Retinal Ganglion Cell Loss In Glaucoma Research Articles

Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma

Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.

CGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

cGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

Interplay between M\xfcller cells and microglia aggravates retinal inflammatory response in experimental glaucoma

BackgroundGlaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown.MethodsA mouse chronic ocular hypertension (COH) experimental glaucoma model was produced. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), transwell co-culture of glial cells, flow cytometry assay, ELISA, Ca2+ image, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the interaction of Müller cells and microglia, and its underlying mechanisms in COH retina.ResultsWe first showed that Müller cell activation in mice with COH induced microglia activation through the ATP/P2X7 receptor pathway. The activation of microglia resulted in a significant increase in mRNA and protein levels of pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-6. These inflammatory factors in turn caused the up-regulation of mRNA expression of pro-inflammatory factors in Müller cells through a positive feedback manner.ConclusionsThese findings provide robust evidence, for the first time, that retinal inflammatory response may be aggravated by an interplay between activated two types of glial cells. These results also suggest that to reduce the interplay between Müller cells and microglia could be a potential effective strategy for preventing the loss of RGCs in glaucoma.

Targeted delivery of LM22A-4 by cubosomes protects retinal ganglion cells in an experimental glaucoma model

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. Statement of significanceRecent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.

Targeted Delivery of Lm22a-4 by Cubosomes Protects Retinal Ganglion Cells in an Experimental Glaucoma Model

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of glaucoma, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss in a mouse model of acute IOP elevation. These results provide evidence that cubosomes-based LM22A-4 delivery may be a useful targeted approached to prevent the progression of RGCs loss in glaucoma.

Activated ephrinA3/EphA4 forward signaling induces retinal ganglion cell apoptosis in experimental glaucoma

Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Müller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca2+-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.

The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.

Glaucoma is a leading cause of irreversible blindness worldwide. Vascular factors play a substantial role in the pathogenesis of glaucoma. Expressed in the vascular endothelium, cytochrome P450 (CYP) 2J2 is one of the CYP epoxygenases that metabolize arachidonic acid to produce epoxyeicosatrienoic acids and exert pleiotropic protective effects on the vasculature. In the present study, we investigated whether endothelium-specific overexpression of CYP2J2 (tie2-CYP2J2-Tr) protects against retinal ganglion cell (RGC) loss induced by glaucoma and in what way retinal vessels are involved in this process. We used a glaucoma model of retinal ischemia-reperfusion (I/R) injury in rats and found that endothelium-specific overexpression of CYP2J2 attenuated RGC loss induced by retinal I/R. Moreover, retinal I/R triggered retinal vascular senescence, indicated by up-regulated senescence-related proteins p53, p16, and β-galactosidase activity. The senescent endothelial cells resulted in pericyte loss and increased endothelial secretion of matrix metallopeptidase 9, which further contributed to RGC loss. CYP2J2 overexpression alleviated vascular senescence, pericyte loss, and matrix metallopeptidase 9 secretion. CYP2J2 suppressed endothelial senescence by down-regulating senescence-associated proteins p53 and p16. These 2 proteins were positively regulated by microRNA-128-3p, which was inhibited by CYP2J2. These results suggest that CYP2J2 protects against endothelial senescence and RGC loss in glaucoma, a discovery that may lead to the development of a potential treatment strategy for glaucoma.-Huang, J., Zhao, Q., Li, M., Duan, Q., Zhao, Y., Zhang, H. The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.

Hyperhomocysteinemia-induced death of retinal ganglion cells: The role of Müller glial cells and NRF2

Hyperhomocysteinemia (Hhcy), or increased levels of the excitatory amino acid homocysteine (Hcy), is implicated in glaucoma, a disease characterized by increased oxidative stress and loss of retinal ganglion cells (RGCs). Whether Hhcy is causative or merely a biomarker for RGC loss in glaucoma is unknown. Here we analyzed the role of NRF2, a master regulator of the antioxidant response, in Hhcy-induced RGC death in vivo and in vitro. By crossing Nrf2−/− mice and two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice), we generated Cbs+/-Nrf2−/− and Mthfr+/-Nrf2−/− mice and performed systematic analysis of retinal architecture and visual acuity followed by assessment of retinal morphometry and gliosis. We observed significant reduction of inner retinal layer thickness and reduced visual acuity in Hhcy mice lacking NRF2. These functional deficits were accompanied by fewer RGCs and increased gliosis. Given the key role of Müller glial cells in maintaining RGCs, we established an ex-vivo indirect co-culture system using primary RGCs and Müller cells. Hhcy-exposure decreased RGC viability, which was abrogated when cells were indirectly cultured with wildtype (WT) Müller cells, but not with Nrf2−/− Müller cells. Exposure of WT Müller cells to Hhcy yielded a robust mitochondrial and glycolytic response, which was not observed in Nrf2−/− Müller cells. Taken together, the in vivo and in vitro data suggest that deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of an intact retinal antioxidant response mechanism.

Early Gene Expression Profile in Retinal Ganglion Cell Layer After Optic Nerve Crush in Mice.

Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss. The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry. At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas. In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.

Visual light effects on mitochondria: The potential implications in relation to glaucoma

Light of different wave-lengths have the potential to interact with four major mitochondrial protein complexes that are involved in the generation of ATP. Neurones of the central nervous system have an absolute dependence on mitochondrial generated ATP. Laboratory studies show that short-wave or blue light (400–480nm) that impinges on the retina affect flavin and cytochrome constituents associated with mitochondria to decrease the rate of ATP formation, stimulate ROS and results in cell death. This suggests that blue light could potentially have a negative influence on retinal ganglion cell (RGC) mitochondria that are abundant and not shielded by macular pigments as occurs for photoreceptor mitochondria. This might be of significance in glaucoma where it is likely that RGC mitochondria are already affected and therefore be more susceptible to blue light. Thus simply filtering out some natural blue light from entering the eye might be beneficial for the treatment of glaucoma.Long-wave or red light (650–800nm) affects mitochondrial complex IV or cytochrome oxidase to increase the rate of formation of ATP and ROS causing the generation of a number of beneficial factors. Significantly, laboratory studies show that increasing the normal amount of natural red light reaching rat RGC mitochondria in situ, subjected to ischemia, proved to be beneficial. A challenge now is to test whether extra red light delivered to the human retina can slow-down RGC loss in glaucoma. Such a methodology has also the advantage of being non-invasive. One very exciting possibility might be in the production of a lens where solar UV light is convertes to add to the amount of natural red light entering the eye.

Caffeine administration prevents retinal neuroinflammation and loss of retinal ganglion cells in an animal model of glaucoma.

Glaucoma is the second leading cause of blindness worldwide, being characterized by progressive optic nerve damage and loss of retinal ganglion cells (RGCs), accompanied by increased inflammatory response involving retinal microglial cells. The etiology of glaucoma is still unknown, and despite elevated intraocular pressure (IOP) being a major risk factor, the exact mechanisms responsible for RGC degeneration remain unknown. Caffeine, which is an antagonist of adenosine receptors, is the most widely consumed psychoactive drug in the world. Several evidences suggest that caffeine can attenuate the neuroinflammatory responses and afford protection upon central nervous system (CNS) injury. We took advantage of a well characterized animal model of glaucoma to investigate whether caffeine administration controls neuroinflammation and elicits neuroprotection. Caffeine or water were administered ad libitum and ocular hypertension (OHT) was induced by laser photocoagulation of the limbal veins in Sprague Dawley rats. Herein, we show that caffeine is able to partially decrease the IOP in ocular hypertensive animals. More importantly, we found that drinking caffeine prevented retinal microglia-mediated neuroinflammatory response and attenuated the loss of RGCs in animals with ocular hypertension (OHT). This study opens the possibility that caffeine or adenosine receptor antagonists might be a therapeutic option to manage RGC loss in glaucoma.

Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration.

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies. Stem Cells 2016;34:1553-1562.

Decorin treatment for reversing trabecular meshwork fibrosis in open-angle glaucoma.

Decorin treatment for reversing trabecular meshwork fibrosis in open-angle glaucoma.

Characteristic patterns of dendritic remodeling in early-stage glaucoma: evidence from genetically identified retinal ganglion cell types.

Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1-2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

Imaging retinal ganglion cells: enabling experimental technology for clinical application.

Recent advances in clinical ophthalmic imaging have enhanced patient care. However, the ability to differentiate retinal neurons, such as retinal ganglion cells (RGCs), would advance many areas within ophthalmology, including the screening and monitoring of glaucoma and other optic neuropathies. Imaging at the single cell level would take diagnostics to the next level. Experimental methods have provided techniques and insight into imaging RGCs, however no method has yet to be translated to clinical application. This review provides an overview of the importance of non-invasive imaging of RGCs and the clinically relevant capabilities. In addition, we report on experimental data from wild-type mice that received an invivo intravitreal injection of a neuronal tracer that labelled RGCs, which in turn were monitored for up to 100 days post-injection with confocal scanning laser ophthalmoscopy. We were able to demonstrate efficient and consistent RGC labelling with this delivery method and discuss the issue of cell specificity. This type of experimental work is important in progressing towards clinically applicable methods for monitoring loss of RGCs in glaucoma and other optic neuropathies. We discuss the challenges to translating these findings to clinical application and how this method of tracking RGCs invivo could provide valuable structural and functional information to clinicians.

Calpain Activation in Experimental Glaucoma

Glaucoma is a neurodegenerative disease in which elevated intraocular pressure (IOP) leads to progressive loss of retinal ganglion cells (RGCs) and blindness. Calcium dyshomeostasis has been suggested to play a role in the pathologic events that lead to RGC loss, though the details of these events are not well understood. Calcium-induced activation of calpain has been shown to contribute to neuronal death in a wide variety of neurodegenerative diseases. The authors hypothesize that similar events occur in glaucoma. The authors used a well-established rat model of experimental glaucoma. Retinal tissues were harvested after 5 or 10 days of elevated IOP and were subjected to immunoblot analysis, immunoprecipitation, and MALDI-ProTOF/MS peptide fingerprint mapping. Immunohistochemistry was used to localize calpain activation. The authors present four independent lines of evidence that calpain is activated in experimental glaucoma. First, they showed that a 55-kDa autocatalytic active form of calpain is detected on immunoblot analysis. Second, they demonstrated the cleavage of two well-established calpain substrates, spectrin and calcineurin, only in eyes with elevated IOP. Third, they used MALDI-ProTOF to analyze cleaved calcineurin and immunoblot analysis of spectrin cleavage products and showed that both substrates were cleaved by calpain in experimental glaucoma. Fourth, they used immunohistochemistry to show that calpain-mediated spectrin cleavage occurs in RGCs under conditions of elevated IOP. These data support the hypothesis that calpain is activated under conditions of elevated intraocular pressure and provide further details of the pathologic events leading to RGC loss in glaucoma.

Modeling individual-specific human optic nerve head biomechanics. Part I: IOP-induced deformations and influence of geometry

Glaucoma, the second most common cause of blindness worldwide, is an ocular disease characterized by progressive loss of retinal ganglion cell (RGC) axons. Biomechanical factors are thought to play a central role in RGC loss, but the specific mechanism underlying this disease remains unknown. Our goal was to characterize the biomechanical environment in the optic nerve head (ONH)--the region where RGC damage occurs--in human eyes. Post mortem human eyes were imaged, fixed at either 5 or 50 mmHg pressure and processed histologically to acquire serial sections through the ONH. Three-dimensional models of the ONH region were reconstructed from these sections and embedded in a generic scleral shell to create a model of an entire eye. We used finite element simulations to quantify the effects of an acute change in intraocular pressure from 5 to 50 mmHg on the ONH biomechanical environment. Computed strains varied substantially within the ONH, with the pre-laminar neural tissue and the lamina cribrosa showing the greatest strains. The mode of strain having the largest magnitude was third principal strain (compression), reaching 12-15% in both the lamina cribrosa and the pre-laminar neural tissue. Shear strains were also substantial. The distribution of strains in all ONH tissues was remarkably similar between eyes. Inter-individual variations in ONH geometry (anatomy) have only modest effects on ONH biomechanics, and may not explain inter-individual susceptibility to elevated intraocular pressure. Consistent with previous results using generic ONH models, the displacements of the vitreo-retinal interface and the anterior surface of the lamina cribrosa can differ substantially, suggesting that currently available optical imaging methods do not provide information of the acute deformations within ONH tissues. Predicted strains within ONH tissues are potentially biologically significant and support the hypothesis that biomechanical factors contribute to the initial insult that leads to RGC loss in glaucoma.

Expression of neuronal nitric oxide synthase in the retina of a rat model of chronic glaucoma

We investigated the expression of neuronal nitric oxide synthase (nNOS) in a rat retina model of chronic glaucoma, which was produced by electrocauterization of the episcleral vessels. Western-blot analysis showed that nNOS expression was significantly increased in cauterized retinas. nNOS immunoreactivity was observed in the cells of both the inner nuclear layer and the ganglion cell layer. Double labeling of retinal ganglion cells (RGCs) revealed that RGCs in the retina of cauterized rat was nNOS-immunopositive. Systemic administration of l-NAME ( N G-nitro- l-arginine-methyl-ester), a non-specific NOS inhibitor, reduced RGC loss in cauterized rat retina, but there was no statistical significance ( P = .06). These results suggest that the cytotoxicity of excessive NO plays a role in selective RGC loss in glaucoma.

Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma

Glaucoma is one of the major neurological disorders in eye leading to irreversible blindness in elderly. Increase in intraocular pressure (IOP) has been considered to be the major risk factor for the progressive loss of retinal ganglion cells (RGCs) in retina. While attenuation of IOP has been a major pharmaceutical target, reduction of IOP cannot prevent progressive loss of RGCs. In this regard, urgent need for alternative treatment has to be investigated. Anti-aging medicinal herb Lycium barbarum L. has been used for centuries in Eastern World to protect the eyes and maintain good health. Using an ocular hypertension (OH) model in rat by laser photocoagulation of episcleral and limbal veins, we attempted to investigate whether L. barbarum can promote RGCs survival against elevated IOP. Oral administration of L. barbarum in Sprague-Dawley rats (250–280 g) significantly reduced the loss of RGCs, although elevated IOP was not significantly altered. Rats fed with the 1 mg/kg extract could nearly totally escape from pressure-induced loss of RGCs. In conclusion, this is the first in vivo report showing the therapeutic function of L. barbarum against neurodegeneration in the retina of rat OH model. The results demonstrate that this extract may be a potential candidate for the development of neuroprotective drug against the loss of RGCs in glaucoma.

Contribution to Ischemic Injury of Rat Optic Nerves by Intracellular Sodium Overload

Ischemic insult to axons of retinal ganglion cells (RGCs) is believed to contribute significantly to preferential loss of RGCs in glaucoma. In this study, we characterized the role of intracellular Na(+) overload in ischemic injury of acutely isolated rat optic nerves by evaluating electrically elicited compound action potentials (CAPs) from the optic nerves. Under control conditions, robust and stable CAPs can be recorded for more than 5 h. One hour of oxygen and glucose deprivation (OGD) that simulates ischemia, virtually eliminated the CAP. Upon returning to control conditions, the CAP gradually recovered. Maximum recovery (35% of control) was obtained by 1 h after returning to normal oxygenated Ringer. When a rapidly reversible Na(+) channel blocker, that completely blocked the CAP under control conditions, was present during OGD, the recovery of the CAP was significantly enhanced to 65% of control. When the Na(+) was replaced with either choline or Li(+) in the Ringer during OGD, CAP recovery was significantly enhanced (65-70% of control). Removing Ca(++) from the Ringer (plus 5 mM EGTA) provided even better preservation of the CAP following OGD (90% of control). Our results are consistent with the hypothesis that intracellular Na(+) overload appears to play a significant role in ischemic injury of optic nerves. This Na(+) overload may depend at least partially upon Ca(++) influx from the extracellular space.