Prevention of retinal ganglion cell swelling by systemic brimonidine in a rat experimental glaucoma model

Background: The objective of this study was to evaluate the neuroprotective effect of brimonidine on retinal ganglion cells in rats with elevated intraocular pressure and to characterize the subpopulation of cells that can be rescued, as well as assess the effect of this drug on retinal ganglion cell soma size. Methods: Episcleral vein cauterization was used to increase intraocular pressure for 5 weeks on left eyes, considering right eyes as intrinsic controls in all cases. All the animals were then given weekly intraperitoneal injections, the experimental group receiving brimonidine, and the control group were administered only phosphate‐buffered saline. Surviving retinal ganglion cells were quantified and their area and distribution measured by retrograde labelling with fluorogold. Results: Brimonidine administered systemically has a neuroprotective effect on retinal ganglion cells, which is unrelated to its capacity to lower intraocular pressure. It prevents the increase of cell size that is associated with stages prior to cell death. This phenomenon is particularly evident in the zones of the retina most susceptible to the damage caused by glaucoma (middle and periphery). Conclusion: This effect of preventing retinal ganglion cell swelling can be considered as a new marker to study neuroprotection from antiglaucomatous drugs in the early stages of neurodegeneration in glaucoma.     

Resveratrol prevents hypoxia-induced retinal ganglion cell death related with ErbB2

AIM: To confirm the changes in proteins related with hypoxia-induced retinal cell death and to assess the effects of resveratrol (Res).METHODS: The therapeutic effect of Res was verified using an ischemic/reperfusion (I/R) model in vivo and a hypoxia modelin retinal ganglion cells (RGCs) in vitro. Death of RGCs were confirmed by TUNEL assay. Protein expression was confirmed by Western blotting and immunohistochemistry. In addition, flow cytometric analysis was used to confirm the response in the cell unit to obtain more accurate data.RESULTS: ErbB2 expression and apoptosis in the ganglion cell layer (GCL) increased after I/R injury. Treatment of Res rescued I/R-induced ganglion cell death, downregulated apoptosis and ErbB2 protein expression in the retina. In subsequent in vitro models, Res affects apoptosis by regulating the phosphorylation and expression of mouse double minute 2 homolog (MDM2), along with those of ErbB2. These results suggest that Res reverses GCL-specific apoptosis via downregulation of ErbB2 in ischemic injury.CONCLUSION: In light of Res favorable properties, it should be evaluated in the treatment of RGC death and related retinal disease characterized by ErbB2 and MDM2 expression. Therefore, Res is appropriate therapeutic agent for treating ischemic injury-related eye diseases by targeting the expression of ErbB2 and MDM2.     

Neuroprotective effects of prostaglandin analogues on retinal ganglion cell death independent of intraocular pressure reduction

Prostaglandin (PG) analogues may have an additional effect to protect neurons independent of IOP reduction. Only a few reports indicated that some PG analogues had neuroprotective effects or increased blood flow in in vivo and in vitro models. However, there is no comparative study using all clinically available PG analogues and also using primary culture of retinal ganglion cell (RGC). Our purpose of study is to investigate the direct neuroprotective effect of PG analogues on glutamate- and hypoxia-induced RGC death using rat purified primary RGC culture with latanoprost acid, travoprost acid, bimatoprost acid, bimatoprost, tafluprost acid, unoprostone, and PGF2α. Purified RGCs cultures were obtained from retinas of 6 days old Wistar rats, following a two-step immuno-panning procedure. After 72 h of cultivation, the neuroprotective effect of PG analogues (1 nM, 10 nM and 100 nM) was investigated by culturing the RGCs in 25 μM glutamate for a further 72 h or 5% O2 hypoxic condition for 24 h. The RGC viability under each condition normalized to that under normal condition without stress was evaluated as live cell percentage based on a total of 15 repeated experiments. As a result, 100 nM of latanoprost acid, tafluprost acid, bimatoprost acid, and bimatoprost significantly increased RGC survival rate by suppressing apoptosis. PG analogues indicated IOP independent neuroprotective effect on glutamate- or hypoxia-induced RGC death using rat primary RGC culture at clinically available intracameral concentration. Since those profiles were different from clinical efficacy in IOP reduction, the mechanism of neuroprotection may be not related to FP receptor stimulation.     

Disruption of the complement cascade delays retinal ganglion cell death following retinal ischemia-reperfusion

Recent reports have indicated that components of the complement cascade are synthesized during the degeneration of retinal ganglion cells (RGC) in glaucoma. While complement deposition in the retina may simply serve to aid phagocytosis of damaged RGC, activation of the complement cascade can also contribute to neuronal loss in neurodegenerative diseases. This study was designed to determine if disruption of the complement cascade affects RGC survival in a murine model of retinal ischemia-reperfusion (I/R) injury. We induced retinal ischemia in the eyes of normal mice and mice with a targeted disruption of the complement component 3 (C3) gene. Tissue was harvested 7 and 21 days after induction of I/R and retinal complement synthesis was determined by quantitative PCR and immunohistochemical methods. RGC death and associated axon loss was evaluated through histological examination of the optic nerve and retina. Our data show that retinal I/R induces the expression and deposition of complement components. C3 deficient mice clearly exhibited reduced optic nerve damage and substantial preservation of RGC 1 week after I/R when compared to normal animals (p = 0.005). Three weeks after the ischemic event C3 deficient mice retained more RGC cell bodies although the degree of optic nerve damage was similar between both groups. These findings demonstrate that inhibition of the complement cascade delays optic nerve axonal and RGC degeneration in retinal I/R. It appears that injured RGC are targeted and actively destroyed through complement mediated processes. These results may have implications for the pathophysiology and clinical management of ischemic retinal conditions.     

Mouse models of retinal ganglion cell death and glaucoma

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.     

GABA A receptors are associated with retinal ganglion cell death induced by oxidative stress

A previously proteomic study from our laboratory showed that the expression of the protein of the GABA A receptor beta1 subunit was significantly increased in the retina of EAAC1-deficient (EAAC1-/-) mice, a mouse model of glaucoma. The purpose of this study was to investigate the role played by GABA A receptor beta1 subunit in the death of retinal ganglion cells (RGCs) caused by oxidative stress. The retinal sites and expression pattern of GABA A receptor beta1 subunit were determined by immunohistochemistry in the retina of ICR mice. A RGC line, RGC-5, was exposed to GABA A receptor agonist and antagonist, and the cell viability was determined by the MTS assay. The effect of GABA A receptor antagonist on the death of RGCs, and the activation of oxidative stress signaling induced by hydrogen peroxide was investigated. Our results showed that the GABA A receptor beta1 subunit was expressed on the RGCs of ICR mice. GABA A receptor agonist induced RGCs death, and this death was inhibited by bicuculline, a GABA A receptor antagonist. The hydrogen peroxide-induced death of RGCs was reduced by GABA A receptor antagonist, and the oxidative stress signaling activated by hydrogen peroxide was also inhibited. These results indicate that GABA A receptors are expressed on RGCs and may play a role in the death of RGCs induced by oxidative stress.     

Stimulation of the P2X7 receptor kills rat retinal ganglion cells in vivo

The P2X₇ receptor is associated with the death of many cell types, and growing evidence supports its presence on neurons. Activation of the P2X₇ receptor on isolated retinal ganglion cells increases intracellular calcium levels and can kill the cells. Within the intact eye, however, glia and other cell types surrounding the ganglion cells may provide protection and attenuate the effects of receptor stimulation. This investigation thus asks whether stimulation of the P2X₇ receptor can actually kill retinal ganglion cells in vivo. Drugs were injected intravitreally into the superior/nasal region of Long Evans rats. Cell survival was determined by counting the number of remaining ganglion cells labeled with aminostilbamidine. The P2X₇ receptor agonist BzATP reduced ganglion cell survival as compared to eyes injected with saline solution. Ganglion cell death was inhibited by co-injection of the P2X₇ antagonists Brilliant Blue G and MRS 2540. The loss of ganglion cells following activation of the P2X₇ receptor was also prevented by the adenosine A₃ adenosine receptor agonist MRS 3558. In conclusion, stimulation of the P2X₇ receptor can kill retinal ganglion cells in vivo. The neuroprotective effects of A₃ receptor activation identified in isolated ganglion cells are also apparent in vivo. This implies that the balance between extracellular ATP and its protective metabolite adenosine can influence ganglion cell survival in the living eye.     

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.     

NgR在糖皮质激素诱导视网膜神经节细胞凋亡中的作用

目的:探讨糖皮质激素对视网膜神经节细胞(retinal ganglion cell,RGC)凋亡的影响及机制.方法:分4组培养RGC,即(1)对照组,(2)激素组(0.1 μmol/L可的松),(3)激素-siNgR组(0.1μmol/L可的松+NgR反义核苷酸病毒),(4)激素-scRNA对照组(0.1μmol/L可的松+阴性核苷酸病毒).3d后四甲基噻唑蓝(Thiazolyl blue tetrazolium bromide,MTT)检测细胞活力变化,倒置显微镜观察细胞形态学变化,Hoechst 33342染色检测细胞凋亡,Western blot检测Nogo受体(Nogo receptor,NgR)表达.结果:对照组、激素组、激素-scRNA组及激素-siNgR组细胞活力分别为(100.0±0.0)％,(76.3±6.8)％,(79.4±9.0)％及(96.7±9.8)％,与对照组相比,激素组及激素-scRNA组细胞活力明显降低,细胞密度降低,体积缩小,NgR表达增加(P＜0.01),而激素-siNgR组无明显变化(P＞0.05).Hoechst 33342染色显示,对照组及激素-siNgR组细胞淡蓝色,激素组及激素-scRNA组可见大量呈亮蓝色的凋亡细胞.结论:糖皮质激素能通过NgR表达增加诱导RGC凋亡.     

Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study

The purpose of this study was to investigate the dose-response effects of topically administered brimonidine (BMD) on retinal ganglion cell (RGC) survival, short and long periods of time after transient retinal ischemia. In adult Sprague-Dawley rats, RGCs were retrogradely labeled with the fluorescent tracer fluorogold (FG) applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 90 min. One hr prior to retinal ischemia, two 5 microl drops of saline alone or saline containing 0.0001, 0.001, 0.01 or 0.1% BMD were instilled on the left eye. Rats were processed 7, 14 or 21 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 12 standard regions of each retina. The following have been found. (1) Seven days after 90 min of transient ischemia there is loss of approximately 46% of the RGC population. (2) topical pre-treatment with BMD prevents ischemia-induced RGC death in a dose-dependent manner. Administration of 0.0001% BMD resulted in the loss of approximately 37% of the RGC population and had no significant neuroprotective effects. Administration of higher concentrations of BMD (0.001 or 0.01%) resulted in the survival of 76 or 90%, respectively, of the RGC population, and 0.1% BMD fully prevented RGC death in the first 7 days after ischemia. (3) Between 7 and 21 days after ischemia there was an additional slow cell loss of approximately 25% of the RGC population. Pre-treatment with 0.1% BMD also reduced significantly this slow cell death. These results indicate that the neuroprotective effects of BMD, when administered topically, are dose-dependent and that the 0.1% concentration achieves optimal neuroprotective effects against the early loss of RGCs. Furthermore, this concentration is also effective to diminish the protracted loss of RGCs that occurs with time after transient ischemia.     

P2X7受体对缺氧诱发小鼠视网膜神经节细胞凋亡的影响

目的：：探讨嘌呤受体P2X7对缺氧诱发小鼠视网膜神经节细胞凋亡的影响。方法：以小鼠视网膜神经节细胞株RGC-5为研究对象,按照不同处理因素将细胞随机分为4组：正常对照组( G1)、缺氧组( G2)、缺氧+激动剂( BzATP )组( G3)、缺氧+拮抗剂(BBG)组(G4);采用四甲基偶氮唑蓝(MTT)法检测细胞的存活率;用Annxin V/PI染色流式细胞术检测细胞凋亡率;Western Blot检测细胞内cleave-caspase-3和cleave-PARP蛋白的表达。结果：与正常对照组相比, RGC-5细胞经缺氧处理后,细胞存活率明显降低;凋亡率显著升高;细胞内 cleave-caspase-3和cleave-PARP蛋白表达增加;P2X7受体激动剂BzATP能明显加重缺氧诱发的细胞凋亡,而BBG预处理可以显著拮抗缺氧所致的细胞凋亡。结论：缺氧能激活视网膜神经节细胞嘌呤受体P2X7,并参与视网膜神经节细胞的凋亡。     

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

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 100 mmHg for 1 h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h and 5d after injury. Hydrogen peroxide (H₂O₂), 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 H₂O₂, 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 relevant for the neuroprotection of inner retina from I/R-related diseases.     

Three experimental glaucoma models in rats: comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death

Glaucoma is a chronic and progressive optic nerve neuropathy involving the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is considered to be the major risk factor associated with the development of this neuropathy. The objective of the present study was to compare the effects on RGC survival of three different experimental methods to induce chronic elevation of IOP in rats. These methods were: (i) injections of latex microspheres into the eye anterior chamber; (ii) injections into the anterior chamber of a mixture of microspheres plus hydroxypropylmethylcellulose (HPM) and (iii) cauterization of three episcleral veins. The IOP of right (control) and left (glaucomatous) eyes was measured with an applanation tonometer in awake animals. Thirteen to 30 weeks later, RGCs were retrogradely labeled with 3% fluorogold. Subsequently, we analyzed the density of RGCs, as well as the major axis length and area of RGC soma resulting from the application of each method. A significant increase in IOP was found following application of each of the three methods. Cell death was evident in the glaucomatous eyes as compared to controls. However, no statistical differences were found between the extent of cell death associated with each of the three methods. IOP increase also induced a significant increase in the size of the soma of the remaining RGCs. In conclusion, the three methods used to increase IOP induce a similar degree of RGC death. Moreover, the extent of cell death was similar when the retinas were maintained under conditions of elevated IOP for 24 weeks in comparison to 13 weeks.     

Rabbit retinal ganglion cell survival after optic nerve section and its effect on the inner plexiform layer

Structural modifications of the inner retina were studied after optic nerve section (ONS) in the rabbit. Retinal ganglion cells (RGC) were labelled by injection of Fast Blue into the optic nerve, and counted under fluorescent light in control retina and retina 7, 14, 21 and 26 days post-axotomy. Studies on retinal cross-sections were also performed. For this purpose, retinal sections were stained with haematoxylin-eosin and immunohystochemistry for alpha1 and beta2/beta3 sub-units of the GABA(A) receptors.One week after axotomy, there was no significant loss in the number of ganglion cells with respect to control counts (1086+/-173cellsmm(-2) in the visual streak and 119+/-46cellsmm(-2) in the periphery, mean+/-SD, n=5). At 14 days post-axotomy, 271+/-46cellsmm(-2) remained in the visual streak and 33+/-6cellsmm(-2) in the periphery, corresponding to a mean survival of 27%. The number of ganglion cells decreased further on the following days, reaching 7.54% 1 month after ONS. A significant reduction in the thickness of the inner plexiform and ganglion cell layers was also observed in retinal cross-sections. Immunocytochemical studies show a remarkable disorganization of the layer stratification in the inner plexiform layer (IPL). We conclude that after ONS, RGC death occurs mainly between 7 and 14 days post-axotomy and a progressive death up to 26 days, causing a decrease in the thickness of the IPL and subsequent disorganization of its layers.     

促红细胞生成素对大鼠急性缺血视网膜节细胞的保护作用

目的:探讨重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对大鼠视网膜缺血再灌注损伤(retina ischemia reperfusion injury,RIR)中视网膜神经节细胞(retinal ganglion cell,RGC)的保护作用。方法:成年雌性SD大鼠20只,采用夹闭视网膜动脉30min造成大鼠双眼缺血再灌注模型。所有大鼠均于建模前1h给予左眼rhEPO10U(6μL),右眼给予同等剂量眼用平衡盐液。按照建模后眼球取材时间不同(1,4,7,14d)分为4组,每组5只,均于取材前4d利用荧光金(fluorogold,FG)逆行标记大鼠RGC,视网膜铺片RGC计数,比较双眼存活RGC数量。结果:rhEPO治疗眼RGC存活数多于平衡盐液对照眼。结论:rhEPO对大鼠视网膜急性缺血后RGC具有保护作用。     

Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant

Glaucoma is a progressive optic neuropathy with characteristic optic disc changes, retinal ganglion cell loss and progressive visual field defects. Elevated intraocular pressure is considered to be a major risk factor in glaucomatous neuropathy. This study aimed to characterize and document a new chronic glaucoma model in the rat with respect to the effect of elevated intraocular pressure on overall retinal dysfunction and retinal ganglion cell loss, and to elucidate the possible mechanisms underlying this cell loss. Intraocular pressure (IOP) was measured in rats using a Tonopen. RGCs were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10 ASP) and quantified on retinal flat mounts using fluorescence microscopy. The optic nerve head was examined fundoscopically. Changes in the histological appearance of the whole eyes was studied in paraffin sections, and immunohistochemistry was carried out on cryostat sections. The levels of mRNA for several genes were compared between control and glaucomatous retinae using semi-quantitative RT-PCR. Mutant animals are affected with either a unilateral or bilateral enlargement of the globes having an IOP that ranged from 25 to 45 mmHg, as compared to control values of 12-16 mmHg. The IOP of glaucomatous eyes increased significantly with age to attain a value of 35+/-7.3 at 1.5 years. Concomitant with the rise in IOP, the number of labeled RGCs continued to decrease in number with age. A total of 1887+/-117RGC mm(-2) could be labeled in wild-type control and juvenile mutant pre-glaucomatous retinas, whereas this number dropped to 92+/-26RGC mm(-2) at 1.5 years. Ophthalmoscopy revealed atrophied optic nerve heads in the affected eyes. The pars plicata and the pars plana of the ciliary body of glaucomatous eyes were hypertrophied and elongated, respectively. The anterior chamber was narrow and the irido-corneal angle open in glaucoma eyes. The mRNA of glial-fibrillary-acidic protein, endothelin-1, STAT-3 and STAT-6 increased in the retinas correlating with the severity and duration of the disease. Changes in the expression of GFAP and endothelin-1 could be confirmed using immunohistochemistry. This model may help to address several fundamental issues in the pathogenesis of glaucoma and aid in the development of neuroprotective strategies.     

From ocular hypertension to ganglion cell death: a theoretical sequence of events leading to glaucoma

Even with exhaustive investigation, however, there is still little understanding of the pathologic events that translate increased IOP into the process of retinal ganglion cell death. New studies, particularly in rat and mouse models of glaucoma, have helped elucidate some of the important events associated with the initiation of glaucoma. This review summarizes a 5-stage series hypothesizing that elevated IOP causes deleterious changes to glia in the optic nerve head (stage 1), which activate the autonomous self-destruction of ganglion cell axons (stage 2), leading to the loss of neurotrophic support and apoptotic death of ganglion cell somas in the retina (stage 3). In the initial wave of ganglion cell death, dying cells may adversely affect their neighbouring cells in a wave of secondary degeneration involving glutamate exposure (stage 4). As ganglion cell structures disappear through the processes of cell death, glia are again involved, but this time to replace the lost neural tissue with a glial scar (stage 5).