isolated cells were incubated in Locke solution with 100 nM of [(3)H]taurine for 25 s. Different concentrations of ZnSO4 (0.5-200 μM) were used. Low concentrations of ZnSO4 (30 and 40 μM) increased the transport, while higher concentrations (100, 150 and 200 μM) decreased it. Various concentrations of TPEN (1-200 μM) were added. Intermediate concentrations of TPEN (10-60 μM) significantly decreased [(3)H]taurine transport. The presence of TPEN, 20 μM, plus ZnSO4 reversed the effect of TPEN alone. Several concentrations of DTPA (1-500 μM) were also investigated. Reduction of transport took place at high concentrations of the chelator (100, 250 and 500 μM). DTPA, 500 μM, plus ZnSO4, did not modify the effect of it. These results indicate that zinc modulates taurine transport in a concentration-dependent manner, directly acting on the transporter or by forming taurine-zinc complexes in cell membranes.

  19. [Metabolic characterization of rat sertoli cell in vitro culture].

    PubMed

    Shi, Bingyang; Zhang, Shuxiang; Guo, Meijin; Wang, Yonghong; Zhang, Siliang; Shi, Xiaolin

    2009-05-01

    Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat's testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine, amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2-4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.

  20. Electrophysiological properties of cultured hippocampal neurons from Wistar Audiogenic Rats.

    PubMed

    Mesquita, Fernando; Aguiar, José F; Oliveira, José A; Garcia-Cairasco, Norberto; Varanda, Wamberto A

    2005-03-15

    The main goal of this work was to analyze the electrophysiological properties of cultured hippocampal neurons from a particular epileptic rat strain, called Wistar Audiogenic Rats (WAR). The whole-cell patch-clamp technique was used to record both active and passive membrane responses in an attempt to detect alterations in their characteristics in relation to controls from Wistar rats. Neurons from WARs show a significant reduction in the magnitude of the inhibitory GABAergic currents ( approximately 45%), in spite of maintaining a normal level of the excitatory glutamatergic currents. In addition, the magnitude of potassium currents, measured at +80 mV, is reduced by about 30% in comparison to controls. Surprisingly, we also found important changes in the passive cellular properties in WAR neurons such as membrane potential (-50.0 mV in WARs and -63.1 mV in controls) and input resistance (647 MOmega in WARs and 408 MOmega in controls). The changes described here, could be the basis of the neurophysiological and behavioral alterations present in these hyperexcitable animals, contributing to a better understanding of epileptogenesis in this particular animal model.

  1. Retinal upregulation of inflammatory and proangiogenic markers in a model of neonatal diabetic rats fed on a high-fat-diet

    PubMed Central

    2013-01-01

    Background The contemporary peak of diabetes seems to be related to obesity, sedentary lifestyle and diet. Diabetic retinopathy is the most leading cause of blindness in adulthood in industrialized countries. Our purpose was to evaluate the effect of a high-fat-diet (HFD) on the retina of diabetic rats. Methods Two groups of Wistar rats were injected with streptozotocin (STZ) two days after birth using 45 and 90 mg/kg, respectively. At 8 weeks the group on lower doses started to be fed on a HFD. Animals were sacrificed at 37 weeks of diabetes. A control group was made up of non-diabetic rats. Retinal flat mounts were examined using the trypsin digestion technique. Pericytes counts were compared between diabetic and control rats. Cross retinal sections were analyzed by histological techniques and immunohistochemistry and immunofluorescent technique. Primary antibodies against inflammatory and proangiogenic mediators such as RAGE, GFAP, 5-LO, VEGF and TNF-α were used for immunohistochemistry and Western Blot (WB) analyses. Results In the two diabetic groups we observed GFAP-positive cells with a morphology and spatial organization similar to those seen in Müller cells. Both diabetic groups had a significantly lower number of pericytes than non-diabetic animals.Increased retinal immunoreactivity of GFAP, RAGE, TNF-α, VEGF and 5-LO was seen in diabetic animals fed on HFD compared to the other groups of animals. WB analysis revealed a higher expression of 5-LO, VEGF, TNF-α and RAGE in the retina of diabetic rats on HFD than in controls and diabetics fed on a normal diet. The percentage of RAGE-stained ganglion cells and ganglion cells was found to be significantly lower in animals on a HFD than in the other animals. Conclusions Diabetic animals fed on a HFD showed an increased upregulation of inflammatory and proangiogenic markers. This animal model may be useful to study mechanisms of diabetic retinopathy and therapeutic targets. PMID:23587252

  2. Characterization of glucose uptake by cultured rat podocytes.

    PubMed

    Lewko, Barbara; Bryl, Ewa; Witkowski, Jacek M; Latawiec, Elzbieta; Gołos, Magdalena; Endlich, Nicole; Hähnel, Brunhilde; Koksch, Claudia; Angielski, Stefan; Kriz, Wilhelm; Stepinski, Jan

    2005-01-01

    The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.

  3. Effect of geranylgeranylacetone on gentamycin ototoxicity in rat cochlea culture.

    PubMed

    Sano, Hajime; Yoneda, Satoshi; Iwase, Hitoo; Itoh, Akihiko; Hashimoto, Daimon; Okamoto, Makito

    2007-03-01

    Geranylgeranylacetone (GGA), an anti-ulcer drug, has been reported to induce heat shock proteins (HSPs) in several animal organs. Purpose of this study was to investigate whether GGA has protective effect on gentamycin (GM) ototoxicity and whether it induces HSP70 in rat cochlea. We used cochlea explant culture from postnatal 5-day rat. Explants were pre-incubated with GGA, then incubated with GM and GGA. The number of surviving outer hair cells (OHCs) labeled by phalloidin was counted to evaluate the protective effect of GGA. The expression of HSP70 in whole cochlea tissue was investigated by immunoblot analysis. The number of surviving OHCs was significantly high in GGA 10(-5)M group compared to GM only group and GGA 10(-6)M group. In the immunoblot analysis, HSP70 levels in GGA added groups were slightly high compared to simple culture group, but much lower than those in heat shock group. It was suggested that GGA-induced HSP70, and had partial protective effects on GM ototoxicity in the cochlea. GGA has possibility to be safe and useful treatment drug for cochlea disorder.

  4. AN ELECTRON MICROSCOPE STUDY OF CULTURED RAT SPINAL CORD

    PubMed Central

    Bunge, Richard P.; Bunge, Mary Bartlett; Peterson, Edith R.

    1965-01-01

    Explants prepared from 17- to 18-day fetal rat spinal cord were allowed to mature in culture; such preparations have been shown to differentiate and myelinate in vitro (61) and to be capable of complex bioelectric activity (14–16). At 23, 35, or 76 days, the cultures were fixed (without removal from the coverslip) in buffered OsO4, embedded in Epon, sectioned, and stained for light and electron microscopy. These mature explants generally are composed of several strata of neurons with an overlying zone of neuropil. The remarkable cytological similarity between in vivo and in vitro nervous tissues is established by the following observations. Cells and processes in the central culture mass are generally closely packed together with little intervening space. Neurons exhibit well developed Nissl bodies, elaborate Golgi regions, and subsurface cisternae. Axosomatic and axodendritic synapses, including synaptic junctions between axons and dendritic spines, are present. Typical synaptic vesicles and increased membrane densities are seen at the terminals. Variations in synaptic fine structure (Type 1 and Type 2 synapses of Gray) are visible. Some characteristics of the cultured spinal cord resemble infrequently observed specializations of in vivo central nervous tissue. Neuronal somas may display minute synapse-bearing projections. Occasionally, synaptic vesicles are grouped in a crystal-like array. A variety of glial cells, many apparently at intermediate stages of differentiation, are found throughout the otherwise mature explant. There is ultrastructural evidence of extensive glycogen deposits in some glial processes and scattered glycogen particles in neuronal terminals. This is the first description of the ultrastructure of cultured spinal cord. Where possible, correlation is made between the ultrastructural data and the known physiological properties of these cultures. PMID:14326105

  5. Regenerative and other responses to injury in the retinal stump of the optic nerve in adult albino rats: transection of the intracranial optic nerve.

    PubMed Central

    Zeng, B Y; Anderson, P N; Campbell, G; Lieberman, A R

    1995-01-01

    The response to injury of the proximal (retinal) stump of the intracranially transected optic nerve in adult rats has been studied by electron microscopy. The central part of the retinal stump of the optic nerve underwent severe ischaemic damage resulting in the formation by 3 days postoperation (dpo) of a cone-shaped region of necrotic tissue which extended from a base occupying most of the cross-sectional area of the nerve at the cut end to an apex within the intraorbital part of the nerve and only 2-3 mm from the eyeball. A mixture of apparently viable and dead or dying cells and axons was present in an intermediate zone surrounding the ischaemic core. Apparently intact nerve fibres occupied most of the periphery of the optic nerve. Small bundles of sprout-like axons were seen in the intermediate zone at 3 dpo, and by 5 dpo such sprouts were present at the periphery of the degenerative core. By 7 dpo, the sprouts were also found in the centre of the degenerative core, accompanied by astrocyte processes. The number of axonal sprouts present in the degenerative core and intermediate zone was much higher at 2 and 4 wk postoperation (wpo) than at 7 dpo, then declined gradually by 6 and 8 wpo. These results show that intracranial transection of the rat optic nerve produces extensive degeneration in the proximal stump and effectively produces an intraorbital axotomy of many retinal ganglion cells. Nevertheless, surviving axons display the ability to produce regenerative sprouts which persist for considerably longer than those produced after intraorbital injury. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 Fig. 17 Fig. 18 PMID:7559123

  6. Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

    PubMed

    Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-09-22

    Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

  7. Retinal detachment

    PubMed Central

    2010-01-01

    Introduction Rhegmatogenous retinal detachment (RRD) is the most common form of retinal detachment, where a retinal "break" allows the ingress of fluid from the vitreous cavity to the subretinal space, resulting in retinal separation. It occurs in about 1 in 10,000 people a year. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of interventions to prevent progression from retinal breaks or lattice degeneration to retinal detachment? What are the effects of different surgical interventions in people with rhegmatogenous retinal detachment? What are the effects of interventions to treat proliferative vitreoretinopathy occurring as a complication of retinal detachment or previous treatment for retinal detachment? We searched: Medline, Embase, The Cochrane Library, and other important databases up to June 2010 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 21 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review, we present information relating to the effectiveness and safety of the following interventions: corticosteroids, cryotherapy, daunorubicin, fluorouracil plus low molecular weight heparin, laser photocoagulation, pneumatic retinopexy, scleral buckling, short-acting or long-acting gas tamponade, silicone oil tamponade, and vitrectomy. PMID:21406128

  8. Retinal detachment

    PubMed Central

    2009-01-01

    Introduction Rhegmatogenous retinal detachment (RRD) is the most common form of retinal detachment, where a retinal "break" allows the ingress of fluid from the vitreous cavity to the subretinal space, resulting in retinal separation. It occurs in about 1 in 10,000 people a year. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of interventions to prevent progression from retinal breaks or lattice degeneration to retinal detachment? What are the effects of different surgical interventions in people with rhegmatogenous retinal detachment? What are the effects of interventions to treat proliferative vitreoretinopathy occurring as a complication of retinal detachment or previous treatment for retinal detachment? We searched: Medline, Embase, The Cochrane Library, and other important databases up to March 2009 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 20 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review, we present information relating to the effectiveness and safety of the following interventions: corticosteroids; cryotherapy; daunorubicin; fluorouracil plus low-molecular-weight heparin; laser photocoagulation; pneumatic retinopexy; scleral buckling; short-acting or long-acting gas tamponade; silicone oil tamponade; and vitrectomy. PMID:19450333

  9. The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells

    PubMed Central

    Song, Yookyung; Hong, Samin; Iizuka, Yoko; Kim, Chan Yun

    2015-01-01

    Purpose Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro. Methods R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis. Results R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-κB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-κB, ERK, and JNK. Conclusions Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act

  10. Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells

    PubMed Central

    Chen, Rui; Grosche, Antje; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2014-01-01

    Purpose Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. Results With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt

  11. Changes in the photoreceptor mosaic of P23H-1 rats during retinal degeneration: implications for rod-cone dependent survival.

    PubMed

    García-Ayuso, Diego; Ortín-Martínez, Arturo; Jiménez-López, Manuel; Galindo-Romero, Caridad; Cuenca, Nicolás; Pinilla, Isabel; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta; Villegas-Pérez, María P

    2013-08-28

    To investigate the spatiotemporal relationship between rod and cone degeneration in the P23H-1 rat. Control Sprague-Dawley (SD) and P23H-1 rats of ages ranging from P30 to P365 were used. Retinas were processed for whole mounts or cross sections and rods and cones were immunodetected. We used newly developed image analysis techniques to quantify the total population of L/M cones (the most abundant cones in the rat) and analyzed the rings of rod-cone degeneration. In P23H-1 rats, rod degeneration occurs rapidly: first the rod outer segment shortens, at P30 there is extensive rod loss, and by P180 rod loss is almost complete except for the most peripheral retina. The numbers of L/M cones are, at all postnatal ages, lower in P23H-1 rats than in control SD rats, and decrease significantly with age (by P180). Rod and cone degeneration is spatiotemporally related and occurs in rings that appear already at P90 and spread throughout the entire retina. At P180, the rings of rod-cone degeneration are more abundant in the equatorial retina and are larger in the dorsal retina. This work describes for the first time that in the P23H-1 rat, rod and cone degeneration is spatiotemporally related and occurs in rings. Cone loss follows rod loss and starts very soon, even before P30, the first age analyzed here. The characteristics of the rings suggest that secondary cone degeneration is influenced by retinal position and/or other intrinsic or extrinsic factors.

  12. Computational tool for morphological analysis of cultured neonatal rat cardiomyocytes.

    PubMed

    Leite, Maria Ruth C R; Cestari, Idágene A; Cestari, Ismar N

    2015-08-01

    This study describes the development and evaluation of a semiautomatic myocyte edge-detector using digital image processing. The algorithm was developed in Matlab 6.0 using the SDC Morphology Toolbox. Its conceptual basis is the mathematical morphology theory together with the watershed and Euclidean distance transformations. The algorithm enables the user to select cells within an image for automatic detection of their borders and calculation of their surface areas; these areas are determined by adding the pixels within each myocyte's boundaries. The algorithm was applied to images of cultured ventricular myocytes from neonatal rats. The edge-detector allowed the identification and quantification of morphometric alterations in cultured isolated myocytes induced by 72 hours of exposure to a hypertrophic agent (50 μM phenylephrine). There was a significant increase in the mean surface area of the phenylephrine-treated cells compared with the control cells (p<;0.05), corresponding to cellular hypertrophy of approximately 50%. In conclusion, this edge-detector provides a rapid, repeatable and accurate measurement of cell surface areas in a standardized manner. Other possible applications include morphologic measurement of other types of cultured cells and analysis of time-related morphometric changes in adult cardiac myocytes.

  13. High resolution optoelectronic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Loudin, Jim; Dinyari, Rostam; Huie, Phil; Butterwick, Alex; Peumans, Peter; Palanker, Daniel

    2009-02-01

    Electronic retinal prostheses seek to restore sight in patients with retinal degeneration by delivering pulsed electric currents to retinal neurons via an array of microelectrodes. Most implants use inductive or optical transmission of information and power to an intraocular receiver, with decoded signals subsequently distributed to retinal electrodes through an intraocular cable. Surgical complexity could be minimized by an "integrated" prosthesis, in which both power and data are delivered directly to the stimulating array without any discrete components or cables. We present here an integrated retinal prosthesis system based on a photodiode array implant. Video frames are processed and imaged onto the retinal implant by a video goggle projection system operating at near-infrared wavelengths (~ 900 nm). Photodiodes convert light into pulsed electric current, with charge injection maximized by specially optimized series photodiode circuits. Prostheses of three different pixel densities (16 pix/mm2, 64 pix/mm2, and 256 pix/mm2) have been designed, simulated, and prototyped. Retinal tissue response to subretinal implants made of various materials has been investigated in RCS rats. The resulting prosthesis can provide sufficient charge injection for high resolution retinal stimulation without the need for implantation of any bulky discrete elements such as coils or tethers. In addition, since every pixel functions independently, pixel arrays may be placed separately in the subretinal space, providing visual stimulation to a larger field of view.

  14. Retinal meteor.

    PubMed

    Venkatesh, Ramesh; Gurav, Prachi; Dave, Prachi Abhishek; Roy, Sankhadeep

    2017-09-01

    We describe a case of a 65-year old man diagnosed with retinal vasoproliferative tumour secondary to posterior uveitis. The fluorescein angiography shows an interesting meteor-like leak emanating from the tumour and rising towards the superior retina in the later frames of the angiogram. Pictorially, we call it the "Retinal Meteor" and also describe the possible mechanism for this pattern of leakage.

  15. Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

    PubMed

    Danesh-Meyer, Helen V; Kerr, Nathan M; Zhang, Jie; Eady, Elizabeth K; O'Carroll, Simon J; Nicholson, Louise F B; Johnson, Cameron S; Green, Colin R

    2012-02-01

    Connexin43 gap junction protein is expressed in astrocytes and the vascular endothelium in the central nervous system. It is upregulated following central nervous system injury and is recognized as playing an important role in modulating the extent of damage. Studies that have transiently blocked connexin43 in spinal cord injury and central nervous system epileptic models have reported neuronal rescue. The purpose of this study was to investigate neuronal rescue following retinal ischaemia-reperfusion by transiently blocking connexin43 activity using a connexin43 mimetic peptide. A further aim was to evaluate the effect of transiently blocking connexin43 on vascular permeability as this is known to increase following central nervous system ischaemia. Adult male Wistar rats were exposed to 60 min of retinal ischaemia. Treatment groups consisted of no treatment, connexin43 mimetic peptide and scrambled peptide. Retinas were then evaluated at 1-2, 4, 8 and 24 h, and 7 and 21 days post-ischaemia. Evans blue dye leak from retinal blood vessels was used to assess vascular leakage. Blood vessel integrity was examined using isolectin-B4 labelling. Connexin43 levels and astrocyte activation (glial fibrillary acidic protein) were assessed using immunohistochemistry and western blot analysis. Retinal whole mounts and retinal ganglion cell counts were used to quantify neurodegeneration. An in vitro cell culture model of endothelial cell ischaemia was used to assess the effect of connexin43 mimetic peptide on endothelial cell survival and connexin43 hemichannel opening using propidium iodide dye uptake. We found that retinal ischaemia-reperfusion induced significant vascular leakage and disruption at 1-2, 4 and 24 h following injury with a peak at 4 h. Connexin43 immunoreactivity was significantly increased at 1-2, 4, 8 and 24 h post ischaemia-reperfusion injury co-localizing with activated astrocytes, Muller cells and vascular endothelial cells. Connexin43 mimetic peptide

  16. An AD-related neuroprotector rescues transformed rat retinal ganglion cells from CoCl₂-induced apoptosis.

    PubMed

    Men, Jie; Zhang, Xiaohui; Yang, Yang; Gao, Dianwen

    2012-05-01

    Some ocular diseases characterized by apoptotic death of retinal ganglion cells (RGCs) and Alzheimer's disease (AD) are chronic neurodegenerative disorders and have similarities in neuropathology. Humanin (HN) is known for its ability to suppress neuronal death induced by AD-related insults. In present study, we investigated the neuroprotective effects of HN on hypoxia-induced toxicity in RGC-5 cells. Hypoxia mimetic compound cobalt chloride (CoCl₂) could increase the cell viability loss and apoptosis, whereas HN can significantly attenuate these effects. This finding may provide new therapeutics for the retinal neurodegenerative diseases targeting neuroprotection.

  17. Effects of Y-39983, a selective Rho-associated protein kinase inhibitor, on blood flow in optic nerve head in rabbits and axonal regeneration of retinal ganglion cells in rats.

    PubMed

    Tokushige, Hideki; Waki, Mitsunori; Takayama, Yoshiko; Tanihara, Hidenobu

    2011-10-01

    To investigate the effects of Y-39983, a selective Rho-associated coiled coil-forming protein kinase inhibitor, on blood flow in the optic nerve head (ONH) in rabbits and axonal regeneration of retinal ganglion cells (RGCs) in rats. Blood flow in ONH was measured by the laser speckle method after topical administration of 0.05% Y-39983 solution or its vehicle in rabbit eyes. To investigate the effects of Y-39983 on axonal regeneration of RGCs, RGCs purified from rat eyes were cultured with or without 10 μM Y-39983 and morphologically observed by phase-contrast microscopy. Moreover, the effects of intravitreal administration of Y-39983 were evaluated using an in vivo model of axotomized RGCs in peripheral nerve-grafted rats. Topical administration of 0.05% Y-39983 solution significantly increased blood flow in ONH compared with the vehicle group in rabbits. Maximum increase in blood flow in the 0.05% Y-39983 group was 122.84 ± 5.98 % (Mean ± S.E.) at 90 minutes after administration compared with before administration. Neurites in rat RGCs treated with 10 μM Y-39983 were extended compared with those without Y-39983 treatment of RGCs in vitro. Y-39983 dose-dependently increased the number of RGCs with regenerating axons in vivo. The numbers of RGCs with regenerating axons in 10 and 100 μM Y-39983-treated rats were 99.3 ± 10.5 and 169.5 ± 43.3 cells/mm(2) (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm(2)). Y-39983 may be a candidate drug not only for lowering of IOP but also for increasing of blood flow in ONH in the treatment of glaucoma. Moreover, Y-39983 may have therapeutic potential for axonal regeneration of RGCs in the treatment of diseases with degenerating axons of RGCs including glaucoma, although improvements of formulation or route of administration are needed in order to reach an effective concentration in retina.

  18. Retinal remodeling.

    PubMed

    Jones, B W; Kondo, M; Terasaki, H; Lin, Y; McCall, M; Marc, R E

    2012-07-01

    Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and

  19. [Kurloff's thymic inclusion : action on rat gonads in culture].

    PubMed

    De Graeve, P; Vincent, M F; Amiel, S; Moatti, J P; Guilhem, A; Bimes, C

    1981-12-01

    Thymic and splenic extracts rich in FOA-KURLOFF (F.K.) body cells, obtained from guinea-pigs treated with oestrogen, were added to rat testis or ovaries in culture. Controls were prepared with extracts from thymus and spleen of non treated animals and from kidneys of treated or non treated animals. After five hours the level of sexual hormones and the germinal cells were studied. The F.K. substance has no effect on germinal cells and on progesterone and testosterone secretion. The F.K. substance induces a significative decrease of oestrogen secretion. In an other paper we established that F.K. bodies induced a hyperactivity of internal theca folliculi and of ovarian interstitial cells. It is a false image of activity in connection with a hypersecretion of FSH. The F.K. substance inhibits oestrogen synthesis.

  20. Cultured rat microglia express functional beta-chemokine receptors.

    PubMed

    Boddeke, E W; Meigel, I; Frentzel, S; Gourmala, N G; Harrison, J K; Buttini, M; Spleiss, O; Gebicke-Härter, P

    1999-08-03

    We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

  1. Formation of extracellular matrix by cultured rat mesangial cells.

    PubMed Central

    Ishimura, E.; Sterzel, R. B.; Budde, K.; Kashgarian, M.

    1989-01-01

    Formation of extracellular matrix (ECM) by mesangial cells (MCs) contributes to progressive glomerulosclerosis. The authors investigated the production and distribution of ECM constituents by cultured rat MCs, using immunocytochemistry and immunoelectron microscopy. Staining for all ECM constituents increased after serum feeding. Localization was strictly intracellular until confluency, when extracellular deposition of collagen IV and laminin appeared, followed by fibronectin and collagen III. In parallel, the intracellular staining for these proteins diminished markedly. Neither extracellular deposition nor intracellular loss was observed for collagen I and thrombospondin. On surfaces coated with collagen IV or laminin, extracellular deposition of ECM constituents clearly preceded confluency. These results indicate that synthesis of ECM constituents parallels MC growth, and that extracellular deposition of ECM occurs at cell-cell contact. Collagen IV or laminin secreted by MCs in the substratum accelerates production and facilitates secretion of other ECM constituents in an autocrine fashion. Images Figure 1 Figure 3 Figure 5 Figure 6 Figure 8 PMID:2650558

  2. Regulation of bile acid synthesis in rat hepatocyte monolayer cultures

    SciTech Connect

    Kubaska, W.M.

    1986-01-01

    Primary hepatocyte monolayer cultures (PHC) were prepared and incubated in serum free media. Cells from a cholestyramine fed rat converted exogenous (/sup 14/C)-cholesterol into (/sup 14/C)-bile acids at a 3-fold greater rate than rats fed a normal diet. PHC synthesize bile acids (BA) at a rate of approximately 0.06 ..mu..g/mg protein/h. The major bile acid composition, as determined by GLC, was ..beta..-muricholic acid (BMC) and cholic acid (CA) in a 3:1 ratio, respectively. PHC rapidly converted free BA and BA intermediates into taurine conjugated trihydroxy-BA up to 87h after plating. 3-Hydroxy-3-methylglutaryl-coenzyme A-reductase activity assayed in microsomes prepared from PHC, decreased during the initial 48h, then remained constant. Cholesterol 7..cap alpha..-hydroxylase activity decreased during the initial 48h, then increased during the next 48h. This occurred while whole cells produced BA at a linear rate. The effect of individual BA on bile acid synthesis (BAS) was also studied. Relative rates of BAS were measured as the conversion of (/sup 14/C)-cholesterol into (/sup 14/C)-BA. BA combinations were tested in order to simulate the composition of the enterohepatic circulation. The addition of TCA (525 ..mu..M) plus TCDCA (80..mu..M), in concentrations which greatly exceed the concentration of BA (60..mu..M) in rate portal blood, failed to inhibit BAS. BA plus phospholipid and/or cholesterol also did not inhibit BAS. Surprisingly, crude rat bile with a final concentration comparable to those in the synthetic mix inhibited (/sup 14/C)-cholesterol conversion into (/sup 14/C)-BA.

  3. Homocysteine Induces Glial Reactivity in Adult Rat Astrocyte Cultures.

    PubMed

    Longoni, Aline; Bellaver, Bruna; Bobermin, Larissa Daniele; Santos, Camila Leite; Nonose, Yasmine; Kolling, Janaina; Dos Santos, Tiago M; de Assis, Adriano M; Quincozes-Santos, André; Wyse, Angela T S

    2017-03-02

    Astrocytes are dynamic glial cells associated to neurotransmitter systems, metabolic functions, antioxidant defense, and inflammatory response, maintaining the brain homeostasis. Elevated concentrations of homocysteine (Hcy) are involved in the pathogenesis of age-related neurodegenerative disorders, such as Parkinson and Alzheimer diseases. In line with this, our hypothesis was that Hcy could promote glial reactivity in a model of cortical primary astrocyte cultures from adult Wistar rats. Thus, cortical astrocytes were incubated with different concentrations of Hcy (10, 30, and 100 μM) during 24 h. After the treatment, we analyzed cell viability, morphological parameters, antioxidant defenses, and inflammatory response. Hcy did not induce any alteration in cell viability; however, it was able to induce cytoskeleton rearrangement. The treatment with Hcy also promoted a significant decrease in the activities of Na(+), K(+) ATPase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as in the glutathione (GSH) content. Additionally, Hcy induced an increase in the pro-inflammatory cytokine release. In an attempt to elucidate the putative mechanisms involved in the Hcy-induced glial reactivity, we measured the nuclear factor kappa B (NFκB) transcriptional activity and heme oxygenase 1 (HO-1) expression, which were activated and inhibited by Hcy, respectively. In summary, our findings provide important evidences that Hcy modulates critical astrocyte parameters from adult rats, which might be associated to the aging process.

  4. Functional studies of acid transporter in cultured rat epididymal cell.

    PubMed

    Zuo, Wu-Lin; Huang, Jie-Hong; Shan, Jia-Jie; Li, Sheng; Wong, Patrick Y D; Zhou, Wen-Liang

    2010-05-15

    To explore the functional role of vacuolar H(+)-ATPase in the pH regulation of epididymal fluid and its effect on sperm motility. Experimental study. Physiology laboratory in a university. Immature male Sprague-Dawley rats. The H(+)-ATPase inhibitor was applied to the primary culture of epididymal cells. The intracellular luminal fluid pH and sperm percent motility were recorded. Double immunofluorescence of H(+)-ATPase and carbonic anhydrase II in primary culture of cauda epididymal epithelial cells showed that the system was a suitable model for investigation of acid secretion by clear cells. Clear cells were pharmacologically distinct from principal cells in acid/base transportation. The intracellular pH recovery from cellular acidification was suppressed by the H(+)-ATPase inhibitor bafilomycin A1(100 nM) and the Na(+)/H(+) exchanger inhibitor amiloride (1 mM) by 85% and 54%, respectively. These results suggest that, in addition to Na(+)/H(+) exchanger, clear cells actively pump proton from cytoplasm into extracellular space through H(+)-ATPase. In addition, inhibition of H(+)-ATPase by bafilomycin A1 blocked the acidification of luminal fluid with IC(50) values of 12 nM, which supports that H(+)-ATPase acidifies the luminal fluid. We also confirm that the acid fluid regulates rat cauda sperm motility. The present work shows that clear cells, the minority cell type of epididymal cell population, play an important role in the pH regulation of epididymal fluid by H(+)-ATPase. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    SciTech Connect

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  6. Sex differences in DNA damage produced by the carcinogen 2-acetylaminofluorene in cultured human hepatocytes compared to rat liver and cultured rat hepatocytes.

    PubMed

    Williams, Gary M; Duan, Jian-Dong; Iatropoulos, Michael J; Perrone, Carmen E

    2016-02-01

    Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague-Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.

  7. Retinal detachment

    PubMed Central

    2014-01-01

    Introduction Rhegmatogenous retinal detachment (RRD) is the most common form of retinal detachment, where a retinal 'break' allows the ingress of fluid from the vitreous cavity to the subretinal space, resulting in retinal separation. It occurs in about 1 in 10,000 people a year. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of different surgical interventions in people with rhegmatogenous retinal detachment? What are the effects of interventions to treat proliferative vitreoretinopathy occurring as a complication of retinal detachment or previous treatment for retinal detachment? We searched: Medline, Embase, The Cochrane Library, and other important databases up to September 2013 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 14 studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review, we present information relating to the effectiveness and safety of the following interventions: corticosteroids, daunorubicin, fluorouracil plus low molecular weight heparin, pneumatic retinopexy, scleral buckling, short-acting or long-acting gas tamponade, silicone oil tamponade, and vitrectomy. PMID:24807890

  8. Bisphenol A-induced apoptosis of cultured rat Sertoli cells.

    PubMed

    Iida, Hiroshi; Maehara, Kazue; Doiguchi, Masamichi; Mōri, Takayuki; Yamada, Fumio

    2003-01-01

    Bisphenol A (BPA) was examined for its effects on cultured Sertoli cells established from 18-day-old rat testes. We demonstrated that exposure of cultured Sertoli cells to BPA decreased the cell viability in a dose- and a time-dependent manner and that exposure to BPA brought about morphologic changes of the cells, such as membrane blebs, cell rounding, cytoskeletal collapse, and chromatin condensation or fragmentation, all of which conform to the morphologic criteria for apoptosis. Immunocytochemistry showed that active caspase-3, a major execution caspase, was expressed in round Sertoli cells positively labeled by the TUNEL method. Co-localization of active caspase-3 and aggregated actin fragments was also observed in the round Sertoli cells. Theses results suggest that BPA induces cell death of Sertoli cells by promoting apoptosis. Apoptosis-inducing cell death was observed in cells exposed to 150-200 microM BPA, while BPA at <100 microM had only slight cytotoxic effects on the cells.

  9. Neurotoxicity of trimethyltin in rat cochlear organotypic cultures

    PubMed Central

    Yu, Jintao; Ding, Dalian; Sun, Hong; Salvi, Richard; Roth, Jerome A.

    2015-01-01

    Trimethyltin (TMT), which has a variety of applications in industry and agricultural is a neurotoxin that is known to affect the auditory system as well as central nervous system (CNS) of humans and experimental animals. However, the mechanisms underlying TMT-induced auditory dysfunction are poorly understood. To gain insights into the neurotoxic effect of TMT on the peripheral auditory system, we treated cochlear organotypic cultures with concentrations of TMT ranging from 5 to 100 μM for 24 h. Interestingly, TMT preferentially damaged auditory nerve fibers and spiral ganglion neurons in a dose-dependent manner, but had no noticeable effects on the sensory hair cells at the doses employed. TMT-induced damage to auditory neurons was associated with significant soma shrinkage, nuclear condensation and activation of caspase-3, biomarkers indicative of apoptotic cell death. Our findings show that TMT is exclusively neurotoxicity in rat cochlear organotypic culture and that TMT-induced auditory neuron death occurs through a caspase-mediated apoptotic pathway. PMID:25957118

  10. Effect of ETBE on reproductive steroids in male rats and rat Leydig cell cultures.

    PubMed

    de Peyster, Ann; Stanard, Bradley; Westover, Christian

    2009-10-08

    These experiments were conducted to follow up on a report of testis seminiferous tubular degeneration in Fischer 344 rats treated with high doses of ethyl t-butyl ether (ETBE). Also, high doses of a related compound, methyl t-butyl ether (MTBE), had been shown to reduce circulating testosterone (T) in rats. Isolated rat Leydig cells were used to compare hCG-stimulated T production following exposure to ETBE, MTBE, and their common main metabolite, TBA. In addition, male Fischer 344 rats were gavaged daily with 600 mg/kg, 1200 mg/kg or 1800 mg/kg ETBE in corn oil (n=12) for 14 days, the 1200 mg/kg dose chosen for comparison with a prior 14-day MTBE gavage experiment. In cell culture experiments, TBA was more potent than either ETBE or MTBE, both of which caused similar inhibition of T production at equimolar concentrations. In the in vivo study, no significant plasma T reduction was seen 1h after the final 1200 mg/kg ETBE dose, whereas 1200 mg/kg MTBE had significantly lowered T when administered similarly to Sprague-Dawley rats. Some rats treated with 1800 mg/kg ETBE had noticeably lower T levels, and the group average T level was 66% of corn oil vehicle control (p>0.05) with high variability also evident in ETBE-treated rats. 17beta-Estradiol had been increased by 1200 mg/kg MTBE, and was elevated in the 1200 and 1800 mg/kg ETBE dose groups (p<0.05), both groups also experiencing significantly reduced body weight gain. None of these effects were seen with 600 mg/kg/day ETBE. No definitive evidence of androgen insufficiency was seen in accessory organ weights, and no testicular pathology was observed after 14 days in a small subset of 1800 mg/kg ETBE-treated animals. Like MTBE, ETBE appears to be capable of altering reproductive steroid levels in peripheral blood sampled 1h after treatment, but only with extremely high doses that inhibit body weight gain and may produce mortality.

  11. Retinal Prosthesis

    PubMed Central

    Weiland, James D.; Humayun, Mark S.

    2015-01-01

    Retinal prosthesis have been translated from the laboratory to the clinical over the past two decades. Currently, two devices have regulatory approval for the treatment of