Molecular and cell-based approaches for neuroprotection in glaucoma

A hallmark of glaucomatous optic nerve damage is retinal ganglion cell (RGC) death. RGCs, like other central nervous system neurons, have a limited capacity to survive or regenerate an axon after injury. Strategies that prevent or slow down RGC degeneration, in combination with intraocular pressure management, may be beneficial to preserve vision in glaucoma. Recent progress in neurobiological research has led to a better understanding of the molecular pathways that regulate the survival of injured RGCs. Here we discuss a variety of experimental strategies including intraocular delivery of neuroprotective molecules, viral-mediated gene transfer, cell implants and stem cell therapies, which share the ultimate goal of promoting RGC survival after optic nerve damage. The challenge now is to assess how this wealth of knowledge can be translated into viable therapies for the treatment of glaucoma and other optic neuropathies.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.     

Autophagy in axonal degeneration in glaucomatous optic neuropathy

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.     

Neuroprotection of retinal ganglion cells with GDNF-Loaded biodegradable microspheres in experimental glaucoma

Glaucoma is the second leading cause of blindness worldwide, and also the most common optic neuropathy. The ultimate cause of vision loss in glaucoma is thought to be retinal ganglion cell (RGC) death. Neuroprotection of RGC is therefore an important goal of glaucoma therapy. Currently, glaucoma treatment relies on pharmacologic or surgical reduction of intraocular pressure (IOP). It is critical to develop treatment approaches that actively prevent the death of RGCs at risk in glaucoma. Neurotrophic factors have the ability to promote the survival and influence the growth of neurons. Neurotrophic factor deprivation has been proposed as one mechanism leading to RGC death in glaucoma. Effective neuroprotection in glaucoma likely requires the consistent availability of the active agent for prolonged periods of time. Biodegradable microspheres are especially attractive as drug delivery vehicles for a number of reasons. Sustained GDNF delivery by biodegradable microspheres offers significant neuroprotection to injured RGC in experimental glaucoma. PLGA microsphere-delivered GDNF represents an important neuroprotective strategy in the treatment of glaucomatous optic neuropathy and provides direction for further investigations of this hypothesis.     

Head-up tilt lowers IOP and improves RGC dysfunction in glaucomatous DBA/2J mice

The inbred DBA/2J (D2) mouse strain is a well established model of spontaneously elevated intraocular pressure (IOP), progressive glaucomatous loss of retinal ganglion cells (RGCs), and early damage of RGC axons at the level of optic nerve head. Pattern electroretinogram (PERG) studies have shown that surviving RGCs in mice 6-12-month-old may be dysfunctional. RGC dysfunction seems to be IOP-dependent, since it may be exacerbated by means of acute IOP elevation with head-down body tilt. Here we test the hypothesis that head-up body posture lowers IOP, resulting in improvement of PERG amplitude in aged D2 mice with glaucoma. We show that head-up body tilt induces age-independent IOP lowering whose magnitude increases with the angle of tilt. For a fixed angle (−60°) of head-up tilt, IOP progressively decreases with a time constant of about 5 min and stabilizes at a value lower by about 5-6 mm Hg compared to the baseline. Head-up tilt also results in an improvement of PERG amplitude in older D2 mice with glaucoma but not in younger D2 mice without glaucoma. Improvement of PERG amplitude in aged D2 mice upon head-up-induced IOP lowering is consistent with the idea that RGCs undergo a stage of IOP-dependent, reversible dysfunction before death. The head-up IOP/PERG protocol may represent a non-invasive way to probe the potential for recovery of RGC dysfunction in D2 mice.     

Caspase-independent component of retinal ganglion cell death, in vitro

PURPOSE: Although in vitro and in vivo models demonstrate caspase activation in retinal ganglion cells (RGCs) undergoing apoptosis, the caspase-independent component of RGC death is unclear. Identification of the precise mechanisms of cell death in these distinct neurons is essential for the development of effective neuroprotective strategies in glaucoma. Because TNF-alpha and hypoxia have been implicated in RGC death during glaucomatous optic nerve degeneration, this study was conducted to determine whether RGCs survive exposure to TNF-alpha or hypoxia in the presence of caspase inhibitor treatment, and whether mitochondrial dysfunction is involved in RGC death induced by these glaucomatous stimuli. METHODS: Primary cultures of rat RGCs were exposed to TNF-alpha or hypoxia for up to 48 hours. The temporal relationship of RGC death with the loss of mitochondrial membrane potential and the release of cell death mediators, including cytochrome c and apoptosis-inducing factor (AIF), was studied in the absence and presence of specific inhibitors of caspases. In addition, treatment with a free-radical scavenger, 4-hydroxytetramethylpiperidine-1-oxyl (tempol; 5 mM), was used in some experiments. Cell viability was assessed using calcein assay, and annexin V binding combined with propidium iodide staining was used for the distinction of apoptotic and necrotic cells. Caspase-3-like protease activity was measured using a fluorometric assay, and for the in situ detection of caspase activity, immunocytochemistry was performed with a cleavage-site-specific antibody. The time course of alterations in the mitochondrial membrane potential and the release of cell death mediators in individual cells undergoing cell death were assessed with a fluorescent tracer and subsequent immunocytochemistry. In addition, a fluorescent dye, dihydroethidium was used to assess the generation of reactive oxygen species (ROS). RESULTS: Findings of this study revealed that the loss of mitochondrial membrane potential and the release of cell death mediators accompanied RGC death induced by TNF-alpha or hypoxia. Although caspase inhibitor treatment temporarily decreased the rate of apoptosis, caspase inhibition was not adequate to block RGC death if the mitochondrial membrane potential was lost and mitochondrial mediators were released. Despite the inhibited caspase activity, survival rate was less than 70% after a 48-hour incubation with death stimuli, and both apoptotic and necrotic cells were detectable in these cultures. When combined with caspase inhibition, tempol reduced the production of ROS and provided an additional 20% increase in RGC survival. CONCLUSIONS: Based on these novel findings, RGC death induced by TNF-alpha or hypoxia involves a caspase-independent component, and reducing the free-radical generation provides additional protection of RGCs temporarily saved by caspase inhibition. Therefore, neuroprotective strategies in glaucoma should include tools to improve the ability of these neurons to survive the cytotoxic consequences of mitochondrial dysfunction.     

Gene therapy and transplantation in CNS repair: the visual system

Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.     

Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells

PURPOSE: Interest in neuroprotection for optic neuropathies is, in part, based on the assumption that retinal ganglion cells (RGCs) die, not only as a result of direct (primary) injury, but also indirectly as a result of negative effects from neighboring dying RGCs (secondary degeneration). This experiment was designed to test whether secondary RGC degeneration occurs after orbital optic nerve injury in monkeys. METHODS: The superior one third of the orbital optic nerve on one side was transected in eight cynomolgus monkeys (Macaca fascicularis). Twelve weeks after the partial transection, the number of RGC bodies in the superior and inferior halves of the retina of the experimental and control eyes and the number and diameter of axons in the optic nerve were compared by detailed histomorphometry. Vitreous was obtained for amino acid analysis. A sham operation was performed in three additional monkeys. RESULTS: Transection caused loss of 55% +/- 13% of RGC bodies in the superior retina of experimental compared with fellow control eyes (mean +/- SD, t-test, P < 0.00,001, n = 7). Inferior RGCs, not directly injured by transection, decreased by 22% +/- 10% (P = 0.002). The loss of superior optic nerve axons was 83% +/- 12% (mean +/- SD, t-test, P = 0.0008, n = 5) whereas, the inferior loss was 34% +/- 20% (P = 0.02, n = 5). Intravitreal levels of glutamate and other amino acids in eyes with transected nerves were not different from levels in control eyes 12 weeks after injury. Fundus examination, fluorescein angiography, and histologic evaluation confirmed that there was no vascular compromise to retinal tissues by the transection procedure. CONCLUSIONS: This experiment suggests that primary RGC death due to optic nerve injury is associated with secondary death of surrounding RGCs that are not directly injured.     

Role of alpha-2 adrenergic receptors in neuroprotection and glaucoma

The loss of retinal ganglion cells (RGCs) in glaucoma occurs progressively over many years. A neuroprotective drug should enhance survival of RGCs in the presence of chronic stress/injury. Four criteria are proposed for assessing the likely therapeutic utility in human glaucoma of drugs that have demonstrated neuroprotective activity in animal models: 1) A specific receptor target must be in the retina/optic nerve; 2) Activation of the target must trigger pathways that enhance a neuron's resistance to stress/injury and/or suppresses toxic insults; 3) The drug must reach the retina/vitreous at pharmacologic doses; and 4) The neuroprotective activity should be demonstrated in clinical trials. Data are presented that illustrate how the specific and potent alpha-2 agonist, brimonidine, meets these criteria. The alpha-2A receptor was localized in the inner rat retina by immunohistochemistry. Brimonidine reduced the rate of RGC loss in the calibrated rat optic nerve injury model even when dosed 12 and 24 hours before injury, consistent with a long-term enhancement of RGC resistance to stress. Brimonidine was also neuroprotective in the lasered chronic hypertensive rat model, reducing RGC loss over three weeks from 33% to 15%. A clinical trial has been initiated to determine brimonidine's neuroprotective activity in patients with non-arteritic ischemic optic neuropathy.     

Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement

Glaucoma is a progressive neurodegenerative disease of retinal ganglion cells (RGCs) associated with characteristic axon degeneration in the optic nerve. Clinically, our only method of slowing glaucomatous loss of vision is to reduce intraocular pressure (IOP), but lowering IOP is only partially effective and does not address the underlying susceptibility of RGCs to degeneration. We review the recent steps forward in our understanding of the pathophysiology of glaucoma and discuss how this understanding has given us a next generation of therapeutic targets by which to maintain RGC survival, protect or rebuild RGC connections in the retina and brain, and enhance RGC function.     

Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice

PURPOSE: Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo. METHODS: The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes. RESULTS: In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice. CONCLUSIONS: EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.     

Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat

The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5mul intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500mum concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500mum concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.     

Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma

PURPOSE: In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys. METHODS: Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus. RESULTS: With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes. CONCLUSIONS: Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.     

Immunoregulation of retinal ganglion cell fate in glaucoma

Glaucomatous neurodegeneration has been associated with the activation of multiple pathogenic mechanisms that can result in RGC death and axonal degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in the neurodegenerative process of glaucoma. The roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. It has been proposed that a critical balance between beneficial protective immunity and harmful sequelae of autoimmune neurodegenerative injury determines the ultimate fate of RGCs in response to various stressors in patients with glaucoma. Here, we review the key role for immunoregulation in cell fate decisions regarding RGC survival in response to glaucomatous tissue stress. Furthermore, we review the mechanisms by which autoimmunity to specific antigens such as heat shock proteins may result in RGC demise in some patients with glaucoma. In these patients, we hypothesized that one form of glaucoma may be an autoimmune optic neuropathy in which an individual's immune system facilitates a somatic or axonal degeneration of RGCs by the very system which normally serves to protect it against stress.     

牛磺酸保护视网膜神经节细胞的研究进展

牛磺酸是动物组织内常见的氨基酸,近年来很多研究提示其对视网膜神经节细胞具有重要保护功能,其作用机制可分为以下6种：(1)降低NMDA(N-甲基-D-天冬氨酸)诱导的兴奋毒性;(2)调节Müller细胞的功能;(3)保护线粒体功能;(4)促进视神经再生;(5)促进适应性调节;(6)改善眼底微循环。在各类眼病中,青光眼则是以视网膜神经节细胞损害为主的疾病。目前体内及体外试验证明牛磺酸可从多方面对抗视网膜神经节细胞损伤,为包括青光眼在内的多种眼科疾病的视神经保护治疗开辟了潜在途径。     

Diabetes Exacerbates the Intraocular Pressure-Independent Retinal Ganglion Cells Degeneration in the DBA/2J Model of Glaucoma

PurposeGlaucoma is a multifactorial disease, causing retinal ganglion cells (RGCs) and optic nerve degeneration. The role of diabetes as a risk factor for glaucoma has been postulated but still not unequivocally demonstrated. The purpose of this study is to clarify the effect of diabetes in the early progression of glaucomatous RGC dysfunction preceding intraocular pressure (IOP) elevation, using the DBA/2J mouse (D2) model of glaucoma.MethodsD2 mice were injected with streptozotocin (STZ) obtaining a combined model of diabetes and glaucoma (D2 + STZ). D2 and D2 + STZ mice were monitored for weight, glycemia, and IOP from 3.5 to 6 months of age. In addition, the activity of RGC and outer retina were assessed using pattern electroretinogram (PERG) and flash electroretinogram (FERG), respectively. At the end point, RGC density and astrogliosis were evaluated in flat mounted retinas. In addition, Müller cell reactivity was evaluated in retinal cross-sections. Finally, the expression of inflammation and oxidative stress markers were analyzed.ResultsIOP was not influenced by time or diabetes. In contrast, RGC activity resulted progressively decreased in the D2 group independently from IOP elevation and outer retinal dysfunction. Diabetes exacerbated RGC dysfunction, which resulted independent from variation in IOP and outer retinal activity. Diabetic retinas displayed decreased RGC density and increased glial reactivity given by an increment in oxidative stress and inflammation.ConclusionsDiabetes can act as an IOP-independent risk factor for the early progression of glaucoma promoting oxidative stress and inflammation-mediated RGC dysfunction, glial reactivity, and cellular death.     

Neuroprotective effects of cardiotrophin-like cytokine on retinal ganglion cells

BACKGROUND: Premature neuronal cell death is a feature of numerous central nervous system and eye diseases, including glaucoma. Neurons (including retinal ganglion cells, RGCs) are protected by several neurotrophic factors, among those the IL-6 family of cytokines. Lately, a novel member of the IL-6 family of cytokines has been identified and cloned. This cytokine is known as novel neurotrophin-1/B-cell-stimulating factor-3 (NNT-1/BSF-3) or cardiotrophin-like cytokine (CLC). It shows neurotrophic as well as B-cell stimulatory effects. METHODS: In this study, the neuroprotective properties of CLC on RGC loss in vivo were investigated. RESULTS: CLC significantly protected RGCs from degeneration in both chosen models of retinal neuronal damage: optic nerve crush (P<0.01) and N-methyl-D-aspartate (NMDA) injection (P<0.001). CONCLUSIONS: CLC shows neuroprotective effects on RGCs in vivo and might be a treatment option for chronic neurodegenerative eye diseases such as glaucoma. Clinical feasibility for the substance requires further investigation since the immunomodulatory and possible adverse effects have not yet been thoroughly characterized.     

Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory’s previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.     

Neuroprotective effect of sulfhydryl reduction in a rat optic nerve crush model

PURPOSE: The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy. METHODS: RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia. RESULTS: The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone. CONCLUSIONS: The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.     

Downregulation of Thy1 in retinal ganglion cells in experimental glaucoma

PURPOSE: Thy1 is a surface glycoprotein uniquely expressed in retinal ganglion cells (RGCs) in retina. The aim of this study was to investigate the expression change of Thy1 in a model of experimental glaucoma. METHODS: The change of protein and mRNA levels of Thy1 in the retina were studied using stereological counts of back-labeled RGCs, Western blot analysis, immunohistochemistry, and laser capture microdissection (LCM) of RGCs with quantitative PCR analysis of mRNA in a model of experimental glaucoma. LCM after optic nerve crush was also performed to evaluate Thy1 mRNA levels after a different injury. RESULTS: After 10 days of elevated IOP, there was a 34% loss of RGC number, Thy1 protein decreased 60% in eyes with elevated intraocular pressure (IOP), and Thy1 mRNA levels decreased 51% in RGCs. Both protein and mRNA level change of Thy1 is to a much greater extent than RGC number loss. CONCLUSIONS: The current results confirm that Thy 1 mRNA levels do not reflect the number of RGCs present and extend this to include a parallel decrease in Thy1 protein levels. These results suggest that Thy1 serves as an early marker of RGC stress, but not a marker of RGC loss, in models of retinal damage.