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Sample records for cultured rat retinal

  1. 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.

  2. Uptake and esterification of vitamin A by RCS rat retinal pigment epithelial cells in primary culture.

    PubMed

    Cia, David; Bonhomme, Brigitte; Azaïs-Braesco, Véronique; Cluzel, Jacques; Doly, Michel

    2004-02-01

    We investigated the capacity of Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells to take up all-trans-retinol (ROL) (vitamin A) and to metabolize it into retinyl esters (RE). Cultures of RPE cells were established from RCS and control newborn rats. All-trans-ROL was delivered to the apical surface of the RPE monolayer. Retinoids were analyzed by high-performance liquid chromatography. The cellular retinol-binding protein type I (CRBP-I) was assessed by Western blotting. Before supplementation with ROL, RE were lower in RCS rats. After ROL supplementation, esters increased and reached values that were similar in the two strains, but the increase, expressed relative to the initial value, was higher in RCS rats. The uptake of ROL and the level of CRBP-I were greater in RCS rats. Our results provide evidence of a functional retinol esterifying enzyme in cultured RCS RPE cells and suggest that CRBP-I could play a role in the uptake and esterification of ROL in the RPE cells.

  3. 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.

  4. 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

  5. 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

  6. Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration.

    PubMed

    Inoue, Yuji; Iriyama, Aya; Ueno, Shuji; Takahashi, Hidenori; Kondo, Mineo; Tamaki, Yasuhiro; Araie, Makoto; Yanagi, Yasuo

    2007-08-01

    Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.

  7. 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.

  8. 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.

  9. 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.

  10. Activation of Müller cells occurs during retinal degeneration in RCS rats.

    PubMed

    Zhao, Tong Tao; Tian, Chun Yu; Yin, Zheng Qin

    2010-01-01

    Müller cells can be activated and included in different functions under many kinds of pathological conditions, however, the status of Müller cells in retinitis pigmentosa are still unknown. Using immunohistochemisty, Western blots and co-culture, we found that Müller cells RCS rats, a classic model of RP, could be activated during the progression of retinal degeneration. After being activated at early stage, Müller cells began to proliferate and hypertrophy, while at later stages, they formed a local 'glial seal' in the subretinal space. As markers of Müller cells activation, the expression of GFAP and ERK increased significantly with progression of retinal degeneration. Co-cultures of normal rat Müller cells and mixed RCS rat retinal cells show that Müller cells significantly increase GFAP and ERK in response to diffusable factors from the degenerting retina, which implies that Müller cells activation is a secondary response to retinal degeneration.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Layer-specific blood-flow MRI of retinitis pigmentosa in RCS rats.

    PubMed

    Li, Guang; De La Garza, Bryan; Shih, Yen-Yu I; Muir, Eric R; Duong, Timothy Q

    2012-08-01

    The Royal College of Surgeons (RCS) rat is an established animal model of retinitis pigmentosa, a family of inherited retinal diseases which starts with loss of peripheral vision and progresses to eventual blindness. Blood flow (BF), an important physiological parameter, is intricately coupled to metabolic function under normal physiological conditions and is perturbed in many neurological and retinal diseases. This study reports non-invasive high-resolution MRI (44 × 44 × 600 μm) to image quantitative retinal and choroidal BF and layer-specific retinal thicknesses in RCS rat retinas at different stages of retinal degeneration compared with age-matched controls. The unique ability to separate retinal and choroidal BF was made possible by the depth-resolved MRI technique. RBF decreased with progressive retinal degeneration, but ChBF did not change in RCS rats up to post-natal day 90. We concluded that choroidal and retinal circulations have different susceptibility to progressive retinal degeneration in RCS rats. Layer-specific retinal thickness became progressively thinner and was corroborated by histological analysis in the same animals. MRI can detect progressive anatomical and BF changes during retinal degeneration with laminar resolution.

  19. 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.

  20. 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.

  1. 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

  2. Activation of retinal stem cells in the proliferating marginal region of RCS rats during development of retinitis pigmentosa.

    PubMed

    Jian, Qian; Xu, Haiwei; Xie, Hanping; Tian, Chunyu; Zhao, Tongtao; Yin, ZhengQin

    2009-11-06

    Retinal stem cells (RSCs) have been demonstrated at the proliferating marginal regions from the pars plana of ciliary body to the ciliary marginal zone (CMZ) in adult lower vertebrates and mammals. Investigations in the lower vertebrates have provided some evidence that RSCs can proliferate following retinal damage; however, the evidence that this occurs in mammals is not clear. In this study, we explored RSCs proliferation potential of adult mammalian in proliferating marginal regions of Royal College of Surgeons (RCS) rats, an animal model for retinitis pigmentosa (RP). The proliferation was evaluated using BrdU labeling, and Chx-10 as markers to discern progenitor cell of CMZ in Long-Evan's and RCS rats at different postnatal day (PND) after eye opening. We found that few Chx-10 and BrdU labeled cells in the proliferating marginal regions of Long-Evan's rats, which significantly increased in RCS rats at PND30 and PND60. Consistent with this, Chx-10/Vimentin double staining cells in the center retina of RCS rats increased significantly at PND30 after eye opening. In addition, mRNA expression of Shh, Ptch1 and Smo was up-regulated in RCS rats at PND60 compared to age-matched Long-Evan's rats, which revealed Shh/ptc pathway involving in the activation of RSCs. These results suggest that RSCs in the mammalian retinal proliferating marginal regions has the potential to regenerate following degeneration.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  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. BDNF improves the efficacy ERG amplitude maintenance by transplantation of retinal stem cells in RCS rats.

    PubMed

    Tian, Chunyu; Weng, Chuan Chuang; Yin, Zheng Qin

    2010-01-01

    The aim of this study was to evaluate the efficacy of subretinal transplantation of rat retinal stem cell when combined with Brain-derived neurotrophic factor (BDNF) in a rat model of retinal degeneration - Royal College of Surgeons (RCS) rats. Retinal stem cells were derived from embryonic day 17 Long-Evans rats and pre-labeled with fluorescence pigment-DiI prior to transplant procedures. RCS rats received injections of retinal stem cells, stem cells+BDNF, phosphate buffered saline or BNDF alone (n = 3 eyes for each procedure). At 1, 2 and 3 months after transplantation, the electroretinogram (ERG) was assessed and the outer nuclear layer thickness measured. The eyes receiving retinal stem cell and stem cell+BDNF transplants showed better photoreceptor maintenance than the other groups (P < 0.01) at all time points. One month after retina transplantation, the amplitudes of rod-ERG and Max-ERG b waves were significantly higher the eyes with stem cells+BDNF (P < 0.01), however, this difference was not seen at two and three months post transplantation. BDNF treatment alone group (without transplanted cells) had no effect when compared to buffer injections. The present results indicate that BDNF can enhance the short-term efficacy of the retinal stem cell transplantation in treating retinal degenerative disease.

  14. 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

  15. Vision maintenance and retinal apoptosis reduction in RCS rats with Okayama University-type retinal prosthesis (OUReP™) implantation.

    PubMed

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

    2015-09-01

    Photoelectric dye-coupled polyethylene film, designated Okayama University-type retinal prosthesis or OUReP™, generates light-evoked surface electric potentials and stimulates neurons. In this study, the vision was assessed by behavior tests in aged hereditary retinal dystrophic RCS rats with OUReP™, retinal apoptosis and electroretinographic responses were measured in dystrophic eyes with OUReP™. The dye-coupled films, or plain films as a control, were implanted in subretinal space of RCS rats. On behavior tests, RCS rats with dye-coupled films, implanted at the old age of 14 weeks, showed the larger number of head-turning, consistent with clockwise and anticlockwise rotation of a surrounding black-and-white-striped drum, compared with rats with plain films, under the dim (50 lux) and bright (150 lux) conditions in the observation period until the age of 22 weeks (n = 5, P < 0.05, repeated-measure ANOVA). The number of apoptotic cells in retinal sections at the site of dye-coupled film implantation was significantly smaller, compared with the other retinal sites, neighboring the film, or opposite to the film, 5 months after film implantation at the age of 6 weeks (P = 0.0021, Friedman test). The dystrophic eyes of RCS rats with dye-coupled films showed positive responses to maximal light stimulus at a significantly higher rate, compared with the eyes with no treatment (P < 0.05, Chi-square test). Electroretinograms in normal eyes of Wistar rats with dye-coupled or plain films showed significantly decreased amplitudes (n = 14, P < 0.05, repeated-measure ANOVA). In conclusions, vision was maintained in RCS rats with dye-coupled films implanted at the old age. The dystrophic eyes with dye-coupled films showed electroretinographic responses. Five-month film implantation caused no additional retinal changes.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. Autoimmune responses against photoreceptor antigens during retinal degeneration and their role in macrophage recruitment into retinas of RCS rats.

    PubMed

    Kyger, Madison; Worley, Aneta; Adamus, Grazyna

    2013-01-15

    Autoimmunity may contribute to retinal degeneration. The studies examined the evolution of autoimmune responses against retina in naïve dystrophic RCS rats over the course of retinal degeneration. We showed that anti-retinal autoantibodies and T cells are generated in response to the availability of antigenic material released from dying photoreceptor cells during retinal degeneration but with distinctive activation trends. Passive transfer of anti-retinal antibodies enhanced disease progression by disrupting the BRB, upregulating MCP-1, attracting blood macrophages into retina, and augmenting apoptotic photoreceptor cell death. Our findings directly link anti-retinal autoantibodies to activated macrophage entry and their possible role in neurodegeneration.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  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. 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.

  15. 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.

  16. [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.

  17. 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.

  18. Correlation of cytokine levels and microglial cell infiltration during retinal degeneration in RCS rats.

    PubMed

    Liu, Yong; Yang, Xuesen; Utheim, Tor Paaaske; Guo, Chenying; Xiao, Mingchun; Liu, Yan; Yin, Zhengqin; Ma, Jie

    2013-01-01

    Microglial cells, which are immunocompetent cells, are involved in all diseases of the central nervous system. During their activation in various diseases, a variety of soluble factors are released. In the present study, the correlation between cytokine levels and microglial cell migration in the course of retinal degeneration of Royal College of Surgeons (RCS) rats was evaluated. MFG-E8 and CD11b were used to confirm the microglial cells. In the retina of RCS rats, the mRNA expression of seven genes (MFG-E8 and its integrins αυ and ß5, CD11b and the cytokines TNF-α, IL-1ß, and MCP-1) formed almost similar bimodal peak distributions, which were centred at P7 and P45 to P60. In contrast, in rdy rats, which comprised the control group, a unimodal peak distribution centred at P14 was observed. The gene expression accompanied the activation and migration of microglial cells from the inner to the outer layer of the retina during the process of degeneration. Principal component analysis and discriminant function analysis revealed that the expression of these seven genes, especially TNF-α and CD11b, positively correlated with retinal degeneration and microglial activity during retinal degeneration in RCS rats, but not in the control rats. Furthermore, linear regression analysis demonstrated a significant correlation between the expression of these genes and the activation of microglial cells in the dystrophic retina. Our findings suggest that the suppression of microglial cells and the blockade of their cytotoxic effects may constitute a novel therapeutic strategy for treating photoreceptor death in various retinal disorders.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. The changes of potassium currents in RCS rat Müller cell during retinal degeneration.

    PubMed

    Zhao, TongTao; Li, YaoChen; Weng, ChuanHuang; Yin, ZhengQin

    2012-01-03

    Müller cells are the principal glial cells expressing membrane-bound potassium channel and predominantly mediating the homeostatic regulation of extracellular K+ produced by neuronal activity in retina. It's well known that Müller cells can be activated in many pathological conditions, but little is known about the change of potassium currents of Müller cells during the progression of retinitis pigmentosa. Herein, the Royal College of Surgeons rats (RCS rat) were employed to investigate some phenotypic and functional changes of Müller cells during retinal degeneration such as the expression of Kir4.1, membrane properties and K+ channel currents by using immunohistochemistry, RT-PCR, western blot and whole-cell patch clamping respectively. Compared with Müller cells in control retina, increased glutamine synthetase (GS) mRNA levels were seen at P30 and P60, and then decreased gradually in RCS rat retina. Morphologically, Müller cells showed significant hypertrophy and proliferation after p60. The increased expression of intermediate filament, glial fibrillary acidic protein (GFAP) and vimentin began at P30 and reached a peak at p60. Kir4.1 channels presented a peak expression at P30. Concomitantly, K(+) currents of Müller cells increased at P30 and decreased at P90 significantly. We concluded that retinal Müller cells of RCS rats underwent an activation initiated by the onset of retinal degeneration before p60 and then an obvious reactive gliosis, which led the basic membrane properties to suffer marked changes, and caused the Kir4.1 channels of Müller cells to occur a clear functional shift, even lose their normal electrophysiological properties. This process aggravates the impairment caused by the initial photoreceptor degeneration.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  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. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Mass spectrometric analyses of phospholipids in the S334ter-3 rat model of retinal degeneration

    PubMed Central

    Chen, Caroline Y.; Lam, Byron L.; Bhattacharya, Sanjoy K.

    2014-01-01

    Purpose The purpose of this study was to profile the endogenous phospholipid species in the retinal tissue of the S334ter-3 rat model of retinal degeneration. Retinal tissue was collected from S334ter-3 rats at postnatal day (P) 20, P30, and P60, while control retinal samples were collected from Sprague-Dawley (SD) rats at the same time points for comparison. Methods Lipids were extracted using the Bligh and Dyer method, and resuspended in an acetonitrile/isopropanol (1:1) solution. For lipid analyses, a positive ion–mode precursor ion scan (PIS) was used for phosphatidylcholine (PC; product m/z of 184), a negative ion–mode neutral loss scan (NLS) was used for phosphatidylserine (PS; product m/z of 87.1), and a negative ion–mode PIS was used for phosphatidylinositol (PI; product m/z of 241) and phosphatidylethanolamine (PE; product m/z of 196); the analyses were carried out using a TSQ Quantum Access Max mass spectrometer. The samples were directly infused with a Triversa Nanomate using 1.6 kV and 0.4 psi of pressure for the positive ion mode, and 1.3 kV and 0.6 psi of pressure for the negative ion mode, and scanned for 2 min between 200 m/z and 1000 m/z. Ratiometric quantification was performed using quantitative standards for each lipid class. Results The comparative profiles of PC, PE, PS, and PI between S334ter-3 and control rats showed that there were several lipid species common to both groups, as well as several that were unique to the S334ter-3 group and vice versa. Conclusions It was found that the proportions of PC and PS were higher in the control retina compared to S334ter-3, and that the proportions of PE and PI were higher in the S334ter-3 retina compared to control. PMID:25489232

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Alterations of sodium and potassium channels of RGCs in RCS rat with the development of retinal degeneration.

    PubMed

    Chen, Zhongshan; Song, Yanping; Yao, Junping; Weng, Chuanhuang; Yin, Zheng Qin

    2013-11-01

    All know that retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive dysfunction of photoreceptors and associated with progressive cells loss; nevertheless, little is known about how rods and cones loss affects the surviving inner retinal neurons and networks. Retinal ganglion cells (RGCs) process and convey visual information from retina to visual centers in the brain. The healthy various ion channels determine the normal reception and projection of visual signals from RGCs. Previous work on the Royal College of Surgeons (RCS) rat, as a kind of classical RP animal model, indicated that, at late stages of retinal degeneration in RCS rat, RGCs were also morphologically and functionally affected. Here, retrograde labeling for RGCs with Fluorogold was performed to investigate the distribution, density, and morphological changes of RGCs during retinal degeneration. Then, patch clamp recording, western blot, and immunofluorescence staining were performed to study the channels of sodium and potassium properties of RGCs, so as to explore the molecular and proteinic basis for understanding the alterations of RGCs membrane properties and firing functions. We found that the resting membrane potential, input resistance, and capacitance of RGCs changed significantly at the late stage of retinal degeneration. Action potential could not be evoked in a part of RGCs. Inward sodium current and outward potassium current recording showed that sodium current was impaired severely but only slightly in potassium current. Expressions of sodium channel protein were impaired dramatically at the late stage of retinal degeneration. The results suggested that the density of RGCs decreased, process ramification impaired, and sodium ion channel proteins destructed, which led to the impairment of electrophysiological functions of RGCs and eventually resulted in the loss of visual function.

  4. 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.

  5. 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.

  6. 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

  7. 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

  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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

    PubMed

    Mizukoshi, Sayuri; Nakazawa, Mitsuru; Sato, Kota; Ozaki, Taku; Metoki, Tomomi; Ishiguro, Sei-ichi

    2010-09-01

    The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  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. 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. 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.

  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. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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 ...

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  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. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Effects of Trigonella foenum-graecum (L.) on retinal oxidative stress, and proinflammatory and angiogenic molecular biomarkers in streptozotocin-induced diabetic rats.

    PubMed

    Gupta, Suresh Kumar; Kumar, Binit; Nag, Tapas Chandra; Srinivasan, B P; Srivastava, Sushma; Gaur, Shrikant; Saxena, Rohit

    2014-03-01

    The aim of the present study was to investigate the protective effects of Trigonella foenum-graecum Linn. (fenugreek) in Streptozotocin-induced diabetic rat retina. Fenugreek (100 and 200 mg/kg body weights) treatment was carried out for 24 weeks and evaluated for inflammatory [tumor necrosis factor (TNF)-α and interleukin (IL)-1β] and angiogenic [vascular endothelial growth factor (VEGF) and protein kinase C (PKC)-β] molecular biomarkers. Retinal oxidative stress was evaluated by estimating antioxidant (Glutathione, Superoxide dismutase, and Catalase) parameters. Fluorescein angiography was performed to detect retinal vascular leakage. Electron microscopy was performed to determine basement membrane thickness. In the present study, significant rises in the expressions of retinal inflammatory (TNF-α and IL-1β) and angiogenic (VEGF and PKC-β) molecular biomarkers were observed in diabetic retinae compared with normal retinae. However, fenugreek-treated retinae showed marked inhibition in the expression of inflammatory and angiogenic molecular biomarkers. Moreover, results from the present study showed positive modulatory effects of fenugreek on retinal oxidative stress. Fluorescein angiograms and fundus photographs obtained from diabetic retinae showed retinal vascular leakage. On the other hand, fenugreek-treated retinae did not show vascular leakage. Further, thickened BM was recorded in diabetic retina compared with normal retinae. However, fenugreek-treated retinae showed relatively lesser thickening of capillary BM. In conclusion, it may be postulated that fenugreek has great potential in preventing diabetes-induced retinal degeneration in humans after regular consumption in the specified dosage.

  3. ‘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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  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. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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)

  4. 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 ...

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. Retinal Detachment

    MedlinePlus

    ... men more than women and whites more than African Americans. A retinal detachment is also more likely to occur in people who Are extremely nearsighted Have had a retinal detachment in the other eye Have a family history of retinal detachment Have had cataract surgery Have ...

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  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. Learning about Retinitis Pigmentosa

    MedlinePlus

    ... genetic terms used on this page Learning About Retinitis Pigmentosa What is retinitis pigmentosa? What are the symptoms ... Pigmentosa Additional Resources for Retinitis Pigmentosa What is retinitis pigmentosa? Retinitis pigmentosa (RP) is the name given to ...

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

    PubMed Central

    Thomas, T P; Porcellati, F; Kato, K; Stevens, M J; Sherman, W R; Greene, D A

    1994-01-01

    Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells. Images PMID:8201009

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  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 retinitis pigmentosa. These devices provide partial sight restoration and patients use this improved vision in their everyday lives. Improved mobility and object detection are some of the more notable findings from the clinical trials. However, significant vision restoration will require both better technology and improved understanding of the interaction between electrical stimulation and the retina. This paper reviews the recent clinical trials, highlights technology breakthroughs that will contribute to next generation of retinal prostheses. PMID:24710817

  12. 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.

  13. 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

  14. Retinal Detachment

    MedlinePlus

    ... immediately. Treatment How is retinal detachment treated? Small holes and tears are treated with laser surgery or ... laser surgery tiny burns are made around the hole to “weld” the retina back into place. Cryopexy ...

  15. Retinal Disorders

    MedlinePlus

    ... be serious enough to cause blindness. Examples are Macular degeneration - a disease that destroys your sharp, central vision Diabetic eye disease Retinal detachment - a medical emergency, when the retina is ... children. Macular pucker - scar tissue on the macula Macular hole - ...

  16. A morphometric study of the endocytosis of wheat germ agglutinin-horseradish peroxidase conjugates by retinal ganglion cells in the rat.

    PubMed

    Trojanowski, J Q; Gonatas, N K

    1983-08-08

    In order to elucidate the sequence for the intraneuronal translocation of ligands after internalization in vivo, the adsorptive endocytosis of horseradish peroxidase (HRP) conjugates of the lectin wheat germ agglutinin (WGHRP) by retinal ganglion cells of the rat was studied by ultrastructural morphometry after intravitreal injections of this probe. Retinas were harvested at post-injection survival times of 15 min to 7 days and processed for the electron microscopic visualization of WGHRP in subcellular organelles. The labeled organelles included vesicles, tubules, lysosomes and the cisterns and coated as well as uncoated vesicles of GERL (Golgi Apparatus-Endoplasmic-Reticulum-Lysosomes). For quantitation, labeled organelles were classed as vesicles, lysosomes and GERL. From 15 min to 3 h the number of labeled GERL and vesicles progressively increased to a maximum at 3 h and then declined to zero by 7 days. In contrast, the number of labeled lysosomes continued to increase beyond 3 h to reach a maximum at 24 h before declining to near zero by 7 days. These results are consistent with the hypothesis that the adsorptive endocytosis of WGHRP entails the passage of the ligand through GERL prior to being deposited in lysosomes. They do not exclude the possibility that other endocytic pathways for WGHRP and possible WGHRP-membrane complexes may exist in retinal ganglion cells including a plasma membrane to lysosome route.

  17. 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.

  18. Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

    PubMed

    Ozaki, Taku; Nakazawa, Mitsuru; Yamashita, Tetsuro; Sorimachi, Hiroyuki; Hata, Shoji; Tomita, Hiroshi; Isago, Hitomi; Baba, Ayaka; Ishiguro, Sei-Ichi

    2012-11-01

    Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.

  19. Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells.

    PubMed

    Tomi, Masatoshi; Tajima, Ayumi; Tachikawa, Masanori; Hosoya, Ken-ichi

    2008-10-01

    The purpose of this study was to identify the uptake mechanism of gamma-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na(+)-, Cl(-)-, and concentration-dependent [3H]GABA uptake with a Km of 1.5 mM. Taurine, beta-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [3H]GABA. The uptake of [3H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na(+)-, Cl(-)-, and concentration-dependent with a Km of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and beta-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 microM. [3H]Taurine also exhibited competitive inhibition with a Ki of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.

  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. 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

  2. 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.

  3. 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

  4. 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.

  5. Phloroglucinol protects retinal pigment epithelium and photoreceptor against all-trans-retinal-induced toxicity and inhibits A2E formation.

    PubMed

    Cia, David; Cubizolle, Aurélie; Crauste, Céline; Jacquemot, Nathalie; Guillou, Laurent; Vigor, Claire; Angebault, Claire; Hamel, Christian P; Vercauteren, Joseph; Brabet, Philippe

    2016-09-01

    Among retinal macular diseases, the juvenile recessive Stargardt disease and the age-related degenerative disease arise from carbonyl and oxidative stresses (COS). Both stresses originate from an accumulation of all-trans-retinal (atRAL) and are involved in bisretinoid formation by condensation of atRAL with phosphatidylethanolamine (carbonyl stress) in the photoreceptor and its transformation into lipofuscin bisretinoids (oxidative stress) in the retinal pigment epithelium (RPE). As atRAL and bisretinoid accumulation contribute to RPE and photoreceptor cell death, our goal is to select powerful chemical inhibitors of COS. Here, we describe that phloroglucinol, a natural phenolic compound having anti-COS properties, protects both rat RPE and mouse photoreceptor primary cultures from atRAL-induced cell death and reduces hydrogen peroxide (H2 O2 )-induced damage in RPE in a dose-dependent manner. Mechanistic analyses demonstrate that the protective effect encompasses decrease in atRAL-induced intracellular reactive oxygen species and free atRAL levels. Moreover, we show that phloroglucinol reacts with atRAL to form a chromene adduct which prevents bisretinoid A2E synthesis in vitro. Taken together, these data show that the protective effect of phloroglucinol correlates with its ability to trap atRAL and to prevent its further transformation into deleterious bisretinoids. Phloroglucinol might be a good basis to develop efficient therapeutic derivatives in the treatment of retinal macular diseases.

  6. Retine revisited.

    PubMed

    Douglas, D E

    2002-10-01

    Retine, so named by Albert Szent-Györgyi, an inhibitor of the growth of transplanted malignant tumours in animals, is present in all mammalian tissues and in urine. Its inhibitory activity was extensively investigated by Szent-Györgyi, but its exact chemical identity was not determined. Details of the reported physical and chemical properties of retine and its ubiquitous occurrence identify it as being identical to a complex mixture of lipid 2,4-diketones of similar ubiquitous occurrence. This lipid mixture has been extensively studied, and individual members have been synthesized.

  7. 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.

  8. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies.

  9. Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus

    PubMed Central

    Rotem, Assaf; Moses, Elisha; Neef, Andreas

    2015-01-01

    Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD) curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields. PMID:26186201

  10. Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture

    PubMed Central

    Malik, Deepika; Tarek, Mohamed; Caceres del Carpio, Javier; Ramirez, Claudio; Boyer, David; Kenney, M Cristina; Kuppermann, Baruch D

    2014-01-01

    Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Methods Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Results Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses. Conclusions At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses. PMID:24836865

  11. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  12. The mechanism of zinc uptake by cultured rat liver cells.

    PubMed Central

    Taylor, J A; Simons, T J

    1994-01-01

    1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898

  13. Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

    PubMed Central

    Yin, Zhaoobao; Aschner, Judy L.; Santos, Ana Paula dos; Aschner, Michael

    2008-01-01

    Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson’s disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague–Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethylrhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (ΔΨm). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 hrs) also significantly (p<0.01) dissipates the ΔΨm in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways. PMID:18313649

  14. Cobalt-Induced Ototoxicity in Rat Postnatal Cochlear Organotypic Cultures.

    PubMed

    Li, Peng; Ding, Dalian; Salvi, Richard; Roth, Jerome A

    2015-10-01

    Cobalt (Co) is a required divalent metal used in the production of metal alloys, batteries, and pigments and is a component of vitamin B12. Excessive uptake of Co is neurotoxic causing temporary or permanent hearing loss; however, its ototoxic effects on the sensory hair cells, neurons, and support cells in the cochlea are poorly understood. Accordingly, we treated postnatal day 3 rat cochlear organotypic cultures with various doses and durations of CoCl2 and quantified the damage to the hair cells, peripheral auditory nerve fibers, and spiral ganglion neurons (SGN). Five-day treatment with 250 μM CoCl2 caused extensive damage to hair cells and neurons which increased with dose and treatment duration. CoCl2 caused greater damage to outer hair cells than inner hair cells; damage was greatest in the base of the cochlea and decreased towards the base. CoCl2 increased expression of superoxide radical in hair cells and SGNs and SGN loss was characterized by nuclear condensation and fragmentation, morphological features of apoptosis. CoCl2 treatment increased the expression of caspase-3 indicative of caspase-mediated programmed cell death. These results identify hair cells and spiral ganglion neurons as the main targets of Co ototoxicity in vitro and implicate the superoxide radical as a trigger of caspase-mediated ototoxicity.

  15. Age-related changes in susceptibility of rat brain slice cultures including hippocampus to encephalomyocarditis virus

    PubMed Central

    Su, Weiping; Ueno-Yamanouchi, Aito; Uetsuka, Koji; Nakayama, Hiroyuki; Doi, Kunio

    1999-01-01

    Replication of the D variant of encephalomyocarditis virus (EMC-D) and its cytopathic effects were studied in the brain slice cultures including hippocampus (hippocampal slice) obtained from postnatal 1-, 4-, 7-, 14-, 28-and 56-day-old Fischer 344 rats. At 0, 12, 24, 36 and 48 h after infection, virus titres of the slices and culture media were assayed. Viral replication was observed in cultures from 1-to 28-day-old rats, and the highest titre was recorded in the slice and culture medium from the youngest rat. The peak of virus titre decreased with age and no distinct viral replication was observed in the cultures from 56-day-old rats. Light microscopy revealed that degenerative and necrotic changes appeared in the infected hippocampal slices from 1- to 28-day-old rats, and the changes became less prominent with age. In situ hybridization and indirect immunofluorescence staining showed that positive signals of viral RNA and antigen were prominent in younger rats and decreased with age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC-D is less related to the maturation of the immune system but possibly to that of the neurone. PMID:10632784

  16. Evaluation of Retinal Nerve Fiber Layer Thickness and Axonal Transport 1 and 2 Weeks After 8 Hours of Acute Intraocular Pressure Elevation in Rats

    PubMed Central

    Abbott, Carla J.; Choe, Tiffany E.; Lusardi, Theresa A.; Burgoyne, Claude F.; Wang, Lin; Fortune, Brad

    2014-01-01

    Purpose. To compare in vivo retinal nerve fiber layer thickness (RNFLT) and axonal transport at 1 and 2 weeks after an 8-hour acute IOP elevation in rats. Methods. Forty-seven adult male Brown Norway rats were used. Procedures were performed under anesthesia. The IOP was manometrically elevated to 50 mm Hg or held at 15 mm Hg (sham) for 8 hours unilaterally. The RNFLT was measured by spectral-domain optical coherence tomography. Anterograde and retrograde axonal transport was assessed from confocal scanning laser ophthalmoscopy imaging 24 hours after bilateral injections of 2 μL 1% cholera toxin B-subunit conjugated to AlexaFluor 488 into the vitreous or superior colliculi, respectively. Retinal ganglion cell (RGC) and microglial densities were determined using antibodies against Brn3a and Iba-1. Results. The RNFLT in experimental eyes increased from baseline by 11% at 1 day (P < 0.001), peaked at 19% at 1 week (P < 0.0001), remained 11% thicker at 2 weeks (P < 0.001), recovered at 3 weeks (P > 0.05), and showed no sign of thinning at 6 weeks (P > 0.05). There was no disruption of anterograde transport at 1 week (superior colliculi fluorescence intensity, 75.3 ± 7.9 arbitrary units [AU] for the experimental eyes and 77.1 ± 6.7 AU for the control eyes) (P = 0.438) or 2 weeks (P = 0.188). There was no obstruction of retrograde transport at 1 week (RCG density, 1651 ± 153 per mm2 for the experimental eyes and 1615 ± 135 per mm2 for the control eyes) (P = 0.63) or 2 weeks (P = 0.25). There was no loss of Brn3a-positive RGC density at 6 weeks (P = 0.74) and no increase in microglial density (P = 0.92). Conclusions. Acute IOP elevation to 50 mm Hg for 8 hours does not cause a persisting axonal transport deficit at 1 or 2 weeks or a detectable RNFLT or RGC loss by 6 weeks but does lead to transient RNFL thickening that resolves by 3 weeks. PMID:24398096

  17. Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells

    PubMed Central

    Peng, Rong-mei; Jin, Ying; Sun, Yu-zhao; Sun, Yi-qian; Zhang, Pei

    2017-01-01

    Background Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. Purpose To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. Methods MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT–PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. Results Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. Conclusions BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS. PMID:28210098

  18. [Aging and retinal vascular diseases].

    PubMed

    Takagi, Hitoshi

    2007-03-01

    Ocular vascular diseases such as diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration, whose population increases along with aging, have become leading causes of severe visual disturbance. Macular edema and serous retinal detachment are associated with abnormal vascular leakage and tractional retinal detachment, and neovascular glaucoma is caused by retinal neovascularization. Such ocular vascular diseases are caused by vascular cell aging and vascular damage associated with lifestyle-related diseases including diabetes mellitus, hypertension, hyperlipidemia, and obesity. In the present study, we investigated molecular mechanisms in such vascular deficiencies using vascular cell biology methodology, and we propose novel strategies for the treatment of such vascular diseases. Along with aging, oxidative stress and physical stress, such as mechanical stretch, continuously and directly insult vascular cells. Such stress induces apoptosis by intracellular signaling through stress kinases in cultured retinal vascular cells. Inhibition of such stress kinases could be an effective treatment to protect the vascular cells against age-related damage. In a retinal vascular developmental model, pericyte loss causes pathology mimicking macular edema and proliferative diabetic retinopathy. Angiopoietin 1 (Ang 1) secreted by pericytes suppresses oxidative stress-induced intracellular signaling through stress kinases linked to cell apoptosis and normalizes such retinal pathology. This suggests that the paracrine action of Ang 1 in the pericytes is necessary to sustain normal retinal vasculature, and that Ang 1-triggered intracellular signaling is useful for the treatment of vascular cell pathology associated with pericyte loss. In diabetic retinopathy and retinal vein occlusion, retinal vessels regress along with retinal vascular cell apoptosis, and the retina becomes ischemic followed by pathological retinal neovascularization. VEGF has been

  19. Retinitis pigmentosa

    PubMed Central

    Hamel, Christian

    2006-01-01

    Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by the loss of photoreceptors and characterized by retinal pigment deposits visible on fundus examination. Prevalence of non syndromic RP is approximately 1/4,000. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. Some extreme cases may have a rapid evolution over two decades or a slow progression that never leads to blindness. In some cases, the clinical presentation is a cone-rod dystrophy, in which the decrease in visual acuity predominates over the visual field loss. RP is usually non syndromic but there are also many syndromic forms, the most frequent being Usher syndrome. To date, 45 causative genes/loci have been identified in non syndromic RP (for the autosomal dominant, autosomal recessive, X-linked, and digenic forms). Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs. Molecular diagnosis can be made for some genes, but is not usually performed due to the tremendous genetic heterogeneity of the disease. Genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, so the visual prognosis is poor. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitaminotherapy, treating the complications (cataract and macular edema), and helping patients to cope with the social and psychological impact of blindness. However, new therapeutic strategies are emerging from intensive research (gene therapy, neuroprotection, retinal prosthesis). PMID:17032466

  20. A novel method for toxicology: in vitro culture system of a rat preantral follicle.

    PubMed

    Wan Xuying; Zhu Jiangbo; Zhu Yuping; Xili, Ma; Liu Zhen; Wang Fei; Xu Guifeng; Zhang Tianbao

    2011-08-01

    Preantral follicle in vitro culture systems have been successfully or nearly successfully established for sheep, pig and mouse, and applied on follicle development and regulation research on reproductive biology and physiology. However, there have been few studies concerning rat preantral follicle in vitro development. The objective is to establish an in vitro culture system for rat preantral follicles which can be used for reproductive biology and toxicology research. Rat preantral follicles are mechanically separated, cultured in vitro in single follicle mode for continuous 12 days using 96-well plates, and then administrated ovulation induction. The observation on follicle development, hormone level, and ovum formation are recorded and assessed. Taking in vivo growth and in vitro maturation of oocytes group as control group, in vitro growth and maturation of oocytes group is assessed to see whether this in vitro culture method is successful. The conditions for rat follicle culture are determined based on the mouse pre-antral follicle culture. The in vitro culture system for rat preantral follicles established in this study is feasible and successful, and can serve as model for reproductive biology and toxicology research.

  1. Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells.

    PubMed

    Nakanishi-Ueda, T; Majima, H J; Watanabe, K; Ueda, T; Indo, H P; Suenaga, S; Hisamitsu, T; Ozawa, T; Yasuhara, H; Koide, R

    2013-10-01

    The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm(2). The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm(2) exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm(2), respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm(2)).

  2. Enhancement of proliferation in a rat hepatocyte co-culture model after mitogenic stimulation.

    EPA Science Inventory

    Primary mouse and rat hepatocyte cultures have long been the gold standard for assessment of cellular changes following chemical exposure. While helpful for assessing proliferative and responses in vitro, these cultures are limited to 1 or 2 days of incubation. Our motivation was...

  3. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    SciTech Connect

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  4. Ototoxicity of paclitaxel in rat cochlear organotypic cultures

    SciTech Connect

    Dong, Yang; Ding, Dalian; Jiang, Haiyan; Shi, Jian-rong; Salvi, Richard; Roth, Jerome A.

    2014-11-01

    Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24 h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea. - Highlights: • Paclitaxel was toxic to cochlear hair cells and spiral ganglion neurons. • Paclitaxel-induced spiral ganglion degeneration was apoptotic. • Paclitaxel activated caspase-6, -8 and -8 in spiral ganglion neurons.

  5. Synthesis and migration of /sup 3/H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

    SciTech Connect

    Haddad, A.; Bennett, G.

    1987-03-01

    /sup 3/H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated /sup 3/H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after /sup 3/H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.

  6. Dose-Dependent Protective Effect of Lithium Chloride on Retinal Ganglion Cells Is Interrelated with an Upregulated Intraretinal BDNF after Optic Nerve Transection in Adult Rats

    PubMed Central

    Wu, Ming-Mei; Zhu, Ting-Ting; Wang, Peng; Kuang, Fang; Hao, Ding-Jun; You, Si-Wei; Li, Yao-Yu

    2014-01-01

    Neuroprotection of lithium for axotomized retinal ganglion cells (RGCs) is attributed to upregulated intraretinal Bcl-2. As lithium also upregulates brain-derived neurotrophic factor (BDNF) which can rescue axotomized RGCs, it is hypothesized that lithium could protect RGCs through BDNF. This study investigated this hypothesis and a possible relationship between the dose and protection of lithium. All adult experimental rats received daily intraperitoneal injections of lithium chloride (LiCl) at 30, 60 or 85 mg/kg·bw until they were euthanized 2, 7 or 14 days after left intraorbital optic nerve (ON) transection. Our results revealed that RGC densities promoted and declined with increased dose of LiCl and the highest RGC densities were always in the 60 mg/kg·bw LiCl group at both 7 and 14 day points. Similar promotion and decline in the mRNA and protein levels of intraretinal BDNF were also found at the 14 day point, while such BDNF levels increased in the 30 mg/kg·bw LiCl group but peaked in the 60 and 85 mg/kg·bw LiCl groups at the 7 day point. These findings suggested that lithium can delay the death of axotomized RGCs in a dose-dependent manner within a certain period after ON injury and such beneficial effect is interrelated with an upregulated level of intraretinal BDNF. PMID:25100168

  7. The Apical Localization of Na(+), K(+)-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

    PubMed

    Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na(+), K(+)-ATPase, or the Na(+) pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na(+), K(+)-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na(+) pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na(+), K(+)-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na(+) pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na(+), K(+)-ATPase in RPE cells depends on the expression of the β2 subunit.

  8. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

    PubMed Central

    Lobato-Álvarez, Jorge A.; Roldán, María L.; López-Murillo, Teresa del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit. PMID:27774068

  9. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    SciTech Connect

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah; Cho, Jin Won; Lee, Joon H.

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  10. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  11. Some Rat Sensory Neurons in Culture Express Characteristics of Differentiated Pain Sensory Cells

    NASA Astrophysics Data System (ADS)

    Baccaglini, Paola I.; Hogan, Patrick G.

    1983-01-01

    Sensory neurons were dissociated from trigeminal ganglia or from dorsal root ganglia of rats, grown in culture, and examined for expression of properties of pain sensory cells. Many sensory neurons in culture are excited by low concentrations of capsaicin, reportedly a selective stimulus for pain sensory neurons. Many are excited by bradykinin, sensitized by prostaglandin E2, or specifically stained by an antiserum against substance P. These experiments provide a basis for the study of pain mechanisms in cell culture.

  12. Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

    PubMed Central

    Meucci, Olimpia

    2011-01-01

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1. At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do

  13. Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode.

    PubMed

    Rikihisa, Y; Lin, Y C; Fukaya, T

    1985-06-01

    In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.

  14. Directional Summation in Non-direction Selective Retinal Ganglion Cells

    PubMed Central

    Abbas, Syed Y.; Hamade, Khaldoun C.; Yang, Ellen J.; Nawy, Scott; Smith, Robert G.; Pettit, Diana L.

    2013-01-01

    Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network. PMID:23516351

  15. Retinal pigment epithelium transplantation: concepts, challenges, and future prospects

    PubMed Central

    Alexander, P; Thomson, H A J; Luff, A J; Lotery, A J

    2015-01-01

    The retinal pigment epithelium (RPE) is a single layer of cells that supports the light-sensitive photoreceptor cells that are essential for retinal function. Age-related macular degeneration (AMD) is a leading cause of visual impairment, and the primary pathogenic mechanism is thought to arise in the RPE layer. RPE cell structure and function are well understood, the cells are readily sustainable in laboratory culture and, unlike other cell types within the retina, RPE cells do not require synaptic connections to perform their role. These factors, together with the relative ease of outer retinal imaging, make RPE cells an attractive target for cell transplantation compared with other cell types in the retina or central nervous system. Seminal experiments in rats with an inherited RPE dystrophy have demonstrated that RPE transplantation can prevent photoreceptor loss and maintain visual function. This review provides an update on the progress made so far on RPE transplantation in human eyes, outlines potential sources of donor cells, and describes the technical and surgical challenges faced by the transplanting surgeon. Recent advances in the understanding of pluripotent stem cells, combined with novel surgical instrumentation, hold considerable promise, and support the concept of RPE transplantation as a regenerative strategy in AMD. PMID:26043704

  16. Transient release kinetics of rod bipolar cells revealed by capacitance measurement of exocytosis from axon terminals in rat retinal slices

    PubMed Central

    Oltedal, Leif; Hartveit, Espen

    2010-01-01

    Presynaptic transmitter release has mostly been studied through measurements of postsynaptic responses, but a few synapses offer direct access to the presynaptic terminal, thereby allowing capacitance measurements of exocytosis. For mammalian rod bipolar cells, synaptic transmission has been investigated in great detail by recording postsynaptic currents in AII amacrine cells. Presynaptic measurements of the dynamics of vesicular cycling have so far been limited to isolated rod bipolar cells in dissociated preparations. Here, we first used computer simulations of compartmental models of morphologically reconstructed rod bipolar cells to adapt the ‘Sine + DC’ technique for capacitance measurements of exocytosis at axon terminals of intact rod bipolar cells in retinal slices. In subsequent physiological recordings, voltage pulses that triggered presynaptic Ca2+ influx evoked capacitance increases that were proportional to the pulse duration. With pulse durations ≤100 ms, the increase saturated at ∼10 fF, corresponding to the size of a readily releasable pool of vesicles. Pulse durations ≥400 ms evoked additional capacitance increases, probably reflecting recruitment from additional pools of vesicles. By using Ca2+ tail current stimuli, we separated Ca2+ influx from Ca2+ channel activation kinetics, allowing us to estimate the intrinsic release kinetics of the readily releasable pool, yielding a time constant of ∼1.1 ms and a maximum release rate of 2–3 vesicles (release site)−1 ms−1. Following exocytosis, we observed endocytosis with time constants ranging from 0.7 to 17 s. Under physiological conditions, it is likely that release will be transient, with the kinetics limited by the activation kinetics of the voltage-gated Ca2+ channels. PMID:20211976

  17. Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

    PubMed

    Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

  18. Metabolic Syndrome Triggered by High-Fructose Diet Favors Choroidal Neovascularization and Impairs Retinal Light Sensitivity in the Rat

    PubMed Central

    Thierry, Magalie; Pasquis, Bruno; Acar, Niyazi; Grégoire, Stéphane; Febvret, Valérie; Buteau, Bénédicte; Gambert-Nicot, Ségolène; Bron, Alain M.; Creuzot-Garcher, Catherine P.; Bretillon, Lionel

    2014-01-01

    Diabetic retinopathy and age-related macular degeneration are the leading causes of blindness in Western populations. Although it is a matter of controversy, large-scale population-based studies have reported increased prevalence of age-related macular degeneration in patients with diabetes or diabetic retinopathy. We hypothesized that metabolic syndrome, one of the major risk factors for type 2 diabetes, would represent a favorable environment for the development of choroidal neovascularization, the main complication of age-related macular degeneration. The fructose-fed rat was used as a model for metabolic syndrome in which choroidal neovascularization was induced by laser photocoagulation. Male Brown Norway rats were fed for 1, 3, and 6 months with a standard equilibrated chow diet or a 60%-rich fructose diet (n = 24 per time point). The animals expectedly developed significant body adiposity (+17%), liver steatosis at 3 and 6 months, hyperleptinemia at 1 and 3 months (two-fold increase) and hyperinsulinemia at 3 and 6 months (up to two-fold increase), but remained normoglycemic and normolipemic. The fructose-fed animals exhibited partial loss of rod sensitivity to light stimulus and reduced amplitude of oscillatory potentials at 6 months. Fructose-fed rats developed significantly more choroidal neovascularization at 14 and 21 days post-laser photocoagulation after 1 and 3 months of diet compared to animals fed the control diet. These results were consistent with infiltration/activation of phagocytic cells and up-regulation of pro-angiogenic gene expression such as Vegf and Leptin in the retina. Our data therefore suggested that metabolic syndrome would exacerbate the development of choroidal neovascularization in our experimental model. PMID:25380250

  19. Missed retinal breaks in rhegmatogenous retinal detachment

    PubMed Central

    Takkar, Brijesh; Azad, Shorya; Shashni, Adarsh; Pujari, Amar; Bhatia, Indrish; Azad, Rajvardhan

    2016-01-01

    AIM To evaluate the causes and associations of missed retinal breaks (MRBs) and posterior vitreous detachment (PVD) in patients with rhegmatogenous retinal detachment (RRD). METHODS Case sheets of patients undergoing vitreo retinal surgery for RRD at a tertiary eye care centre were evaluated retrospectively. Out of the 378 records screened, 253 were included for analysis of MRBs and 191 patients were included for analysis of PVD, depending on the inclusion criteria. Features of RRD and retinal breaks noted on examination were compared to the status of MRBs and PVD detected during surgery for possible associations. RESULTS Overall, 27% patients had MRBs. Retinal holes were commonly missed in patients with lattice degeneration while missed retinal tears were associated with presence of complete PVD. Patients operated for cataract surgery were significantly associated with MRBs (P=0.033) with the odds of missing a retinal break being 1.91 as compared to patients with natural lens. Advanced proliferative vitreo retinopathy (PVR) and retinal bullae were the most common reasons for missing a retinal break during examination. PVD was present in 52% of the cases and was wrongly assessed in 16%. Retinal bullae, pseudophakia/aphakia, myopia, and horse shoe retinal tears were strongly associated with presence of PVD. Traumatic RRDs were rarely associated with PVD. CONCLUSION Pseudophakic patients, and patients with retinal bullae or advanced PVR should be carefully screened for MRBs. Though Weiss ring is a good indicator of PVD, it may still be over diagnosed in some cases. PVD is associated with retinal bullae and pseudophakia, and inversely with traumatic RRD. PMID:27990367

  20. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    PubMed

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity.

  1. Human serum teratogenicity studies using in vitro cultures of rat embryos

    SciTech Connect

    Klein, N.W.; Chatot, C.L.; Plenefisch, J.D.; Carey, S.W.

    1982-01-01

    Those conditions that constitute reproductive risks to man are being analyzed. Particular concern is with those conditions that cannot be or have not been identified by present methodologies. These conditions constitute the majority of factors causing fetal wastages and birth defects. The test system uses intact rat embryos that are cultured in vitro for 2 days. Findings to date suggest that this system may have a number of distinct advantages: (1) whole-embryo culture provides the test with the entire repertoire of processes involved in embryonic development; (2) whole-rat embryos can be cultured on high levels of blood serum; and (3) they can be cultured on serum from human subjects, which provides a direct and unique evaluation of the principal organism of concern. In regard to this last point, it is important to recognize that there is a large range of teratogenic responses and sensitivities to teratogens dependent upon both individual and species differences. (ERB)

  2. Dissociation of Retinal Ganglion Cells Without Enzymes

    PubMed Central

    Hayashida, Yuki; Partida, Gloria J.; Ishida, Andrew T.

    2011-01-01

    We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease. PMID:15196824

  3. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  4. Retinal functional development is sensitive to environmental enrichment: a role for BDNF.

    PubMed

    Landi, S; Sale, A; Berardi, N; Viegi, A; Maffei, L; Cenni, M C

    2007-01-01

    Retina has long been considered less plastic than cortex or hippocampus, the very sites of experience-dependent plasticity. Now, we show that retinal development is responsive to the experience provided by an enriched environment (EE): the maturation of retinal acuity, which is a sensitive index of retinal circuitry development, is strongly accelerated in EE rats. This effect is present also in rats exposed to EE up to P10, that is before eye opening, suggesting that factors sufficient to trigger retinal acuity development are affected by EE during the first days of life. Brain derived neurotrophic factor (BDNF) is precociously expressed in the ganglion cell layer of EE with respect to non-EE rats and reduction of BDNF expression in EE animals counteracts EE effects on retinal acuity. Thus, EE controls the development of retinal circuitry, and this action depends on retinal BDNF expression.

  5. Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides.

    PubMed

    Környei, J L; Vértes, Z; Kovács, K A; Göcze, P M; Vértes, M

    2003-06-01

    Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.

  6. Retinal synaptic regeneration via microfluidic guiding channels

    PubMed Central

    Su, Ping-Jung; Liu, Zongbin; Zhang, Kai; Han, Xin; Saito, Yuki; Xia, Xiaojun; Yokoi, Kenji; Shen, Haifa; Qin, Lidong

    2015-01-01

    In vitro culture of dissociated retinal neurons is an important model for investigating retinal synaptic regeneration (RSR) and exploring potentials in artificial retina. Here, retinal precursor cells were cultured in a microfluidic chip with multiple arrays of microchannels in order to reconstruct the retinal neuronal synapse. The cultured retinal cells were physically connected through microchannels. Activation of electric signal transduction by the cells through the microchannels was demonstrated by administration of glycinergic factors. In addition, an image-based analytical method was used to quantify the synaptic connections and to assess the kinetics of synaptic regeneration. The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations. Furthermore, RSR was enhanced by chemical stimulation with potassium chloride. Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation. PMID:26314276

  7. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    SciTech Connect

    Marion, Tracy L.; Perry, Cassandra H.; St Claire, Robert L.; Brouwer, Kim L.R.

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  8. Evidence for Cholinergic Synapses Between Dissociated Rat Sympathetic Neurons in Cell Culture

    PubMed Central

    O'Lague, P. H.; Obata, K.; Claude, P.; Furshpan, E. J.; Potter, D. D.

    1974-01-01

    Sympathetic principal neurons were dissociated from superior cervical ganglia of new-born rats, and grown in cell culture. In electrophysiological experiments two types of excitatory synapses were found. One, which was relatively rare, was shown to operate by electrical transmission. The other, the predominant type, had several characteristics of chemical transmission, and pharmacological evidence indicated it was cholinergic. Images PMID:4372629

  9. The rat whole embryo culture assay using the Dysmorphology Score system.

    PubMed

    Zhang, Cindy; Panzica-Kelly, Julie; Augustine-Rauch, Karen

    2013-01-01

    The rat whole embryo culture (WEC) system has been used extensively for characterizing teratogenic properties of test chemicals. In this chapter, we describe the methodology for culturing rat embryos as well as a new morphological score system, the Dysmorphology Score (DMS) system for assessing morphology of mid gestation (gestational day 11) rat embryos. In contrast to the developmental stage focused scoring associated with the Brown and Fabro score system, this new score system assesses the respective degree of severity of dysmorphology, which delineates normal from abnormal morphology of specific embryonic structures and organ systems. This score system generates an approach that allows rapid identification and quantification of adverse developmental findings, making it conducive for characterization of compounds for teratogenic properties and screening activities.

  10. Antidiabetic effect of taurine in cultured rat skeletal l6 myotubes.

    PubMed

    Cheong, Sun Hee; Chang, Kyung Ja

    2013-01-01

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, is found in all animal cells at millimolar concentrations and has been reported to show various health promoting activities including antidiabetic properties. The beneficial effects of taurine in diabetes mellitus have been known. However, the exact mechanism of hypoglycemic action of taurine is not properly defined. In this study, we investigated antidiabetic effect of taurine in the cell culture system using rat skeletal muscle cells. In cultured rat skeletal L6 myotubes, we studied the effect of taurine (0-100 μM) on glucose uptake to plasma membrane from the aspects of AMP-activated protein kinase (AMPK) signaling. Taurine stimulated glucose uptake in a dose-dependent manner by activating AMPK signaling. From these results, it may suggest that taurine show antidiabetic effect by stimulating insulin-independent glucose uptake in rat skeletal muscle.

  11. Iron in hereditary retinal degeneration: PIXE microanalysis. Preliminary results

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Gouget, B.; Llabador, Y.; Simonoff, M.; Yefimova, M.; Courtois, Y.; Jeanny, J. C.

    1999-10-01

    Several types of hereditary retinal degeneration with progressive alteration of photoreceptors exist in men and animals. Recent immunohistochemical results have shown strong degradation of transferrin, the protein responsible for iron transport, in retinas of rats with hereditary retinal degeneration. Freeze-dried thin sections of rat retinas from different stages of the disease, and respective coeval control sections, have been analyzed using nuclear microprobe. In this first part of the study, the rat retinas at post-natal stages of 35 and 45 days have been analyzed. The sample preparation and the post-irradiation staining to determine precisely the retinal layers involved are described. Preliminary results of element distributions (K, Ca, Fe) in the rat retina layers are discussed. A very high content of calcium in the choriocapillaris of dystrophic rat retinas was observed. Preliminary results on iron distribution in the rat retina layers are presented.

  12. Functional alterations in immature cultured rat hippocampal neurons after sustained exposure to static magnetic fields.

    PubMed

    Hirai, Takao; Yoneda, Yukio

    2004-01-15

    In cultured rat hippocampal neurons, gradual increases were seen in the expression of microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN) and growth-associated protein-43 (GAP-43), in proportion to increased duration, up to 9 days in vitro (DIV). Sustained exposure to static magnetic fields at 100 mT for up to 9 DIV significantly decreased expression of MAP-2 and NeuN in cultured rat hippocampal neurons without markedly affecting GAP-43 expression. Although a significant increase was seen in the expression of glial fibrillary acidic protein (GFAP) in hippocampal neuronal preparations cultured for 6-9 DIV under sustained magnetism, GFAP and proliferating cell nuclear antigen expression were not affected markedly in cultured astrocytes prepared from rat hippocampus and neocortex, irrespective of cellular maturity. No significant alteration was seen in cell survivability of hippocampal neurons or astrocytes cultured under sustained magnetism. In hippocampal neurons cultured for 3 DIV under sustained magnetism, marked mRNA expression was seen for N-methyl-D-aspartate (NMDA) receptor subunits, NR1, NR2A-2C, NR2D, and NR3A. In addition, significant potentiation of the ability of NMDA to increase intracellular free Ca(2+) ions was observed. Differential display analysis revealed a significant decrease in mRNA expression for the transcription factor ALF1 in response to sustained magnetism for 3 DIV. These results suggest that sustained exposure to static magnetic fields may affect cellular functionality and maturity in immature cultured rat hippocampal neurons through modulation of expression of particular NMDA receptor subunits.

  13. Neonatal hypothyroidism affects the adenine nucleotides metabolism in astrocyte cultures from rat brain.

    PubMed

    Braganhol, Elizandra; Bruno, Alessandra Nejar; Bavaresco, Luci; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas; Battastini, Ana Maria Oliveira

    2006-04-01

    Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.

  14. Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

    PubMed Central

    Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

    1985-01-01

    Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

  15. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  16. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    PubMed

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  17. Berberine inhibits aldose reductase and oxidative stress in rat mesangial cells cultured under high glucose.

    PubMed

    Liu, Weihua; Liu, Peiqinq; Tao, Sha; Deng, Yanhui; Li, Xuejuan; Lan, Tian; Zhang, Xiaoyan; Guo, Fenfen; Huang, Wenge; Chen, Fengying; Huang, Heqing; Zhou, Shu-Feng

    2008-07-15

    Diabetic nephropathy (DN), one of the most serious microvascular complications of diabetes mellitus, is a major cause of end-stage renal disease. Berberine is one of the main constituents of Coptidis rhizoma and Cortex phellodendri. In the present study, we examined effects of berberine (BBR) on renal injury in streptozotocin-induced diabetic rats, and on the changes of aldose reductase (AR) and oxidative stress in cultured rat mesangial cells exposed to high glucose. Fasting blood glucose, blood urea nitrogen, creatinine, and urine protein over 24 h were detected by using the commercially available kits. Cell proliferation, collagen synthesis, aldose reductase (AR), superoxide anion, superoxide dismutase (SOD), and malondialdehyde (MDA) were detected, respectively, by different methods. In streptozotocin-induced diabetic rats, fasting blood glucose, blood urea nitrogen, creatinine, and urine protein over 24 h were significantly decreased in rats treated with 200 mg/kg berberine for 12 weeks compared with diabetic control rats (P < 0.05). This was accompanied by a reduced AR activity and gene expression at both mRNA and protein levels. In cultured rat mesangial cells exposed to high glucose, incubation of BBR significantly decreased cell proliferation, collagen synthesis and AR activity as well as its mRNA and protein levels compared with control cells (P < 0.05). In vitro, BBR also significantly increased SOD activity and decreased superoxide anion and MDA compared with control cells (P < 0.05). These results suggested that BBR could ameliorate renal dysfunction in DN rats, which may be ascribed to inhibition of AR in mesangium, reduction of oxidative stress, and amelioration of extracellular matrix synthesis and cell proliferation. Further studies are warranted to explore the role of AR in DN and the therapeutic implications by AR inhibitors such as BBR.

  18. Notch Signaling Functions in Retinal Pericyte Survival

    PubMed Central

    Arboleda-Velasquez, Joseph F.; Primo, Vincent; Graham, Mark; James, Alexandra; Manent, Jan; D'Amore, Patricia A.

    2014-01-01

    Purpose. Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a master regulator of cell fate decisions, in retinal pericyte survival. Methods. A coculture system of ligand-dependent Notch signaling was developed using primary cultured retinal pericytes and a mesenchymal cell line derived from an inducible mouse model expressing the Delta-like 1 Notch ligand. This model was used to examine the effect of Notch activity on pericyte survival using quantitative PCR (qPCR) and a light-induced cell death assay. The effect of Notch gain- and loss-of-function was analyzed in monocultures of retinal pericytes using antibody arrays to interrogate the expression of apoptosis-related proteins. Results. Primary cultured retinal pericytes differentially expressed key molecules of the Notch pathway and displayed strong expression of canonical Notch/RBPJK (recombination signal-binding protein 1 for J-kappa) downstream targets. A gene expression screen using gain- and loss-of-function approaches identified genes relevant to cell survival as downstream targets of Notch activity in retinal pericytes. Ligand-mediated Notch activity protected retinal pericytes from light-induced cell death. Conclusions. Our results have identified signature genes downstream of Notch activity in retinal pericytes and suggest that tight regulation of Notch signaling is crucial for pericyte survival. PMID:25015359

  19. Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

    PubMed

    Julien, Sylvie; Peters, Tobias; Ziemssen, Focke; Arango-Gonzalez, Blanca; Beck, Susanne; Thielecke, Hagen; Büth, Heiko; Van Vlierberghe, Sandra; Sirova, Milada; Rossmann, Pavel; Rihova, Blanka; Schacht, Etienne; Dubruel, Peter; Zrenner, Eberhart; Schraermeyer, Ulrich

    2011-06-01

    Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

  20. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  1. Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Takemoto, N; Sanae, F; Koshiura, R

    1985-05-01

    The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

  2. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    PubMed

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  3. Estradiol synthesis and release in cultured female rat bone marrow stem cells.

    PubMed

    Zhang, Dalei; Yang, Bei; Zou, Weiying; Lu, Xiaying; Xiong, Mingdi; Wu, Lei; Wang, Jinglei; Gao, Junhong; Xu, Sifan; Zou, Ting

    2013-01-01

    Bone marrow stem cells (BMSCs) have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17 β (E2) under high glucose culture conditions with or without retinoic acid (RA). The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency.

  4. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    PubMed Central

    Lepsch, Lucilia B.; Planeta, Cleopatra S.; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine. PMID:26295051

  5. Effect of alginate culture and mechanical stimulation on cartilaginous matrix synthesis of rat dedifferentiated chondrocytes.

    PubMed

    Wang, Yun; de Isla, Natalia; Huselstein, Céline; Wang, Binghua; Netter, Patrick; Stoltz, Jean-François; Muller, Sylvaine

    2008-01-01

    To investigate whether the application of alginate culture and mechanical stimulation will improve the synthesis of cartilaginous matrix in dedifferentiated chondrocytes, rat chondrocytes underwent dedifferentiation upon serial monolayer culture up to passage 6, and then were encapsulated in 2% alginate gel and subject to static culture. After 28 days culture in static, the beads were exposed to 48 h of mechanical stimulation with continuous agitation. The sGAG content in alginate bead was measured by alcian blue staining. The expression of collagen protein was detected using immunofluorescence. After 28 days culture in alginate bead, the dedifferentiated chondrocytes remained round in shape and re-synthesized the chondrocyte-specific matrix. Compared with static culture, mechanical stimulation induced statistically increases in the production of glycosaminoglycan (p< or =0.01), as well as in the synthesis of collagen type II protein (p< or =0.05). On the contrary, no positive expression of collagen type I protein was observed at the end of culture. Our results demonstrated that both of alginate culture and mechanical stimulation help to restore chondrocyte phenotype and promotes the synthesis of cartilaginous matrix.

  6. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  7. Increased oxidative stress in the placenta tissue and cell culture of tumour-bearing pregnant rats.

    PubMed

    Toledo, M T; Ventrucci, G; Gomes-Marcondes, M C C

    2011-11-01

    Placental dysfunction leads to foetal damage, which jeopardises the exchange between the maternal and foetal systems. We evaluated the effects of tumour growth on the activity of antioxidant enzymes and oxidative stress in placental tissue and cell culture from tumour-bearing pregnant rats compared to non-tumour-bearing pregnant rats that were ascitic fluid injected. Ascitic fluid is obtained from Walker tumour-bearing rats and contains a cytokine called Walker factor (WF), which is a molecule similar to proteolysis-inducing factor (PIF), and induces changes in protein metabolism and oxidative stress. Pregnant Wistar rats were distributed into control (C), tumour-bearing (W) and ascitic fluid injected (A) groups and were sacrificed on days 16, 19 and 21 of pregnancy to analyse the profile of enzyme activities (glutathione-S-transferase (GST), catalase (CAT), alkaline phosphatase (AP)) and malondialdehyde (MDA) content in placental tissue. Meanwhile, placenta samples from all groups were obtained on day 21, placed in primary culture and treated with WF for 72 h. The presence of tumour or ascitic fluid reduced the protein content of the placental tissue. On day 16 there was a significant reduction in AP activity in W rats, and on day 19, CAT activity and MDA content significantly increased. These results indicate that the presence of cancer decreased antioxidant enzyme capacity in the placenta, increasing the amount of oxidation in these cells, which may contribute to irreversible placental damage and compromisefoetal development. WF treatment induces similar changes in placental cells in primary culture, resulting in less cell viability and increased oxidative stress. These results indicate that WF, provided by the tumour or inoculation of ascitic fluid, has negative effects on placental homeostasis, which impairs foetal health.

  8. Toxic effect of the glycoalkaloids solanine and tomatine on cultured neonatal rat heart cells.

    PubMed

    Bergers, W W; Alink, G M

    1980-06-01

    The toxic effects of the glycoalkaloids, alpha-solanine and tomatine, were studied in beating heart cell cultures from 1--2-day-old rats. After addition of alpha-solanine (80 microgram/ml) and tomatine (40 microgram/ml) to the culture medium, the cells ceased beating within a few minutes. At a concentration of 40 microgram/ml alpha-solanine and 20 microgram/ml tomatine, both compounds caused a pronounced increase of the contraction frequency, lasting for at least 2h. K-strophantin, a reference heart glycoside, caused arrhythmic beating at 20 microgram/ml and complete cessation of contractions at 160 microgram/ml.

  9. Trophic effects of nootropic peptide preparations cerebrolysin and semax on cultured rat pheochromocytoma.

    PubMed

    Safarova, E R; Shram, S I; Grivennikov, I A; Myasoedov, N F

    2002-04-01

    Trophic characteristics of neuroprotectors cerebrolysin and semax were evaluated by their capacity to induce differentiation and improve survival of cultured rat pheochromocytoma (PC12) cells. Morphological signs of cell differentiation (enlargement and formation of processes) were seen 24 h after addition of cerebrolysin into culture medium. Cerebrolysin improved survival of PC12 cells in serum-free medium. In a concentration of 100 microg/ml cerebrolysin decreased the content of apoptotic cells from 32% (control) to 10%. Semax produced no trophic effect on PC12 cells. hence, the neuroprotective effect of cerebrolysin in vivo probably results from trophic activity, while the protective effects of semax are mediated by other mechanisms.

  10. Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

    PubMed Central

    Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

    2009-01-01

    Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

  11. Regulation of period 1 expression in cultured rat pineal

    NASA Technical Reports Server (NTRS)

    Fukuhara, Chiaki; Dirden, James C.; Tosini, Gianluca

    2002-01-01

    The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

  12. Changes in expression of a functional G sub i protein in cultured rat heart cells

    SciTech Connect

    Allen, I.S.; Gaa, S.T.; Rogers, T.B. )

    1988-07-01

    The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G{sub i}) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for G{sub i} in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated {sup 32}P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G{sub i} is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G{sub i} is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G{sub i} in heart.

  13. Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts.

    PubMed

    Moriguchi, N; Hinoi, E; Tsuchihashi, Y; Fujimori, S; Iemata, M; Takarada, T; Yoneda, Y

    2006-09-01

    Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

  14. Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside.

    PubMed

    Yim, S H; Farrer, R G; Hammer, J A; Yavin, E; Quarles, R H

    1994-06-15

    Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12-14 days in vitro. Addition of 50 micrograms/ml exogenous Neu-NAc alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [14C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [14C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [14C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but

  15. Effects of pure 2,4-dichlorophenoxyacetic acid on cultured rat embryos.

    PubMed

    Sameshima, Kiyoko; Kobae, Hidehiko; Fofana, Djibril; Yoshidome, Kouichi; Nishi, Junichiro; Miyata, Koichiro

    2004-06-01

    2,4-dichlorophenoxyacetic acid (2,4-D), a plant growth regulator, has been used worldwide as a herbicide. Previously we evaluated the prenatal developmental effects of 2,4-D by feeding it to pregnant rats and found that it is maternally toxic and embryolethal, and it induces urogenital malformations in rat fetuses. In the study presented here, we investigated the effects of pure 2,4-D on rat embryos in whole embryo culture. Rat embryos on day 9.5 of gestation were cultured for 48 h at several concentration levels with pure 2,4-D (50-500 microg/mL). 2,4-D caused a concentration-related increase in the incidence of each malformation. Significant decreases in the number of somites were observed at a concentration of 100 microg/mL or more. At the concentration of 100 microg/mL, there was normal yolk sac circulation. This result suggests that 2,4-D has a detrimental effect on somite development and directly damages developing embryos.

  16. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

    PubMed

    Ji, LiLi; Liu, TianYu; Wang, ZhengTao

    2010-04-01

    Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

  17. Identification and pharmacological characterization of the histamine H3 receptor in cultured rat astrocytes.

    PubMed

    Mele, Tina; Jurič, Damijana Mojca

    2013-10-24

    Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex, cerebellum, hippocampus and striatum. Double-antigen immunofluorescence staining and [(3)H]N-α-methylhistamine ([(3)H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes. Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations. [(3)H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes (KD=4.55±0.46nM; Bmax=5.63±0.21fmol/mg protein) and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor (range of pKi values 7.50-8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors. The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

  18. Identification and pharmacological characterization of the histamine H3 receptor in cultured rat astrocytes.

    PubMed

    Mele, Tina; Jurič, Damijana Mojca

    2013-11-15

    Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex,cerebellum, hippocampus and striatum.Double-antigen immunofluorescence staining and[3H]N-α-methylhistamine([3H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes.Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations.[3H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes(KD¼4.5570.46 nM; Bmax¼5.6370.21 fmol/mg protein)and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor(range of pKi values 7.50–8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors.The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

  19. The effect of supplementation of amino acids and taurine to modified KSOM culture medium on rat embryo development.

    PubMed

    Nakamura, Kazuomi; Morimoto, Kayoko; Shima, Kaoru; Yoshimura, Yuki; Kazuki, Yasuhiro; Suzuki, Osamu; Matsuda, Junichiro; Ohbayashi, Tetsuya

    2016-11-01

    The rat is widely used as a laboratory animal for research. In particular, genetically engineered rats are essential for production of animal models of several diseases. Although embryo manipulation techniques are needed to produce them, such technology for rat preimplantation embryos is not as advanced as it is for mouse embryos. One reason is that in vitro culture systems for preimplantation embryos are limited in rats. Therefore, we intended to develop a new culture system for rat preimplantation embryos focusing on supplementation of amino acids as nutrition to the culture media. First, we found that taurine, glycine, glutamate, and alanine were abundant in the oviductal fluid of Wistar rats. The profile of taurine and these three amino acids was unchanged during the estrous cycle and from Days 0 to 3 of pregnancy (Day 0; vaginal plug was confirmed). Second, we assessed the effect of phosphate and phenol red on the development of rat zygotes and confirmed that they caused two-cell block. Third, we examined the effect of changing the medium on zygote development because addition of amino acids into culture medium causes ammonium accumulation, which is detrimental to embryo development. Blastocyst formation was suppressed in cultures with no medium change (P = 0.004; decreased to approximately one-fourth of that with medium change). Fourth, we examined the effect of supplementation of these three amino acids and taurine to modified potassium simplex optimized medium (KSOM). The zygote development rates were increased by the three amino acids and taurine in a concentration-dependent manner at 48, 72, and 96 hours (P = 0.001, 0.005, and 0.009, respectively) in culture. Finally, we confirmed that blastocysts cultured in modified KSOM had the capacity to develop to full term after implantation. These results showed that not only the supply of nutrients but also removal of wastes and toxicants is important for culture of rat preimplantation embryos.

  20. Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

    PubMed

    Ito, D; Tamate, H; Nagayama, M; Uchida, T; Kudoh, S N; Gohara, K

    2010-11-24

    To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

  1. Effects of Cultured Adrenal Chromaffin Cell Implants on Hindlimb Reflexes of the 6-OHDA Lesioned Rat

    PubMed Central

    Pulford, Bruce E.; Mihajlov, Andrea R.; Nornes, Howard O.; Whalen, L. Ray

    1994-01-01

    The effects of implantation of cultured adrenal medullary cells on the recovery of neurotransmitter specific reflex activity were studied in the rat spinal cord using electrophysiological testing methods. Cell suspensions of cultured neonatal adrenal medullary chromaffin (AM) cells (which produce catecholamines), or Schwann (Sc) cells (controls) were implanted into the lumbar region of the spinal cord 2 weeks after catecholamine (CA) denervation by intracisternal injection of 6-hydroxydopamine (6-OHDA). All cells were taken from 7 day neonates and cultured for 10 days in the presence of nerve growth factor (NGF). Three months after implantation, the extent of implant-associated recovery of reflex activity was determined by measuring electromyogram (EMG) activity and force associated with the long latency component of the hindlimb withdrawal reflex (which is CA modulated). After the electrophysiological testing, rats were anesthetized, and the spinal cords were rapidly removed and frozen. Spinal cords were sectioned longitudinally, and implanted cells were visualized using glyoxylic acid techniques. Labelled sections were examined to determine cell survival. Results indicate that 1) chromaffin cells survive for 3 months in the segments of the cord into which they have been implanted and 2) rats implanted with AM cells have significantly more forceful withdrawal reflexes than those that received Sc cells or received no implant after lesioning. PMID:7703294

  2. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

    Kumar, S.; McGinnis, J.F.; de Vellis, J.

    1980-03-25

    The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

  3. Retinal image quality in the rodent eye.

    PubMed

    Artal, P; Herreros de Tejada, P; Muñoz Tedó, C; Green, D G

    1998-01-01

    Many rodents do not see well. For a target to be resolved by a rat or a mouse, it must subtend a visual angle of a degree or more. It is commonly assumed that this poor spatial resolving capacity is due to neural rather than optical limitations, but the quality of the retinal image has not been well characterized in these animals. We have modified a double-pass apparatus, initially designed for the human eye, so it could be used with rodents to measure the modulation transfer function (MTF) of the eye's optics. That is, the double-pass retinal image of a monochromatic (lambda = 632.8 nm) point source was digitized with a CCD camera. From these double-pass measurements, the single-pass MTF was computed under a variety of conditions of focus and with different pupil sizes. Even with the eye in best focus, the image quality in both rats and mice is exceedingly poor. With a 1-mm pupil, for example, the MTF in the rat had an upper limit of about 2.5 cycles/deg, rather than the 28 cycles/deg one would obtain if the eye were a diffraction-limited system. These images are about 10 times worse than the comparable retinal images in the human eye. Using our measurements of the optics and the published behavioral and electrophysiological contrast sensitivity functions (CSFs) of rats, we have calculated the CSF that the rat would have if it had perfect rather than poor optics. We find, interestingly, that diffraction-limited optics would produce only slight improvement overall. That is, in spite of retinal images which are of very low quality, the upper limit of visual resolution in rodents is neurally determined. Rats and mice seem to have eyes in which the optics and retina/brain are well matched.

  4. Retinal remodeling in human retinitis pigmentosa.

    PubMed

    Jones, B W; Pfeiffer, R L; Ferrell, W D; Watt, C B; Marmor, M; Marc, R E

    2016-09-01

    Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.

  5. Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

    PubMed

    Viollon-Abadie, C; Bigot-Lasserre, D; Nicod, L; Carmichael, N; Richert, L

    2000-12-01

    Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T(4)-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), beta-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T(4)-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T(4)-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T(4)-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T(4)-UGT activities.

  6. Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: a study of retinal regeneration in a novel animal model.

    PubMed

    Miyake, Ayumi; Araki, Masasuke

    2014-07-01

    Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three-dimensional (3-D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin-positive cells nor 3-D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ.

  7. Genetics Home Reference: retinitis pigmentosa

    MedlinePlus

    ... Me Understand Genetics Home Health Conditions retinitis pigmentosa retinitis pigmentosa Enable Javascript to view the expand/collapse boxes. Download PDF Open All Close All Description Retinitis pigmentosa is a group of related eye disorders that ...

  8. Antibiotic Supplements Affect Electrophysiological Properties and Excitability of Rat Hippocampal Pyramidal Neurons in Primary Culture

    PubMed Central

    Bahrami, Farideh; Janahmadi, Mahyar

    2013-01-01

    Introduction: Antibiotic supplements are regularly used in neuronal culture media to control contamination; however, they can interfere with the neuronal excitability and affect electrophysiological properties. Therefore, in this study, the effect of penicillin/streptomycin supplements on the spontaneous electrophysiological activity of hippocampal pyramidal neurons was examined. Methods: Electrophysiological whole-cell patch-clamp recordings from rat hippocampal pyramidal cells in primary culture were performed to investigate the effects of antibiotic supplements on the intrinsic excitability of cultured cells. Results: The present findings indicated that presence of antibiotic supplements (penicillin/streptomycin) in the culture medium altered the intrinsic electrical activity of hippocampal pyramidal neurons in primary culture. These alterations included: 1) depolarized resting membrane potential; 2) a significant enhancement in the after-hyperpolarization amplitude; 3) a significant increase in the area under the action potential and in the decay and rise time of the action potential; 4) a significant broadening of action potential and 5) a significant reduction in the firing frequency. Conclusion: These findings suggest that addition of antibiotic supplements to culture media influences the neuronal excitability and alters the electrophysiological properties of cultured neurons, possibly through changing the ionic conductance underlying neuronal excitability. PMID:23567852

  9. In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure

    PubMed Central

    1995-01-01

    Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from

  10. Expression of precerebellins in cultured rat calvaria osteoblast-like cells.

    PubMed

    Rucinski, Marcin; Zok, Agnieszka; Guidolin, Diego; De Caro, Raffaele; Malendowicz, Ludwik K

    2008-10-01

    Cerebellin (CER), originally isolated from rat cerebellum, is a hexadecapeptide derived from the larger precursor called precerebellin 1 (Cbln1). At present 4 propeptides designated as Cbln1, Cbln2, Cbln3 and Cbln4 are recognized. They belong to precerebellin subfamily of the C1q family proteins. Precerebellins act as transneuronal regulators of synapse development and synaptic plasticity in various brain regions. Initially CER was thought to be a cerebellum specific peptide, however subsequent studies revealed its presence in other brain regions as well as in extraneuronal tissues. We investigated whether precerebellins are expressed and involved in regulation of cultured rat calvarial osteoblast-like (ROB) cells. Classic RT-PCR revealed the presence of Cbln1 and Cbln3 mRNA in fragments of rat calvaria, in freshly isolated ROB cells and in ROB cells cultured for 7, 14 and 21 days. Cbln2 and Cbln4 mRNA, on the other hand, could not be demonstrated in ROB cells but was found to be present in the brain. In freshly isolated ROB cells expression of Cbln1 gene was very low and gradually increased in relation to the duration of culture. Expression of Cbln3, on the other hand, was very low in fragments of rat calvaria, and increased notably after digestion with collagenase-I. The highest expression of this precerebellin was observed at day 14 of culture while at days 7 and 21 levels of expressions were notably lower. Neither Cbln2 nor Cbln4 was found to be expressed in the ROB cells. Neither CER nor des-Ser1-CER (10(-10)-10(-6)M) affect osteocalcin production and proliferation rate of studied cells. The above findings suggest that CER, which theoretically would be derived from Cbln1, modulate neither differentiated (osteocalcin secretion) nor basic (proliferation) functions of cultured rat osteoblast-like cells. The obtained data raise an intriguing hypothesis that precerebellins may be involved in regulating of spatial organization of osteoblastic niches in the bone.

  11. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

    PubMed Central

    Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

    2016-01-01

    STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no

  12. Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells.

    PubMed

    Seki, Masaaki; Tanaka, Takayuki; Nawa, Hiroyuki; Usui, Tomoaki; Fukuchi, Takeo; Ikeda, Kazuhito; Abe, Haruki; Takei, Nobuyuki

    2004-09-01

    Although neurotrophins have been assessed as candidate therapeutic agents for neural complications of diabetes, their involvement in diabetic retinopathy has not been fully characterized. We found that the protein and mRNA levels of brain-derived neurotrophic factor (BDNF) in streptozotocin-induced diabetic rat retinas were reduced to 49% (P < 0.005) and 74% (P < 0.05), respectively, of those of normal control animals. In addition, dopaminergic amacrine cells appeared to be degenerating in the diabetic rat retinas, as revealed by tyrosine hydroxylase (TH) immunoreactivity. Overall TH protein levels in the retina were decreased to one-half that of controls (P < 0.01), reflecting reductions in the density of dopaminergic amacrine cells and the intensity of TH immunoreactivity within them. To confirm the neuropathological implications of BDNF reduction, we administered BDNF protein into the vitreous cavities of diabetic rats. Intraocular administration of BDNF rescued dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of TH expression, demonstrating its therapeutic potential. These findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of BDNF and can be ameliorated by an exogenous supply of this neurotrophin.

  13. Retinal detachment repair

    MedlinePlus

    Scleral buckling; Vitrectomy; Pneumatic retinopexy; Laser retinopexy; Rhegmatogenous retinal detachment repair ... it meets the hole in the retina. Scleral buckling can be done using numbing medicine while you ...

  14. Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

    PubMed

    Nims, R W; Lubet, R A; Fox, S D; Jones, C R; Thomas, P E; Reddy, A B; Kocarek, T A

    1998-03-27

    In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

  15. Leptin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland.

    PubMed

    Peliciari-Garcia, Rodrigo Antonio; Andrade-Silva, Jéssica; Cipolla-Neto, José; Carvalho, Carla Roberta de Oliveira

    2013-01-01

    Pineal melatonin synthesis can be modulated by many peptides, including insulin. Because melatonin appears to alter leptin synthesis, in this work we aimed to investigate whether leptin would have a role on norepinephrine- (NE-)mediated melatonin synthesis in cultured rat pineal glands. According to our data, cultured rat pineal glands express leptin receptor isoform b (Ob-Rb). Pineal expression of Ob-Rb mRNA was also observed in vivo. Administration of leptin (1 nM) associated with NE ( 1 µM) reduced melatonin content as well as arylalkylamine-N-acetyl transferase (AANAT) activity and expression in cultured pineal glands. Leptin treatment per se induced the expression of STAT3 in cultured pineal glands, but STAT3 does not participate in the leptin modulation of NE-mediated pineal melatonin synthesis. In addition, the expression of inducible cAMP early repressor (ICER) was further induced by leptin challenge when associated with NE. In conclusion, leptin inhibition of pineal melatonin synthesis appears to be mediated by a reduction in AANAT activity and expression as well as by increased expression of Icer mRNA. Peptidergic signaling within the pineal gland appears to be one of the most important signals which modulates melatonin synthesis; leptin, as a member of this system, is not an exception.

  16. Induction of autophagy in rat hippocampus and cultured neurons by iron.

    PubMed

    He, Y; Hua, Y; Song, S; Liu, W; Keep, R F; Xi, G

    2008-01-01

    Autophagy occurs in the brain after intracerebral hemorrhage (ICH). Iron is an important factor causing neuronal death and brain atrophy after ICH. In this study, we examined whether iron can induce autophagy in the hippocampus and in cultured neurons. For in vivo studies, rats received an infusion of either saline or ferrous iron into the right hippocampus and were killed 1, 3, or 7 days later for Western blot analysis of microtubule-associated protein light chain-3 (LC3). For in vitro studies, primary cultured cortex neurons from rat embryos were exposed to ferrous iron. Cells were used for Western blot analysis of LC3 and monodansylcadaverine (MDC) staining 24h later. Intrahippocampal injection of ferrous iron resulted in an increased conversion of LC3-I to LC3-II. Exposure of primary cultured neurons to ferrous iron also induced an enhanced conversion of LC3-I to LC3-II. MDC labeling showed an accumulation of MDC in cultured neurons exposed to ferrous iron. These results indicate that autophagy is induced by iron in neurons and that iron-induced autophagy may contribute to brain injury after ICH.

  17. Protective Effect of Edaravone against Carbon Monoxide Induced Apoptosis in Rat Primary Cultured Astrocytes

    PubMed Central

    Xu, Xiaodan; Zhang, Hong; Wang, Ke; Tu, Tao

    2017-01-01

    Objective. To observe the protective effect of edaravone (Eda) on astrocytes after prolonged exposure to carbon monoxide (CO) and further to investigate the potential mechanisms of Eda against CO-induced apoptosis. Methods. The rat primary cultured astrocytes were cultured in vitro and exposed to 1% CO for 24 h after being cultured with different concentrations of Eda. MTT assay was used to detect the cytotoxicity of CO. Flow cytometry was used to detect the apoptosis rate, membrane potential of mitochondria, and ROS level. The mRNA and protein expressions of Bcl-2, Bax, and caspase-3 were assessed by real-time PCR and Western blotting analysis, respectively. Results. Eda can significantly suppress cytotoxicity of CO, and it can significantly increase membrane potential of mitochondria and Bcl-2 expressions and significantly suppress the apoptosis rate, ROS level, Bax, and caspase-3 expressions. Conclusion. Eda protects against CO-induced apoptosis in rat primary cultured astrocytes through decreasing ROS production and subsequently inhibiting mitochondrial apoptosis pathway. PMID:28261501

  18. Basic Fibroblast Growth Factor Contributes to a Shift in the Angioregulatory Activity of Retinal Glial (Müller) Cells

    PubMed Central

    Yafai, Yousef; Iandiev, Ianors; Lange, Johannes; Yang, Xiu Mei; Wiedemann, Peter; Bringmann, Andreas; Eichler, Wolfram

    2013-01-01

    Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK−1/−2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under

  19. Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

    PubMed

    Simão, Ana Maria S; Beloti, Márcio M; Cezarino, Rodrigo M; Rosa, Adalberto Luiz; Pizauro, João M; Ciancaglini, Pietro

    2007-04-01

    Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

  20. A simple method for isolating and culturing the rat brain microvascular endothelial cells.

    PubMed

    Liu, Yang; Xue, Qiang; Tang, Qing; Hou, Min; Qi, Hongyi; Chen, Gang; Chen, Weihai; Zhang, Jifen; Chen, Yi; Xu, Xiaoyu

    2013-11-01

    Brain microvascular endothelial cells (BMECs), a main component of the blood-brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (>99%) and yield (about 2.2×10(7) per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood-brain barrier and neurovascular units, etc.

  1. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    SciTech Connect

    Nilsson, Mats F; Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma; Cebers, Gvido; Hellmold, Heike; Gustafson, Anne-Lee; Webster, William S

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  2. Optimization of neuronal cultures from rat superior cervical ganglia for dual patch recording

    PubMed Central

    Amendola, Julien; Boumedine, Norah; Sangiardi, Marion; El Far, Oussama

    2015-01-01

    Superior cervical ganglion neurons (SCGN) are often used to investigate neurotransmitter release mechanisms. In this study, we optimized the dissociation and culture conditions of rat SCGN cultures for dual patch clamp recordings. Two weeks in vitro are sufficient to achieve a significant CNTF-induced cholinergic switch and to develop mature and healthy neuronal profiles suited for detailed patch clamp analysis. One single pup provides sufficient material to prepare what was formerly obtained from 12 to 15 animals. The suitability of these cultures to study neurotransmitter release mechanisms was validated by presynaptically perturbing the interaction of the v-SNARE VAMP2 with the vesicular V-ATPase V0c subunit. PMID:26399440

  3. Detection of retinitis pigmentosa by differential interference contrast microscopy.

    PubMed

    Oh, Juyeong; Kim, Seok Hwan; Kim, Yu Jeong; Lee, Hyunho; Cho, Joon Hyong; Cho, Young Ho; Kim, Chul-Ki; Lee, Taik Jin; Lee, Seok; Park, Ki Ho; Yu, Hyeong Gon; Lee, Hyuk-Jae; Jun, Seong Chan; Kim, Jae Hun

    2014-01-01

    Differential interference contrast microscopy is designed to image unstained and transparent specimens by enhancing the contrast resulting from the Nomarski prism-effected optical path difference. Retinitis pigmentosa, one of the most common inherited retinal diseases, is characterized by progressive loss of photoreceptors. In this study, Differential interference contrast microscopy was evaluated as a new and simple application for observation of the retinal photoreceptor layer and retinitis pigmentosa diagnostics and monitoring. Retinal tissues of Royal College of Surgeons rats and retinal-degeneration mice, both well-established animal models for the disease, were prepared as flatmounts and histological sections representing different elapsed times since the occurrence of the disease. Under the microscopy, the retinal flatmounts showed that the mosaic pattern of the photoreceptor layer was irregular and partly collapsed at the early stage of retinitis pigmentosa, and, by the advanced stage, amorphous. The histological sections, similarly, showed thinning of the photoreceptor layer at the early stage and loss of the outer nuclear layer by the advanced stage. To count and compare the number of photoreceptors in the normal and early-retinitis pigmentosa-stage tissues, an automated cell-counting program designed with MATLAB, a numerical computing language, using a morphological reconstruction method, was applied to the differential interference contrast microscopic images. The number of cells significantly decreased, on average, from 282 to 143 cells for the Royal College of Surgeons rats and from 255 to 170 for the retinal-degeneration mouse. We successfully demonstrated the potential of the differential interference contrast microscopy technique's application to the diagnosis and monitoring of RP.

  4. Rat Hippocampal Neurons Express Genes for Both Rod Retinal and Olfactory Cyclic Nucleotide-Gated Channels: Novel Targets for cAMP/cGMP Function

    NASA Astrophysics Data System (ADS)

    Kingston, Paul A.; Zufall, Frank; Barnstable, Colin J.

    1996-09-01

    Cyclic nucleotide-gated (CNG) channels are Ca2+-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

  5. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

    PubMed

    Parkinson, Fiona E; Hacking, Cindy

    2005-07-05

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  6. Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures.

    PubMed

    Leboy, P S; Beresford, J N; Devlin, C; Owen, M E

    1991-03-01

    We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the

  7. Arsenite decreases CYP3A23 induction in cultured rat hepatocytes by transcriptional and translational mechanisms

    SciTech Connect

    Noreault, Trisha L.; Nichols, Ralph C.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Peter R.; Evans, Ronald M.; Sinclair, Jacqueline F. . E-mail: JSINC@dartmouth.edu

    2005-12-01

    Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 {mu}M arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.

  8. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  9. Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

    PubMed

    Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2007-10-01

    We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

  10. Protective effect of alpha-melanocyte-stimulating hormone (α-MSH) on the recovery of ischemia/reperfusion (I/R)-induced retinal damage in a rat model.

    PubMed

    Varga, Balazs; Gesztelyi, Rudolf; Bombicz, Mariann; Haines, David; Szabo, Adrienn Monika; Kemeny-Beke, Adam; Antal, Miklos; Vecsernyes, Miklos; Juhasz, Bela; Tosaki, Arpad

    2013-07-01

    The present study demonstrates capacity of α-MSH to augment recovery from ischemia/reperfusion (I/R)-induced retinal damage in vivo and correlation of its protective effects with expression of heme oxygenase-1 (HO-1). Used techniques include ocular ischemia and reperfusion, electroretinography, histology, electron microscopy, and molecular-biological techniques. The results demonstrate the α-MSH-mediated inhibition of I/R-induced functional deterioration of the retina. Outcomes suggest that the protective effects of α-MSH occur mainly through HO-1-dependent pathways but HO-1-independent mechanisms may also contribute to protection. The observation that post-ischemic treatment with α-MSH exhibits therapeutic efficacy in the same range as pre-ischemic treatment, is a novel result. This outcome suggests a highly conserved protective role for α-MSH as a major stress response mechanism--and offers the possibility for development of novel therapeutic strategies utilizing this hormone, in particular in treatment of conditions resulting from I/R injury, such as deterioration of retinal microcirculation. The merit of the study lies in the fact that I/R injury contribute significantly to the severity of retinopathies. However, currently there are no evidence-based treatments for retinal I/R injury available for clinical use. Our finding suggests that α-MSH may have a very wide range of uses in the prevention of I/R-mediated pathologies.

  11. Time-Dependent Nerve Growth Factor Signaling Changes in the Rat Retina During Optic Nerve Crush-Induced Degeneration of Retinal Ganglion Cells

    PubMed Central

    Mesentier-Louro, Louise A.; De Nicolò, Sara; Rosso, Pamela; De Vitis, Luigi A.; Castoldi, Valerio; Leocani, Letizia; Mendez-Otero, Rosalia; Santiago, Marcelo F.; Tirassa, Paola; Rama, Paolo; Lambiase, Alessandro

    2017-01-01

    Nerve growth factor (NGF) is suggested to be neuroprotective after nerve injury; however, retinal ganglion cells (RGC) degenerate following optic-nerve crush (ONC), even in the presence of increased levels of endogenous NGF. To further investigate this apparently paradoxical condition, a time-course study was performed to evaluate the effects of unilateral ONC on NGF expression and signaling in the adult retina. Visually evoked potential and immunofluorescence staining were used to assess axonal damage and RGC loss. The levels of NGF, proNGF, p75NTR, TrkA and GFAP and the activation of several intracellular pathways were analyzed at 1, 3, 7 and 14 days after crush (dac) by ELISA/Western Blot and PathScan intracellular signaling array. The progressive RGC loss and nerve impairment featured an early and sustained activation of apoptotic pathways; and GFAP and p75NTR enhancement. In contrast, ONC-induced reduction of TrkA, and increased proNGF were observed only at 7 and 14 dac. We propose that proNGF and p75NTR contribute to exacerbate retinal degeneration by further stimulating apoptosis during the second week after injury, and thus hamper the neuroprotective effect of the endogenous NGF. These findings might aid in identifying effective treatment windows for NGF-based strategies to counteract retinal and/or optic-nerve degeneration. PMID:28067793

  12. Time-Dependent Nerve Growth Factor Signaling Changes in the Rat Retina During Optic Nerve Crush-Induced Degeneration of Retinal Ganglion Cells.

    PubMed

    Mesentier-Louro, Louise A; De Nicolò, Sara; Rosso, Pamela; De Vitis, Luigi A; Castoldi, Valerio; Leocani, Letizia; Mendez-Otero, Rosalia; Santiago, Marcelo F; Tirassa, Paola; Rama, Paolo; Lambiase, Alessandro

    2017-01-05

    Nerve growth factor (NGF) is suggested to be neuroprotective after nerve injury; however, retinal ganglion cells (RGC) degenerate following optic-nerve crush (ONC), even in the presence of increased levels of endogenous NGF. To further investigate this apparently paradoxical condition, a time-course study was performed to evaluate the effects of unilateral ONC on NGF expression and signaling in the adult retina. Visually evoked potential and immunofluorescence staining were used to assess axonal damage and RGC loss. The levels of NGF, proNGF, p75(NTR), TrkA and GFAP and the activation of several intracellular pathways were analyzed at 1, 3, 7 and 14 days after crush (dac) by ELISA/Western Blot and PathScan intracellular signaling array. The progressive RGC loss and nerve impairment featured an early and sustained activation of apoptotic pathways; and GFAP and p75(NTR) enhancement. In contrast, ONC-induced reduction of TrkA, and increased proNGF were observed only at 7 and 14 dac. We propose that proNGF and p75(NTR) contribute to exacerbate retinal degeneration by further stimulating apoptosis during the second week after injury, and thus hamper the neuroprotective effect of the endogenous NGF. These findings might aid in identifying effective treatment windows for NGF-based strategies to counteract retinal and/or optic-nerve degeneration.

  13. Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture.

    PubMed

    Montagne, Kevin; Huang, Hongyun; Ohara, Keikou; Matsumoto, Kunio; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2011-11-01

    Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.

  14. Screening retinal transplants with Fourier-domain OCT

    NASA Astrophysics Data System (ADS)

    Rao, Bin

    2009-02-01

    Transplant technologies have been studied for the recovery of vision loss from retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In several rodent retinal degeneration models and in patients, retinal progenitor cells transplanted as layers to the subretinal space have been shown to restore or preserve vision. The methods for evaluation of transplants are expensive considering the large amount of animals. Alternatively, time-domain Stratus OCT was previously shown to be able to image the morphological structure of transplants to some extent, but could not clearly identify laminated transplants. The efficacy of screening retinal transplants with Fourier-domain OCT was studied on 37 S334ter line 3 rats with retinal degeneration 6-67 days after transplant surgery. The transplants were morphologically categorized as no transplant, detachment, rosettes, small laminated area and larger laminated area with both Fourier-domain OCT and histology. The efficacy of Fourier-domain OCT in screening retinal transplants was evaluated by comparing the categorization results with OCT and histology. Additionally, 4 rats were randomly selected for multiple OCT examinations (1, 5, 9, 14 and 21days post surgery) in order to determine the earliest image time of OCT examination since the transplanted tissue may need some time to show its tendency of growing. Finally, we demonstrated the efficacy of Fourier-domain OCT in screening retinal transplants in early stages and determined the earliest imaging time for OCT. Fourier-domain OCT makes itself valuable in saving resource spent on animals with unsuccessful transplants.

  15. Development of Animal Models of Local Retinal Degeneration

    PubMed Central

    Lorach, Henri; Kung, Jennifer; Beier, Corinne; Mandel, Yossi; Dalal, Roopa; Huie, Philip; Wang, Jenny; Lee, Seungjun; Sher, Alexander; Jones, Bryan William; Palanker, Daniel

    2015-01-01

    Purpose Development of nongenetic animal models of local retinal degeneration is essential for studies of retinal pathologies, such as chronic retinal detachment or age-related macular degeneration. We present two different methods to induce a highly localized retinal degeneration with precise onset time, that can be applied to a broad range of species in laboratory use. Methods A 30-μm thin polymer sheet was implanted subretinally in wild-type (WT) rats. The effects of chronic retinal separation from the RPE were studied using histology and immunohistochemistry. Another approach is applicable to species with avascular retina, such as rabbits, where the photoreceptors and RPE were thermally ablated over large areas, using a high power scanning laser. Results Photoreceptors above the subretinal implant in rats degenerated over time, with 80% of the outer nuclear layer disappearing within a month, and the rest by 3 months. Similar loss was obtained by selective photocoagulation with a scanning laser. Cells in the inner nuclear layer and ganglion cell layer were preserved in both cases. However, there were signs of rewiring and decrease in the size of the bipolar cell terminals in the damaged areas. Conclusions Both methods induce highly reproducible degeneration of photoreceptors over a defined area, with complete preservation of the inner retinal neurons during the 3-month follow-up. They provide a reliable platform for studies of local retinal degeneration and development of therapeutic strategies in a wide variety of species. PMID:26207299

  16. High speed optical holography of retinal blood flow

    NASA Astrophysics Data System (ADS)

    Pellizzari, M.; Simonutti, M.; Degardin, J.; Sahel, J.-A.; Fink, M.; Paques, M.; Atlan, M.

    2016-08-01

    We performed non-invasive video imaging of retinal blood flow in a pigmented rat by holographic interferometry of near-infrared laser light backscattered by retinal tissue, beating against an off-axis reference beam sampled at a frame rate of 39 kHz with a high throughput camera. Local Doppler contrasts emerged from the envelopes of short-time Fourier transforms and the phase of autocorrelation functions of holograms rendered by Fresnel transformation. This approach permitted imaging of blood flow in large retinal vessels (30 microns diameter) over 400 by 400 pixels with a spatial resolution of 8 microns and a temporal resolution of 6.5 ms.

  17. Pharmacotherapies for Retinal Detachment.

    PubMed

    Wubben, Thomas J; Besirli, Cagri G; Zacks, David N

    2016-07-01

    Retinal detachment is an important cause of visual loss. Currently, surgical techniques, including vitrectomy, scleral buckle, and pneumatic retinopexy, are the only means to repair retinal detachment and restore vision. However, surgical failure rates may be as high as 20%, and visual outcomes continue to vary secondary to multiple processes, including postoperative cystoid macular edema, epiretinal membrane formation, macular folds, and, ultimately, photoreceptor death. Therefore, pharmacotherapies are being sought to aid the success rates of modern surgical techniques and reduce or slow the degeneration of photoreceptors during retinal detachment. This review discusses potential therapeutic avenues that aid in retinal reattachment, reduce the rate of retinal redetachment by limiting proliferative vitreoretinopathy, and protect against photoreceptor cell death.

  18. Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

    PubMed

    Eldred, Megan K; Charlton-Perkins, Mark; Muresan, Leila; Harris, William A

    2017-03-15

    To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

  19. Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination

    PubMed Central

    Eldred, Megan K.; Charlton-Perkins, Mark; Muresan, Leila

    2017-01-01

    ABSTRACT To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process. PMID:28174240

  20. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    SciTech Connect

    Shen Chong; Meng Qin Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 {mu}M which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 {mu}M. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to {beta}-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  1. Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

    PubMed

    Mezey, E; Potter, J J; Diehl, A M

    1986-01-15

    Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

  2. Dual signal transduction pathways activated by TSH receptors in rat primary tanycyte cultures.

    PubMed

    Bolborea, Matei; Helfer, Gisela; Ebling, Francis J P; Barrett, Perry

    2015-06-01

    Tanycytes play multiple roles in hypothalamic functions, including sensing peripheral nutrients and metabolic hormones, regulating neurosecretion and mediating seasonal cycles of reproduction and metabolic physiology. This last function reflects the expression of TSH receptors in tanycytes, which detect photoperiod-regulated changes in TSH secretion from the neighbouring pars tuberalis. The present overall aim was to determine the signal transduction pathway by which TSH signals in tanycytes. Expression of the TSH receptor in tanycytes of 10-day-old Sprague Dawley rats was observed by in situ hybridisation. Primary ependymal cell cultures prepared from 10-day-old rats were found by immunohistochemistry to express vimentin but not GFAP and by PCR to express mRNA for Dio2, Gpr50, Darpp-32 and Tsh receptors that are characteristic of tanycytes. Treatment of primary tanycyte/ependymal cultures with TSH (100  IU/l) increased cAMP as assessed by ELISA and induced a cAMP-independent increase in the phosphorylation of ERK1/2 as assessed by western blot analysis. Furthermore, TSH (100  IU/l) stimulated a 2.17-fold increase in Dio2 mRNA expression. We conclude that TSH signal transduction in cultured tanycytes signals via Gαs to increase cAMP and via an alternative G protein to increase phosphorylation of ERK1/2.

  3. Pharmacological induction of heat shock protein 68 synthesis in cultured rat astrocytes.

    PubMed

    Nishimura, R N; Dwyer, B E

    1995-12-15

    The induction of the highly inducible 70-kDa heat shock protein (HSP 70) is associated with thermotolerance and survival from many other types of stress. This investigation studied the pharmacological induction of HSP 68 (HSP 68 is the rat homolog of human HSP 70) by 1,10-phenanthroline in cultured rat astrocytes under conditions that activated heat shock transcription factor-1 without inducing HSP 68 synthesis. Two conditions that activate heat shock transcription factor-1 and promote its binding to the heat shock element without subsequent transcription of HSP 68 mRNA, intracellular acidosis and exposure to salicylate, showed synthesis of HSP 68 when 1,10-phenanthroline was added to culture medium after the activation of heat shock transcription factor-1. 1,10-phenanthroline mimicked heat shock by inducing HSP 68 mRNA and protein under both conditions. 1,10-phenanthroline added alone to culture medium did not induce the synthesis of HSP 68 or activate heat shock transcription factor-1. These findings strongly suggest a multistep activation for HSP 68 synthesis and also demonstrate that the synthesis of HSP 68 can be pharmacologically regulated.

  4. Activator protein-1 complex expressed by magnetism in cultured rat hippocampal neurons.

    PubMed

    Hirai, Takao; Nakamichi, Noritaka; Yoneda, Yukio

    2002-03-22

    Brief exposure for 15 min to static magnetic filed at 100 mT led to marked but transient potentiation of binding of a radiolabeled probe for activator protein-1 (AP1) in immature cultured rat hippocampal neurons with high expression of growth-associated protein-43. Immunoblotting and supershift analyses revealed that brief exposure to static magnetic field increased AP1 DNA binding through expression of Fra-2, c-Jun, and Jun-D proteins in immature cultured hippocampal neurons. Significantly less potent increases were seen in both intracellular free Ca(2+) concentration and AP1 binding following the addition of N-methyl-d-aspartate in these immature neurons exposed to magnetism 24 h before. These results suggest that brief exposure to weak static magnetic field may lead to desensitization of NMDA receptor channels through modulation of de novo synthesis of particular inducible target proteins at the level of gene transcription by the AP1 complex expressed in the nucleus of immature cultured rat hippocampal neurons.

  5. Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

    SciTech Connect

    Vance, J.E.; Vance, D.E.

    1986-05-01

    The role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine (PC) is made both by the CDP-choline pathway and by the methylation of phosphatidyl-ethanolamine (PE), which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanol-amine). Monolayer cultures of rat hepatocytes were incubated in the presence of (methyl-/sup 3/H)choline, (2-/sup 3/H)ethanolamine or (3-/sup 3/H)serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the culture medium. The specific radioactivities of PC and PE derived from (1-/sup 3/H)ethanolamine were markedly lower (approximately 1/2 and less than 1/10, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of PC made from (methyl-/sup 3/H)choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with (3-/sup 3/H)serine, the specific radioactivities of PC and PE were significantly higher in the lipoproteins than in the cells. These data indicate that specific pools of phospholipids are selected on the basis of their routes of biosynthesis, for secretion into lipoproteins.

  6. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  7. Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

    SciTech Connect

    Cervos-Navarro, J.; Hamdorf, G. )

    1993-05-01

    Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a [sup 60]Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor[sup [trademark]2350] using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals.

  8. Morphology and albumin secretion of adult rat hepatocytes cultured on a hydrophobic porous expanded polytetrafluoroethylene membrane.

    PubMed

    Kurosawa, Hiroshi; Yuminamochi, Eri; Yasuda, Ruri; Amano, Yoshifumi

    2003-01-01

    Primary culture of rat hepatocytes was performed on a hydrophobic porous expanded polytetrafluoroethylene (ePTFE) membrane incorporated into the base of a culture dish. Two types of ePTFE membranes, a uniaxially expanded type (ePTFE-1) and a biaxially expanded type (ePTFE-2), could be used as the culture surfaces for hepatocytes. The formation of multicellular aggregates was observed in the culture dish when each membrane type was used. A pore size of 1 mum or higher was adequate for cell adhesion and albumin secretion for both membrane types. The activity of albumin secretion in the dish with the ePTFE membrane was markedly higher than that in the polystyrene dish. Spheroidal multicellular aggregates (spheroids) were observed when hepatocytes were cultured on the ePTFE-1 membrane. The ePTFE-1 membrane maintained the albumin secretion activity for a longer period than the non-expanded PTFE film. It was assumed that the cooperative action of membrane structure and oxygen permeability promoted the formation of cell aggregates and increased the albumin secretion activity.

  9. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    PubMed Central

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-01-01

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of long-term (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain. PMID:27782041

  10. Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture

    SciTech Connect

    Kispert, J.; Mogayzel, P.J. Jr.; Pratt, C.A.; Toselli, P.; Wolfe, B.L.; Faris, B.; Franzblau, C.

    1986-05-01

    The effect of porcine pancreatic elastase on the extracellular matrix (ECM) of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Initially, the elastin appeared as non-coalesced material closely associated with filaments, presumably microfibrils. The insoluble elastin accumulated in the ECM of cells in culture for 6 weeks accounted for 40-45% of the total protein. After exposure to elastase for 30-60 minutes, the elastin content was reduced to 14-20%. The reduction in the total protein content of the cultures after elastase treatment was due primarily to the loss of elastin. Although the amino acid compositions of the elastin isolated from cultures both before and after elastase treatment were similar, there were striking ultrastructural differences in the amorphous elastin. The elastin assumed a mottled appearance after elastase exposure, similar to that seen in in vivo emphysema models. Pulse experiments with /sup 3/H-valine demonstrated an increase in protein synthesis by the cells 20 hours after elastase exposure, suggesting the potential for elastin repair. The use of this culture system will aid in clarifying the role of elastolysis in pulmonary and vascular injuries.

  11. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

    PubMed

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-10-04

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  12. Surfactant and varespladib co-administration in stimulated rat alveolar macrophages culture.

    PubMed

    De Luca, Daniele; Vendittelli, Francesca; Trias, Joaquim; Fraser, Heather; Minucci, Angelo; Gentile, Leonarda; Perez-Gil, Jesus; Conti, Giorgio; Antonelli, Massimo; Capoluongo, Ettore D

    2013-01-01

    Acute lung injury is a life-threatening condition characterized by surfactant dysfunction and raised secretory phospholipase A2 (sPLA2) activity. Varespladib is a sPLA2 inhibitor shown to be effective in animal models of acute lung injury. We aimed at investigating the effect of co-administration of surfactant and varespladib on sPLA2 activity. Alveolar macrophages were cultured and stimulated with lipopolysaccharide and then treated with either varespladib, surfactant, varespladib followed by surfactant or nothing. sPLA2 activity, free fatty acids, tumour necrosis factor-α (TNF-α) and protein concentrations were measured in culture supernatants. Treatment with varespladib (p=0.019) and varespladib + surfactant (p=0.013), reduced the enzyme activity by approximately 15% from the basal level measured in the untreated cultures. Surfactant, varespladib and varespladib + surfactant, respectively decreased free fatty acids by -45% (p=0.045), - 62% (p=0.009) and -48% (p=0.015), from the baseline concentration of the untreated cultures. Varespladib and poractant- α co-administration reduces sPLA2 activity and free fatty acids release in cultured rat alveolar macrophages, although a clear drug synergy was not evident. Since co-administration may be useful to reduce inflammation and surfactant inactivation in acute lung injury, further in vivo studies are warranted to verify its clinical usefulness.

  13. Effects of dehulled adlay on the culture count of some microbiota and their metabolism in the gastrointestinal tract of rats.

    PubMed

    Chiang, W; Cheng, C y; Chiang, M t; Chung, K T

    2000-03-01

    Experiments were conducted to study the effect of a dietary supplement of dehulled adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) on the culture counts of some important groups of intestinal bacteria and their metabolism in the gastrointestinal (GI) tract of Sprague-Dawley rats. Rats were divided into four groups, and each group was fed a diet containing different levels of dehulled adlay for 30 days as follows: 0% (control), 5%, 20%, and 40%. All animals fed with adlay had normal healthy intestinal walls and no pathogenic signs whatsoever. There were no significant differences in body weight gain or the cecal pH between different groups of rats. Both the 20% and 40% groups had lower culture counts of enterics in their feces than the 5% and control groups, whereas the culture counts of fecal lactic acid bacteria were higher in feces of rats fed with adlay than in the control group. Cecal total short-chain fatty acid (SCFA) content and fecal SCFA were significantly higher in the 20% and 40% groups than in the control and 5% groups. All the adlay-fed rats had a higher fecal butyric acid concentration than the control rats. It is concluded that adlay has a significant influence on the growth of intestinal bacteria, which may ultimately affect the physiology and other functions of GI tracts of rats.

  14. Ultrastructural and biochemical alterations induced in human, rat and mouse hepatocytes in primary culture exposed to selected carcinogens

    SciTech Connect

    Cole, K.H.

    1987-01-01

    Aflatoxin B1 (AFB{sub 1}), dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-AAF) and actinomycin D are all potential human liver carcinogens. In order to investigate carcinogenic susceptibility of human liver to these agents, primary cultures of normal human hepatocytes were exposed to the four carcinogens. In the first series of experiments, human, rat, and mouse hepatocytes in primary culture were exposed to actinomycin D, AFB{sub 1}, and DMN for 24 h and examined for ultrastructural alterations. In an effort to relate the ultrastructural effects with total covalent binding of the carcinogen to DNA, human, rat and mouse hepatocytes were exposed to 2.0 {times} 10{sup {minus}7} M ({sup 3}H)AFB{sub 1} for 24 h. Hepatocytes from male rats had the highest degree of ({sup 3}H)AFB{sub 1}-DNA binding. Human hepatocytes contained the next highest binding level, while hepatocytes from female rats bound 38 pmoles/mg DNA. The AFB{sub 1}-DNA binding level in mouse hepatocytes was 1.4 pmoles/mg DNA. In similar experiments, human, and male and female rat hepatocytes in primary culture were exposed to the carcinogen 2-acetylamino (9-{sup 14}C)fluorene for 24 h. It was determined that male rat hepatocytes had the highest amount of radiolabeled 2-AAF bound to their DNA, female rats contained 0.57 nmoles/mg DNA, while human hepatocytes contained 0.29 nmoles/mg DNA.

  15. Toxic effects of anabolic-androgenic steroids in primary rat hepatic cell cultures.

    PubMed

    Welder, A A; Robertson, J W; Melchert, R B

    1995-08-01

    Hepatic complications in athletes and bodybuilders after abusing anabolic-androgenic steroids (AAS) have been reported. Hepatic injury, including cholestasis, peliosis hepatis, hyperplasia, and tumors, have been attributed to abuse of the 17 alpha-alkylated AAS. Some of these pathological conditions have been reversed when individuals were converted to nonalkylated AAS regimens. The purpose of this study was to determine and compare the direct toxic effects of commonly abused AAS (both 17 alpha-alkylated and nonalkylated) in primary hepatic cell cultures. Primary cultures, established from 60-day-old Sprague-Dawley rats, were exposed to doses of 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4)M 19-nortestosterone, fluoxymesterone, testosterone cypionate, stanozolol, danazol, oxymetholone, testosterone, estradiol, and methyltestosterone for 1, 4, and 24 hr. Lactate dehydrogenase (LDH) release, neutral red (NR) retention, and glutathione (GSH) depletion were evaluated to determine plasma membrane damage, cell viability, and possible oxidative injury, respectively. Those cultures exposed to the 17 alpha-alkylated AAS, methyltestosterone and stanozolol, at doses of 1 x 10(-4) M for 24 hr and the 17 alpha-alkylated AAS, oxymetholone, at 1 x 10(-4) M for 4 and 24 hr showed significant increased in LDH release and decreases in NR retention while there were no significant differences with the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol). GSH depletion was evaluated in cultures treated with 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4) M concentrations of methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr. Cultures exposed to 1 x 10(-4) M oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to 1 x 10(-4) M methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested. These

  16. Digestion of an exogenous protein by rat yolksac cultured in vitro

    PubMed Central

    Williams, K. E.; Lloyd, J. B.; Davies, M.; Beck, F.

    1971-01-01

    Yolk-sacs were removed from 17.5-day pregnant rats injected 2–5h previously with 125I-labelled bovine serum albumin. The specific activities of acid phosphatase and acid proteinase, and the specific radioactivities (trichloroacetic acid-insoluble and trichloroacetic acid-soluble) were measured in subcellular fractions prepared by homogenization and differential centrifugation. The conversion of acid-insoluble into acid-soluble radioactivity within cultured tissue was followed and the nature of the liberated products was investigated by gel chromatography. The results are consistent with the protein entering lysosomes and being digested there. The radiolabel was released chiefly as free iodotyrosine. PMID:5158915

  17. Different biocompatibility of crystalline triamcinolone deposits on retinal cells in vitro and in vivo.

    PubMed

    Szurman, Peter; Sierra, Ana; Kaczmarek, Radoslaw; Jaissle, Gesine B; Wallenfels-Thilo, Barbara; Grisanti, Salvatore; Lüke, Matthias; Bartz-Schmidt, Karl U; Spitzer, Martin S

    2007-07-01

    Epiretinal deposits of triamcinolone acetonide (TA) can be detrimental to retinal cells in vitro as several laboratory studies have shown. This contrasts with the good clinical experience of intravitreal TA use. We investigated the effect of TA crystals on retinal cells concerning the critical dose range, a potential cell recovery, the drug-tissue interaction and what protective biological factors could explain the discrepancy between in vivo and in vitro results. A human retinal pigment epithelium cell line (ARPE19) and transformed rat retinal ganglion cells (RGC5) were used. Purified TA crystals were either added directly on top of the cell cultures or on top of membrane filter inserts, basement membrane sheets or porcine vitreous with the cells growing underneath. To determine the number of live versus dead cells fluorescent stains were used. Proliferation and viability were measured using the MTT assay and the mean inhibitory dose (ID(50)) calculated with or without a filter. Cell recovery was measured after transient TA exposure (0.01-1 mg/ml) compared to continuous exposure after 7 days. To exclude a mere mechanical effect of epicellular deposits the TA crystals were replaced by glass pearls in a serum-free medium and the MTT toxicity assay was performed after 24 h. Without direct contact of TA crystals with the cells only a moderate decrease of mitochondrial activity was observed that fully recovered after transient exposure and showed a clinically safe ID(50) of 7.7 mg/ml. In contrast, direct exposure to even minute crystalline deposits for 7 days caused a rapid progressive and irreversible cell death being significant far below clinically used concentrations (ID(50) 0.058 mg/ml). Direct exposure to glass pearls did not show any loss of viability. Both basement membrane sheets and vitreous reliably prevented direct cytotoxicity to underlying retinal ganglion cells. Our findings suggest that irreversible TA cytotoxicity in a cell culture setting occurs

  18. Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development

    PubMed Central

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S.

    2012-01-01

    Background Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot. PMID:22916200

  19. Prolonged Minocycline Treatment Impairs Motor Neuronal Survival and Glial Function in Organotypic Rat Spinal Cord Cultures

    PubMed Central

    Pinkernelle, Josephine; Fansa, Hisham; Ebmeyer, Uwe; Keilhoff, Gerburg

    2013-01-01

    Background Minocycline, a second-generation tetracycline antibiotic, exhibits anti-inflammatory and neuroprotective effects in various experimental models of neurological diseases, such as stroke, Alzheimer’s disease, amyotrophic lateral sclerosis and spinal cord injury. However, conflicting results have prompted a debate regarding the beneficial effects of minocycline. Methods In this study, we analyzed minocycline treatment in organotypic spinal cord cultures of neonatal rats as a model of motor neuron survival and regeneration after injury. Minocycline was administered in 2 different concentrations (10 and 100 µM) at various time points in culture and fixed after 1 week. Results Prolonged minocycline administration decreased the survival of motor neurons in the organotypic cultures. This effect was strongly enhanced with higher concentrations of minocycline. High concentrations of minocycline reduced the number of DAPI-positive cell nuclei in organotypic cultures and simultaneously inhibited microglial activation. Astrocytes, which covered the surface of the control organotypic cultures, revealed a peripheral distribution after early minocycline treatment. Thus, we further analyzed the effects of 100 µM minocycline on the viability and migration ability of dispersed primary glial cell cultures. We found that minocycline reduced cell viability, delayed wound closure in a scratch migration assay and increased connexin 43 protein levels in these cultures. Conclusions The administration of high doses of minocycline was deleterious for motor neuron survival. In addition, it inhibited microglial activation and impaired glial viability and migration. These data suggest that especially high doses of minocycline might have undesired affects in treatment of spinal cord injury. Further experiments are required to determine the conditions for the safe clinical administration of minocycline in spinal cord injured patients. PMID:23967343

  20. Correlation between cell attachment areas after 2 h of culture and osteogenic differentiation activity of rat mesenchymal stem cells on hydroxyapatite substrates with various surface properties.

    PubMed

    Cheng, Kan; Hirose, Motohiro; Wang, Xiupeng; Sogo, Yu; Yamazaki, Atsushi; Ito, Atsuo

    2013-01-04

    The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter.

  1. [3H]ouabain binding to cultured rat vascular smooth muscle cells.

    PubMed

    Khalil, F; Hopp, L; Searle, B M; Tokushige, A; Tamura, H; Kino, M; Aviv, A

    1984-05-01

    The number of Na+ pump units (Bmax) and the equilibrium dissociation constant (Kd) for ouabain as well as parameters of K+ binding to the Na+ pump were examined in in vitro-grown vascular smooth muscle cells ( VSMC ) derived from Sprague-Dawley rats. The technique to measure these variables utilizes analyses of [3H]ouabain displacement from its VSMC receptors by nonlabeled ouabain and K+. The mean values for Bmax and Kd in the cultured VSMCs were 1.95 X 10(5) receptor sites per single VSMC and 2.68 X 10(-6) M, respectively. The equilibrium dissociation constant for K+ (Ki) was 0.92 mM. K+ binding to the cultured VSMCs demonstrated positive cooperativity with a Hill coefficient (n) of 1.78.

  2. Effect of Semax peptide on survival of cultured rat pheochromocytoma cells during oxidative stress.

    PubMed

    Safarova, E R; Shram, S I; Zolotarev, Yu A; Myasoedov, N F

    2003-03-01

    We studied the effects of Semax (antiinsulin peptide with neuroprotective effect) on the survival of cultured rat pheochromocytoma cell after oxidative stress induced by short-term incubation with hydrogen peroxide. Studies with fluorescent dyes propidium iodide and Hoechst 33258 showed that cell incubation with hydrogen peroxide led to the formation of damaged cells with characteristic signs of necrosis. Semax dose-dependently reduced the number of cells damaged by oxidative stress. The efficiency of Semax depended on the time of its addition to the culture medium. The results suggest that the neuroprotective effect of Semax in ischemic stroke can be due to its capacity to protect neurons from damage caused by oxidative stress.

  3. Quantitative analysis of the toxicity of human amniotic fluid to cultured rat spinal cord.

    PubMed

    Drewek, M J; Bruner, J P; Whetsell, W O; Tulipan, N

    1997-10-01

    It has been proposed that the myelodysplastic components of a myelomeningocele are secondarily damaged as the result of exposure to amniotic fluid, the so-called 'two-hit' hypothesis. The critical time at which this secondary insult might occur has not been clearly defined. The present study addresses this issue by quantitatively assessing the toxic effects of human amniotic fluid of various gestational ages upon organotypic cultures of rat spinal cord. Using an assay for lactate dehydrogenase efflux to evaluate toxicity in such spinal cord cultures, we found that the amniotic fluid became toxic at approximately 34 weeks' gestation. This toxic effect of amniotic fluid appears to emerge rather suddenly. Accordingly, it seems reasonable to suggest that prevention of exposure of vulnerable spinal cord tissue to this toxicity by surgical closure of a myelomeningocele defect prior to the emergence of toxicity in amniotic fluid may prevent injury to vulnerable myelodysplastic spinal cord tissue.

  4. Distribution of phospholipase C isozymes in various rat tissues and cultured cells

    SciTech Connect

    Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

    1987-05-01

    Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts.

  5. Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels

    PubMed Central

    Korkotian, Eduard; Bombela, Tatyana; Odegova, Tatiana; Zubov, Petr; Segal, Menahem

    2013-01-01

    The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25–0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor. PMID:24260098

  6. The isolation and culture of endothelial colony-forming cells from human and rat lungs.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Zhong, Shumei; Zong, Shumei; McConaghy, Suzanne; Ohls, Robin; Yoder, Mervin C; Thébaud, Bernard

    2015-11-01

    Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human

  7. Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

    PubMed

    Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

    2016-05-01

    Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

  8. Neural Stem Cell-based Intraocular Administration of Pigment Epithelium-derived Factor Promotes Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Crush Injury in Rat: An Experimental Study

    PubMed Central

    Zhang, Wei-Min; Zhang, Zhi-Ren; Zhang, Yong-Gang; Gao, Yan-Sheng

    2016-01-01

    Background: Pigment epithelium-derived factor (PEDF) is regarded as a multifunctional protein possessing neurotrophic and neuroprotective properties. PEDF has a very short half-life, and it would require multiple injections to maintain a therapeutically relevant level without a delivery system. However, multiple injections are prone to cause local damage or infection. To overcome this, we chose a cell-based system that provided sustained delivery of PEDF and compared the effect of weekly injections of PEDF and neural stem cell (NSC)-based intraocular administration of PEDF on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve injury. Methods: Seventy-two rats were randomly assigned to 3 groups: group with injections of phosphate buffered saline (PBS) (n=24), group with weekly injections of PEDF (n=24), and group with NSC-based administration of PEDF (n=24). Western blot was used to analyze the PEDF protein level 2 weeks after injection. Retinal flat mounts and immunohistochemistry were employed to analyze RGC survival and axon regeneration 2 weeks and 4 weeks after injection. The data were analyzed with one-way ANOVA in SPSS (version 19.0). A P<0.05 was considered significant. Results: The PEDF protein level in the group with NSC-based administration of PEDF increased compared with that in the groups with injections of PEDF and PBS (P<0.05). The PEDF-modified NSCs differentiated into GFAP-positive astrocytes andβ-tubulin-III-positive neurons. NSC-based administration of PEDF effectively increased RGC survival and improved the axon regeneration of the optic nerve compared with weekly injections of PEDF. Conclusion: Subretinal space transplantation of PEDF-secreting NSCs sustained high concentrations of PEDF, differentiated into neurons and astrocytes, and significantly promoted RGC survival and axon regeneration after optic nerve injury. PMID:27582587

  9. Retinal vascular regeneration.

    PubMed

    Otani, Atsushi; Friedlander, Martin

    2005-01-01

    We discuss the potential use of stem cells for therapeutic angiogenesis in the treatment of retinal diseases. We demonstrate that the clinical utility of these EPC may be not limited in the treatment of ischemic retinal diseases but may also have application for the treatment of retinal degenerative disorders and for a form of cell-based gene therapy. One of the greatest potential benefits of bone marrow derived EPC therapy is the possible use of autologous grafts. Nonetheless, potential toxicities and unregulated cell growth will need to be carefully evaluated before this approach is brought to the clinics.

  10. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  11. The generation of rhythmic activity in dissociated cultures of rat spinal cord.

    PubMed

    Streit, J; Tscherter, A; Heuschkel, M O; Renaud, P

    2001-07-01

    Locomotion in vertebrates is controlled by central pattern generators in the spinal cord. The roles of specific network architecture and neuronal properties in rhythm generation by such spinal networks are not fully understood. We have used multisite recording from dissociated cultures of embryonic rat spinal cord grown on multielectrode arrays to investigate the patterns of spontaneous activity in randomised spinal networks. We were able to induce similar patterns of rhythmic activity in dissociated cultures as in slice cultures, although not with the same reliability and not always with the same protocols. The most reliable rhythmic activity was induced when a partial disinhibition of the network was combined with an increase in neuronal excitability, suggesting that both recurrent synaptic excitation and neuronal excitability contribute to rhythmogenesis. During rhythmic activity, bursts started at several sites and propagated in variable ways. However, the predominant propagation patterns were independent of the protocol used to induce rhythmic activity. When synaptic transmission was blocked by CNQX, APV, strychnine and bicuculline, asynchronous low-rate activity persisted at approximately 50% of the electrodes and approximately 70% of the sites of burst initiation. Following the bursts, the activity in the interval was transiently suppressed below the level of intrinsic activity. The degree of suppression was proportional to the amount of activity in the preceding burst. From these findings we conclude that rhythmic activity in spinal cultures is controlled by the interplay of intrinsic neuronal activity and recurrent excitation in neuronal networks without the need for a specific architecture.

  12. Ethanol inhibits development of dendrites and synapses in rat hippocampal pyramidal neuron cultures.

    PubMed

    Yanni, P A; Lindsley, T A

    2000-04-14

    Evidence suggests that some neuropathologic manifestations of Fetal Alcohol Syndrome (FAS) result from the disruption of neuromorphogenesis and synapse formation in the hippocampus. Prior research in this laboratory has shown that ethanol in the medium during the first 24 h in culture increases the number of minor processes (the precursors of axons and dendrites) and accelerates the rate at which axons are formed in low-density cultures of embryonic rat hippocampal neurons. The current study examined the effects of ethanol on the subsequent development of dendrites and synapses in these cultures. Quantitative morphometric analysis utilized double-immunofluorescent staining for MAP2 and synapsin I to visualize dendrites and synaptic specializations, respectively. Six days of ethanol (200, 400 or 600 mg/dl) in the medium, beginning at the time of plating, resulted in decreases in total dendritic length per cell, dendrite number per cell, length of individual dendrites and synapse number per innervated dendrite but had no effect on cell survival. The decrease in synapse number was correlated with dendrite length, suggesting that ethanol's effects on synapse number are secondary to its effects on dendritogenesis. Taken together with our previous findings, these results are the first to demonstrate that ethanol has differential effects on axonal and dendritic growth in a culture model of neurons that are vulnerable to ethanol-induced cytoarchitectural abnormalities during development in vivo.

  13. Rat vascular smooth muscle cells in culture contract upon Ca2+ repletion after depletion.

    PubMed Central

    Kobayashi, S.; Kanaide, H.; Hasegawa, M.; Yamamoto, H.; Nakamura, M.

    1985-01-01

    We investigated the effects of Ca2+-repletion following depletion on cultured vascular smooth muscle cells (SMCs) from the rat aorta. With Ca2+-repletion, the cells in primary cultures contracted, as indicated by a decrease in cell area. The process was slow (30 min to maximum effect) and reversible (relaxation completed by 120 min). Contraction during Ca2+-repletion was never observed in subcultured cells. The SMCs in primary culture after treatment maintained the ability to grow and to exclude dye, with a normal plating efficiency. There was no treatment-related additional leakage of intracellular enzymes, LDH and CPK, into the medium. Ca2+-repletion at first accelerated the 45Ca uptake by SMCs (1-5 min after repletion) and then increased Ca2+ efflux after about 10 min of Ca2+-repletion. We conclude that Ca2+-repletion after depletion induces a transient and reversible contraction of vascular SMCs in primary culture, without cell injury and in association with a transient increase in Ca2+ influx and then efflux. This phenomenon may relate to the decrease in perfusion flow in hearts and kidneys during Ca2+-repletion after depletion (Ca2+-paradox). Images Fig. 1 Fig. 3 PMID:4084451

  14. Sotalol permeability in cultured-cell, rat intestine and PAMPA system

    PubMed Central

    LIU, WEI; OKOCHI, HIDEAKI; BENET, LESLIE Z.; ZHAI, SUO-DI

    2014-01-01

    Purpose To clarify sotalol’s classification in the BCS versus BDDCS systems through cellular, rat everted sac and PAMPA permeability studies. Methods Studies were carried out in Madin Darby canine kidney (MDCK) and MDR1-transfected MDCK (MDCK-MDR1) cell lines, rat everted gut sacs and the Parallel Artificial Membrane Permeability Assay (PAMPA) system. Three-hour transport studies were conducted in MDCK cell lines (with apical pH changes) and MDCK-MDR1 cells (with and without the P-glycoprotein inhibitor GG918); male Sprague-Dawley rats (300 ~ 350 g) were used to prepare everted sacs. In the PAMPA studies, drug solutions at different pH’s were dosed in each well and incubated for 5 hours. Samples were measured by LC-MS/MS, or liquid scintillation counting and apparent permeability (Papp) was calculated. Results Sotalol showed low permeability in all of the cultured-cell lines, everted sacs and PAMPA systems. It might be a border line P-glycoprotein substrate. The PAMPA study showed that sotalol’s permeability increased with a higher apical pH, while much less change was found in MDCK cells. Conclusion The low permeability rate for sotalol correlates with its Class 3 BDDCS assignment and lack of in vivo metabolism. PMID:22350803

  15. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.

    PubMed

    Igoudjil, Anissa; Massart, Julie; Begriche, Karima; Descatoire, Véronique; Robin, Marie-Anne; Fromenty, Bernard

    2008-06-01

    The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c

  16. Evaluation of Cisplatin Neurotoxicity in Cultured Rat Dorsal Root Ganglia via Cytosolic Calcium Accumulation

    PubMed Central

    Erol, Kevser; Yiğitaslan, Semra; Ünel, Çiğdem; Kaygısız, Bilgin; Yıldırım, Engin

    2016-01-01

    Background: Calcium homeostasis is considered to be important in antineoplastic as well as in neurotoxic adverse effects of cisplatin. Aims: This study aimed to investigate the role of Ca2+ in cisplatin neurotoxicity in cultured rat dorsal root ganglia (DRG) cells. Study Design: Cell culture study. Methods: DRG cells prepared from 1-day old Sprague-Dawley rats were used to determine the role of Ca2+ in the cisplatin (10–600 μM) neurotoxicity. The cells were incubated with cisplatin plus nimodipine (1–3 μM), dizocilpine (MK-801) (1–3 μM) or thapsigargin (100–300 nM). Toxicity of cisplatinon DRG cells was determined by the MTT assay. Results: The neurotoxicity of cisplatin was significant when used in high concentrations (100–600 μM). Nimodipine (1 μM) but not MK-801 or thapsigargin prevented the neurotoxic effects of 200 μM of cisplatin. Conclusion: Voltage-dependent calcium channels may play a role in cisplatin neurotoxicity. PMID:27403382

  17. Toxicity of organotin compounds in primary cultures of rat cortical astrocytes.

    PubMed

    Röhl, C; Gülden, M; Seibert, H

    2001-01-01

    The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 micromol/L (TBT), 30 micromol/L (TET), and 800 micromol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals.

  18. Ghrelin-Induced Enhancement of Vasopressin and Oxytocin Secretion in Rat Neurohypophyseal Cell Cultures.

    PubMed

    Gálfi, M; Radács, M; Molnár, Zs; Budai, I; Tóth, G; Pósa, A; Kupai, K; Szalai, Z; Szabó, R; Molnár, H A; Gardi, J; László, Ferenc A; Varga, Cs

    2016-12-01

    The effects of ghrelin on vasopressin and oxytocin secretion were studied in 13-14-day cell cultures of isolated rat neurohypophyseal tissue. The vasopressin and oxytocin contents of the supernatant were determined by radioimmunoassay after a 1- or 2-h incubation. Significantly increased levels of vasopressin and oxytocin production were detected in the cell culture media following ghrelin administration, depending on the ghrelin doses. The oxytocin level proved to be more elevated than that of vasopressin. The increase of vasopressin and oxytocin secretion could be totally blocked by previous administration of the ghrelin receptor antagonist ([D-Lys(3)]-growth hormone-releasing peptide-6). Application of the ghrelin receptor antagonist after ghrelin administration proved ineffective. The results indicate that vasopressin and oxytocin release is influenced directly by the ghrelin system, and the effects of ghrelin on vasopressin and oxytocin secretion from the neurohypophyseal tissue in rats can occur at the level of the posterior pituitary. Our observations lend support to the view that neurohypophysis contains ghrelin receptors.

  19. Isolated rat dental pulp cell culture and transplantation with an alginate scaffold.

    PubMed

    Fujiwara, Shiro; Kumabe, Shunji; Iwai, Yasutomo

    2006-05-01

    Many studies have been conducted on tissue stem cells in the field of regenerative medicine, and cultured dental pulp mesenchymal cells have been reported to secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured rat dental-pulp-derived cells subcutaneously into the back of nude mice. We found that when beta-glycerophosphate was added to the culture medium, the mRNA of the dentin sialophosphoprotein (DSPP) gene coding dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) was expressed, and an increase in alkaline phosphatase, an early marker of odontoblast differentiation, was also demonstrated. Six weeks after implantation, subcutaneous formation of radiopaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants, and isolated odontoblast-like cells began to form dentin-like hard tissue formation. Scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured rat dental-pulp-derived cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

  20. Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen.

    PubMed Central

    Dolphin, A. C.; Scott, R. H.

    1986-01-01

    Voltage-dependent inward calcium currents (ICa) activated in cultured rat dorsal root ganglion neurones were reversibly reduced in a dose-dependent manner by (-)-baclofen (10 microM to 100 microM). Baclofen (100 microM) reduced the calcium-dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium-free medium and by 300 microM cadmium chloride. The action of baclofen on IK(Ca) was reduced when the calcium concentration in the medium was increased from 5 mM to 30 mM. The calcium independent fast transient voltage-dependent outward current (IK(Vt] was also reduced by baclofen; this effect remained present when Ca2+-free medium was used to prevent contamination by IK(Ca). 4-Aminopyridine (500 microM) reduced IK(Vt) and induced a small increase in ICa. The action of baclofen on ICa was partially antagonized by 4-aminopyridine. GABAB receptor-mediated inhibition of ICa in cultured rat dorsal root ganglion neurones involves a direct mechanism rather than resulting indirectly from an increase in the residual outward potassium currents activated by depolarization. The reduction in ICa by baclofen was variable and dependent on the amplitude of control ICa, larger currents being more resistant to the baclofen-induced inhibition. PMID:2423173

  1. Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

    PubMed

    Ebneter, Andreas; Agca, Cavit; Dysli, Chantal; Zinkernagel, Martin S

    2015-01-01

    Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

  2. Investigation of Retinal Morphology Alterations Using Spectral Domain Optical Coherence Tomography in a Mouse Model of Retinal Branch and Central Retinal Vein Occlusion

    PubMed Central

    Ebneter, Andreas; Agca, Cavit; Dysli, Chantal; Zinkernagel, Martin S.

    2015-01-01

    Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions. PMID:25775456

  3. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    PubMed

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  4. Retinal oximetry in patients with ischaemic retinal diseases.

    PubMed

    Rilvén, Sandra; Torp, Thomas Lee; Grauslund, Jakob

    2017-03-01

    The retinal oximeter is a new tool for non-invasive measurement of retinal oxygen saturation in humans. Several studies have investigated the associations between retinal oxygen saturation and retinal diseases. In the present systematic review, we examine whether there are associations between retinal oxygen saturation and retinal ischaemic diseases. We used PubMed and Embase to search for retinal oxygen saturation and retinal ischaemic diseases. Three separate searches identified a total of 79 publications. After two levels of manual screening, 10 studies were included: six about diabetic retinopathy (DR) and four about retinal vein occlusion. No studies about retinal artery occlusion were included. In diabetes, all studies found that increases in retinal venous oxygen saturation (rvSatO2 ) were associated with present as well as increasing levels of DR. Four of six studies also found increased retinal arterial oxygen saturation (raSatO2 ) in patients with DR. In patients with central retinal vein occlusion (CRVO), all studies found that rvSatO2 was reduced, but raSatO2 remained unchanged. Branch retinal vein occlusion was not associated with changes in retinal oxygen saturation, but this was based on a single study. In conclusion, DR is associated with increased rvSatO2 and might also be related to increased raSatO2 . Central retinal vein occlusion (CRVO) is correlated with increased rvSatO2 but unrelated to raSatO2 . Prospective studies are needed to expand these findings. These would tell whether retinal oximetry could be a potential tool for screening or a biomarker of treatment outcome in patients with ischaemic retinal diseases.

  5. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  6. Signaling by TGF-betas in tubule cultures of adult rat testis

    PubMed Central

    Chan, Kai-Hui; Galuska, Sebastian P; Kudipudi, Pradeep Kumar; Riaz, Mohammad Assad; Loveland, Kate L; Konrad, Lutz

    2017-01-01

    Although signal transduction of transforming growth factor-betas (TGF-βs) is well characterized in individual cell types, data about TGF-β signaling in a cellular context is still scarce. In this study, we used ex vivo tubule cultures from adult rat testis to investigate TGF-β signaling. We show for the first time in testicular tubules, that TGF-βs also signal via the BMP type I receptors, with ALK2 used by TGF-β1 and ALK3 and ALK6 by TGF-β2. This signal transduction is mediated via Smad3 as well as via Smad1. In contrast, BMPs (BMP2 and BMP7) do not signal via the high-affinity type I and type II TGFβ receptors, TBR1 or TBR2. Furthermore, treatment of tubule cultures with either TGF-β1 or TGF-β2 had profound significant stimulatory effects on secretion of plasminogen activator-1 (PAI-1) through utilization of TGF-β and BMP receptors. Specific inhibitors for either TBR1 or BMP receptors yielded nearly complete inhibition of TGF-β signaling. The TBR1-TBR2 signalosome was detected with Duolink upon stimulation with either TGF-β1 or TGF-β2, predominantly in spermatogenic cells of the adult rat testis, particularly in elongated spermatids. In summary, this examination of intact rat testicular tubules demonstrated for the first time that TGF-βs signal mainly through TBR1 and TBR2 but also use BMP receptors, including for secretion of PAI-1. Whereas ALK2 participates in the TGF-β1-induced TBR1-TBR2 signalosome, ALK3 and ALK6 are involved in signaling of TGF-β2. Detection of the TBR1-TBR2 signalosome in late spermiogenic cells indicates a post-meiotic activity. PMID:28386343

  7. Mycolactone-mediated neurite degeneration and functional effects in cultured human and rat DRG neurons

    PubMed Central

    Sinisi, M; Fox, M; MacQuillan, A; Quick, T; Korchev, Y; Bountra, C; McCarthy, T; Anand, P

    2016-01-01

    Background Mycolactone is a polyketide toxin secreted by the mycobacterium Mycobacterium ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients with Buruli ulcer. A recent pre-clinical study proposed that mycolactone may produce analgesia via activation of the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shown analgesic efficacy in animal models and clinical trials for neuropathic pain. We therefore investigated the morphological and functional effects of mycolactone in cultured human and rat dorsal root ganglia (DRG) neurons and the role of AT2R using EMA401. Primary sensory neurons were prepared from avulsed cervical human DRG and rat DRG; 24 h after plating, neurons were incubated for 24 to 96 h with synthetic mycolactone A/B, followed by immunostaining with antibodies to PGP9.5, Gap43, β tubulin, or Mitotracker dye staining. Acute functional effects were examined by measuring capsaicin responses with calcium imaging in DRG neuronal cultures treated with mycolactone. Results Morphological effects: Mycolactone-treated cultures showed dramatically reduced numbers of surviving neurons and non-neuronal cells, reduced Gap43 and β tubulin expression, degenerating neurites and reduced cell body diameter, compared with controls. Dose-related reduction of neurite length was observed in mycolactone-treated cultures. Mitochondria were distributed throughout the length of neurites and soma of control neurons, but clustered in the neurites and soma of mycolactone-treated neurons. Functional effects: Mycolactone-treated human and rat DRG neurons showed dose-related inhibition of capsaicin responses, which were reversed by calcineurin inhibitor cyclosporine and phosphodiesterase inhibitor 3-isobutyl-1-Methylxanthine, indicating involvement of cAMP/ATP reduction. The morphological and functional effects of mycolactone were not altered by Angiotensin II or AT2R antagonist EMA401. Conclusion Mycolactone

  8. Retinal detachment in pseudophakia.

    PubMed

    Galin, M A; Poole, T A; Obstbaum, S A

    1979-07-01

    In a series of cataract patients excluding myopic individuals, under age 60 years, and cases in which vitreous loss occurred, retinal detachment was no less frequent after intracapsular cataract extraction and Sputnik iris supported lenses than in controls. Both groups were followed up for a minimum of two years. The detachments predominantly occurred from retinal breaks in areas of the retina that looked normal preoperatively.

  9. Retinal dysplasia of holoprosencephaly.

    PubMed

    Gorovoy, Ian R; Layer, Noelle; de Alba Campomanes, Alejandra G

    2014-03-04

    Retinal dysplasia occurs in the setting of sporadic and syndromic holoprosencephaly, which often has associated ocular malformations. The pathology of this dysplasia, which includes rosettes, has been previously described. However, its funduscopic findings have not been well documented. The authors present the fundus images of a patient with severe holoprosencephaly with retinal dysplasia and bilateral optic nerve colobomas that resulted in death 2 weeks after birth.

  10. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

    PubMed Central

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  11. Therapy for acute retinal necrosis.

    PubMed

    Kawaguchi, Tatsushi; Spencer, Doran B; Mochizuki, Manabu

    2008-01-01

    Acute retinal necrosis is a progressive necrotizing retinopathy caused by herpes simplex virus (HSV) or varicella zoster virus (VZV). The mainstay of its treatment is antiviral therapy against these pathogenic organisms, such as intravenous acyclovir or oral valacyclovir. Systemic and topical corticosteroids together with antiviral therapy are used as an anti-inflammatory treatment to minimize damages to the optic nerve and retinal blood vessels. Because the majority of severe cases of the disease show occlusive retinal vasculitis, a low dosage of aspirin is used as anti-thrombotic treatment. Vitreo-retinal surgery is useful to repair rhegmatogenous retinal detachment, one of the main late-stage complications. Moreover, recent articles have reported some encouraging results of prophylactic vitrectomy before rhegmatogenous retinal detachment occurs. The efficacy of laser photocoagulation to prevent the development or extension of rhegmatogenous retinal detachment is controversial. Despite these treatments, the visual prognosis of acute retinal necrosis is still poor, in particular VZV-induced acute retinal necrosis.

  12. Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro.

    PubMed

    Gianinazzi, C; Schild, M; Müller, N; Leib, S L; Simon, F; Nuñez, S; Joss, P; Gottstein, B

    2005-12-01

    The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

  13. Effects of intracellular calcium on sodium current density in cultured neonatal rat cardiac myocytes.

    PubMed Central

    Chiamvimonvat, N; Kargacin, M E; Clark, R B; Duff, H J

    1995-01-01

    1. Na+ channel mRNA levels in the heart can be modulated by changes in intracellular Ca2+ ([Ca2+]i). We have investigated whether this regulation of Na+ channel biosynthesis by cytosolic Ca2+ translates into functional Na+ channels that can be detected electrophysiologically. 2. Whole-cell Na+ currents (INa) were recorded using patch-clamp techniques from single ventricular myocytes isolated from neonatal rats and maintained in tissue culture for 24 h. Na+ current density, measured at a membrane potential of -10 mV, was significantly decreased in the cells which were exposed for 24 h to culture medium containing 10 mM of both external Ca2+ and K+ in order to raise [Ca2+]i compared with control cells which were maintained in culture medium containing 2 and 5 mM of Ca2+ and K+, respectively. In contrast, Na+ current density (at -10 mV) was significantly increased in cells exposed for 24 h to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetraacetoxymethyl ester (BAPTA AM; a cell membrane-permeable Ca2+ chelator) which lowered the average [Ca2+]i compared with control. 3. Changes in current density were not associated with changes in the voltage dependence of activation and inactivation of INa. There were no changes in single-channel conductances. 4. It is concluded that Na+ current density in neonatal rat cardiac myocytes is modulated by [Ca2+]i. The findings suggest that the differences in current density are attributable to a change in Na+ channel numbers rather than to changes in single-channel conductance or gating. These changes are consistent with the previously documented modulation of Na+ channel biosynthesis by cytosolic Ca2+. PMID:7650605

  14. Photovoltaic Retinal Prosthesis with High Pixel Density

    PubMed Central

    Mathieson, Keith; Loudin, James; Goetz, Georges; Huie, Philip; Wang, Lele; Kamins, Theodore I.; Galambos, Ludwig; Smith, Richard; Harris, James S.; Sher, Alexander; Palanker, Daniel

    2012-01-01

    Retinal degenerative diseases lead to blindness due to loss of the “image capturing” photoreceptors, while neurons in the “image processing” inner retinal layers are relatively well preserved. Electronic retinal prostheses seek to restore sight by electrically stimulating surviving neurons. Most implants are powered through inductive coils, requiring complex surgical methods to implant the coil-decoder-cable-array systems, which deliver energy to stimulating electrodes via intraocular cables. We present a photovoltaic subretinal prosthesis, in which silicon photodiodes in each pixel receive power and data directly through pulsed near-infrared illumination and electrically stimulate neurons. Stimulation was produced in normal and degenerate rat retinas, with pulse durations from 0.5 to 4 ms, and threshold peak irradiances from 0.2 to 10 mW/mm2, two orders of magnitude below the ocular safety limit. Neural responses were elicited by illuminating a single 70 μm bipolar pixel, demonstrating the possibility of a fully-integrated photovoltaic retinal prosthesis with high pixel density. PMID:23049619

  15. Photovoltaic retinal prosthesis with high pixel density

    NASA Astrophysics Data System (ADS)

    Mathieson, Keith; Loudin, James; Goetz, Georges; Huie, Philip; Wang, Lele; Kamins, Theodore I.; Galambos, Ludwig; Smith, Richard; Harris, James S.; Sher, Alexander; Palanker, Daniel

    2012-06-01

    Retinal degenerative diseases lead to blindness due to loss of the `image capturing' photoreceptors, while neurons in the `image-processing' inner retinal layers are relatively well preserved. Electronic retinal prostheses seek to restore sight by electrically stimulating the surviving neurons. Most implants are powered through inductive coils, requiring complex surgical methods to implant the coil-decoder-cable-array systems that deliver energy to stimulating electrodes via intraocular cables. We present a photovoltaic subretinal prosthesis, in which silicon photodiodes in each pixel receive power and data directly through pulsed near-infrared illumination and electrically stimulate neurons. Stimulation is produced in normal and degenerate rat retinas, with pulse durations of 0.5-4 ms, and threshold peak irradiances of 0.2-10 mW mm-2, two orders of magnitude below the ocular safety limit. Neural responses were elicited by illuminating a single 70 µm bipolar pixel, demonstrating the possibility of a fully integrated photovoltaic retinal prosthesis with high pixel density.

  16. Prolonged Inner Retinal Photoreception Depends on the Visual Retinoid Cycle

    PubMed Central

    Zhao, Xiwu; Pack, Weston; Khan, Naheed W.

    2016-01-01

    In addition to rods and cones, mammals have inner retinal photoreceptors called intrinsically photosensitive retinal ganglion cells (ipRGCs), which use the photopigment melanopsin and mediate nonimage-forming visual responses, such as pupil reflexes and circadian entrainment. After photic activation, photopigments must be reverted to their dark state to be light-sensitive again. For rods and to some extent cones, photopigment regeneration depends on the retinoid cycle in the adjacent retinal pigment epithelium (RPE). By contrast, ipRGCs are far from the RPE, and previous work suggests that melanopsin is capable of light-dependent self-regeneration. Here, we used in vitro ipRGC recording and in vivo pupillometry to show that the RPE is required for normal melanopsin-based responses to prolonged light, especially at high stimulus intensities. Melanopsin-based photoresponses of rat ipRGCs were remarkably sustained when a functional RPE was attached to the retina, but became far more transient if the RPE was removed, or if the retinoid cycle was inhibited, or when Müller glia were poisoned. Similarly, retinoid cycle inhibition markedly reduced the steady-state amplitude of melanopsin-driven pupil reflexes in both mice and rats. However, melanopsin photoresponses in RPE-separated rat retinas became more sustained in the presence of an 11-cis-retinal analog. In conclusion, during prolonged illumination, melanopsin regeneration depends partly on 11-cis-retinal from the RPE, possibly imported via Müller cells. Implications for RPE-related eye diseases and the acne drug isotretinoin (a retinoid cycle inhibitor) are discussed. SIGNIFICANCE STATEMENT Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and drive subconscious physiological responses to light, e.g., pupillary constriction and neuroendocrine regulation. In darkness, each photopigment molecule in ipRGCs, as well as rod/cone photoreceptors, contains 11-cis-retinal (a

  17. Specific inhibition of TRPV4 enhances retinal ganglion cell survival in adult porcine retinal explants.

    PubMed

    Taylor, Linnéa; Arnér, Karin; Ghosh, Fredrik

    2017-01-01

    Signaling through the polymodal cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) has been implicated in retinal neuronal degeneration. To further outline the involvement of this channel in this process, we here explore modulation of Transient Receptor Potential Vanilloid 4 (TRPV4) activity on neuronal health and glial activation in an in vitro model of retinal degeneration. For this purpose, adult porcine retinal explants were cultured using a previously established standard protocol for up to 5 days with specific TRPV4 agonist GSK1016790A (GSK), or specific antagonist RN-1734, or culture medium only. Glial and neuronal cell health were evaluated by a battery of immunohistochemical markers, as well as morphological staining. Specific inhibition of TRPV4 by RN-1734 significantly enhanced ganglion cell survival, improved the maintenance of the retinal laminar architecture, reduced apoptotic cell death and attenuated the gliotic response as well as preserved the expression of TRPV4 in the plexiform layers and ganglion cells. In contrast, culture controls, as well as specimens treated with GSK, displayed rapid remodeling and neurodegeneration as well as a downregulation of TRPV4 and the Müller cell homeostatic mediator glutamine synthetase. Our results indicate that TRPV4 signaling is an important contributor to the retinal degeneration in this model, affecting neuronal cell health and glial homeostasis. The finding that pharmacological inhibition of the receptor significantly attenuates neuronal degeneration and gliosis in vitro, suggests that TRPV4 signaling may be an interesting pharmaceutical target to explore for treatment of retinal degenerative disease.

  18. Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model.

    PubMed

    Hidaka, Saburo; Okamoto, Yoshizo; Uchiyama, Satoshi; Nakatsuma, Akira; Hashimoto, Ken; Ohnishi, S Tsuyoshi; Yamaguchi, Masayoshi

    2006-09-01

    Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17beta-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17beta-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5-2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17beta-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  19. Neurotoxicity evaluation of three root canal sealers on cultured rat trigeminal ganglion neurons

    PubMed Central

    Ayar, Ahmet; Kalkan, Omer-Faruk; Canpolat, Sinan; Tasdemir, Tamer; Ozan, Ulku

    2017-01-01

    Background The aim of this study was to investigate the possible neurotoxic effects of 3 root canal sealers (RCSs) (AH Plus, GuttaFlow, iRoot SP) on cultured rat trigeminal ganglion (TG) neurons. Material and Methods Primary cultures of TG neurons were obtained from 1 to 2-day old rats. Freshly mixed RCSs were incubated in sterile phosphate buffered saline and cells were incubated with supernatants of the RCSs for different time intervals (1-, 3-, 6- and 24-h; 1 or 1/10 diluted) and viability/cytotoxicity was tested by counting the number of live cells. Pair of dishes with cells from the same culture incubated with only culture medium was considered as negative controls. Cell images were captured and acquired at x200 magnification using a microscope equipped with a camera using special image program. The viable cells were manually counted assigned from the images for each dose and incubation duration. Data was analysed by using 1-way analysis of variance with Tukey post hoc tests. Results There was no significant change in cell viability after short duration of incubation (1- and 3-h) with the supernatant of any of RCSs, except for undiluted-AH Plus at 3-h. When AH Plus was compared with other RCSs, for diluted supernatants, there was only significant difference between iRoot SP and AH Plus at 24-h (P<0.05). Whereas undiluted-AH Plus was significantly more cytotoxic for 3-, 6- and 24-h periods as compared to respective incubation periods of undiluted other groups (P<0.05). GuttaFlow groups had similar neurotoxic effect on cells for all test periods. Conclusions All tested RCSs exhibited a variable degree of neurotoxicity on these primary sensory neurons of orofacial tissues, depending on their chemical compositions. GuttaFlow and iRoot SP evoked a less toxic response to TG cells than AH Plus. Key words:Neurotoxicity, trigeminal ganglia, cell culture, root canal sealer, AH Plus, GuttaFlow, iRoot SP. PMID:28149460

  20. Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture.

    PubMed Central

    Bhattacharyya, M H; Whelton, B D; Stern, P H; Peterson, D P

    1988-01-01

    Loss of bone mineral after ovariectomy was studied in mice exposed to dietary cadmium at 0.25, 5, or 50 ppm. Results show that dietary cadmium at 50 ppm increased bone mineral loss to a significantly greater extent in ovariectomized mice than in sham-operated controls. These results were obtained from two studies, one in which skeletal calcium content was determined 6 months after ovariectomy and a second in which 45Ca release from 45Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 45Ca-prelabeled fetal rat limb bones in culture demonstrated that Cd at 10 nM in the medium, a concentration estimated to be in the plasma of mice exposed to 50 ppm dietary Cd, strikingly increased bone resorption, from 27 +/- 2% (mean +/- SEM) 45Ca release in cultures with no added cadmium to 68 +/- 6% release in cultures containing cadmium (n = 4). These in vitro results indicate that cadmium may enhance bone mineral loss by a direct action on bone. Results of the in vivo studies are consistent with a significant role of cadmium in the etiology of Itai-Itai disease among postmenopausal women in Japan and may in part explain the increased risk of postmenopausal osteoporosis among women who smoke. Images PMID:3186759

  1. Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Moriarity, D. M.; Campbell, P. S.

    1993-01-01

    During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

  2. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    SciTech Connect

    Brouwer, A.; Knook, D.L.

    1982-10-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA /sup 125/I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA /sup 125/I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA /sup 125/I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA /sup 125/I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA /sup 125/I. The intracellular degradation of CA /sup 125/I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA /sup 125/I occurred within the Kupffer cell lysosomes.

  3. Impact of an autologous oxygenating matrix culture system on rat islet transplantation outcome.

    PubMed

    Schaschkow, A; Mura, C; Bietiger, W; Peronet, C; Langlois, A; Bodin, F; Dissaux, C; Bruant-Rodier, C; Pinget, M; Jeandidier, N; Juszczak, M T; Sigrist, S; Maillard, E

    2015-06-01

    Disruption of the pancreatic islet environment combined with the decrease in oxygen supply that occurs during isolation leads to poor islet survival. The aim of this study was to validate the benefit of using a plasma-based scaffold supplemented with perfluorodecalin to improve islet transplantation outcome. Rat islets were cultured in three conditions: i) control group, ii) plasma based-matrix (P-matrix), and iii) P-matrix supplemented with emulsified perfluorodecalin. After 24 h culture, matrix/cell contacts (Integrinβ1, p-FAK/FAK, p-Akt/Akt), survival (caspase 3, TUNEL, FDA/PI), function, and HIF-1α translocation were assessed. Afterwards, P-matrices were dissolved and the islets were intraportally transplanted. Graft function was monitored for 31 days with glycaemia and C-peptide follow up. Inflammation was assessed by histology (macrophage and granulocyte staining) and thrombin/anti-thrombin complex measurement. Islet survival correlated with an increase in integrin, FAK, and Akt activation in P-matrices and function was maintained. Perfluorodecalin supplementation decreased translocation of HIF-1α in the nucleus and post-transplantation islet structure was better preserved in P-matrices, but a quicker activation of IBMIR resulted in early loss of graft function. "Oxygenating" P-matrices provided a real benefit to islet survival and resistance in vivo. However, intraportal transplantation is not suitable for this kind of culture due to IBMIR; thus, alternative sites must be explored.

  4. Progressive outer retinal necrosis-like retinitis in immunocompetent hosts.

    PubMed

    Chawla, Rohan; Tripathy, Koushik; Gogia, Varun; Venkatesh, Pradeep

    2016-08-10

    We describe two young immunocompetent women presenting with bilateral retinitis with outer retinal necrosis involving posterior pole with centrifugal spread and multifocal lesions simulating progressive outer retinal necrosis (PORN) like retinitis. Serology was negative for HIV and CD4 counts were normal; however, both women were on oral steroids at presentation for suspected autoimmune chorioretinitis. The retinitis in both eyes responded well to oral valaciclovir therapy. However, the eye with the more fulminant involvement developed retinal detachment with a loss of vision. Retinal atrophy was seen in the less involved eye with preservation of vision. Through these cases, we aim to describe a unique evolution of PORN-like retinitis in immunocompetent women, which was probably aggravated by a short-term immunosuppression secondary to oral steroids.

  5. Photovoltaic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Loudin, James; Mathieson, Keith; Kamins, Ted; Wang, Lele; Galambos, Ludwig; Huie, Philip; Sher, Alexander; Harris, James; Palanker, Daniel

    2011-03-01

    Electronic