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

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 12 weeks 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.     

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.     

α-硫辛酸对青光眼模型大鼠视网膜神经节细胞的保护作用及其机制

目的　探讨α-硫辛酸对青光眼模型大鼠视网膜神经节细胞（RGC）的保护作用及其机制。方法　选取SPF 级雄性SD大鼠 60只,取30只大鼠,选右眼为实验眼,采用烙闭表层巩膜静脉的方法制作大鼠青光眼模型。将造模成功的大鼠随机分为两组:青光眼对照组和青光眼硫辛酸组,每组各15眼。另取30只大鼠,右眼行假手术操作,术后随机分为两组:正常眼压对照组和正常眼压硫辛酸组,每组各15眼。于造模第3周末,正常眼压硫辛酸组和青光眼硫辛酸组大鼠腹腔注射α-硫辛酸150 mg·kg^-1。分别于造模前、造模后第1周末、第2周末、第3周末、第4周末、第5周末、第6周末应用手持式眼压计测量并记录大鼠双眼眼压。分别用相应测试盒检测丙二醛(MDA)和活性氧簇(ROS)含量,超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶（GSH-Px）活性。通过TUNEL法检测RGC凋亡情况,通过免疫组织化学法检测视网膜Caspase-3 蛋白表达情况。应用GraphPadPrism6软件作图,采用SPSS 20.0统计软件分析所有数据。结果　造模后3周末开始腹腔注射α-硫辛酸,青光眼硫辛酸组大鼠右眼眼压在造模后4周、5周、6周与青光眼对照组比较,差异均无统计学意义（均为P＞0.05）。MDA和ROS含量:四组间比较差异均有统计学意义（均为P＜0.05）,均为正常眼压硫辛酸组＜正常眼压对照组＜青光眼硫辛酸组＜青光眼对照组（均为P＜0.05）。GSH-Px及SOD酶活性:四组间比较差异均有统计学意义（均为P＜0.05）,均为正常眼压硫辛酸组＞正常眼压对照组＞青光眼硫辛酸组＞青光眼对照组（均为P＜0.05）。青光眼硫辛酸组和青光眼对照组的凋亡细胞数明显高于正常眼压硫辛酸组和正常眼压对照组。正常眼压硫辛酸组RGC 凋亡指数和正常眼压对照组差异无统计学意义（P>0.05）,但均小于青光眼硫辛酸组和青光眼对照组（均为P＜0.05）;青光眼硫辛酸组RGC 凋亡指数小于青光眼对照组（P＜0.05）。青光眼硫辛酸组和青光眼对照组Caspase-3蛋白表达明显高于正常眼压硫辛酸组和正常眼压对照组。青光眼硫辛酸组Caspase-3蛋白平均光密度值小于青光眼对照组（P＜0.05）。结论　α-硫辛酸通过抗氧化作用降低Caspase-3的表达,抑制RGC凋亡,对青光眼模型大鼠RGC具有一定的保护作用。     

Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death

PURPOSE: To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. METHODS: RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. RESULTS: Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an alpha(2)-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an alpha(2)-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death. CONCLUSIONS: Pretreatment and early posttreatment with an alpha(2)-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.     

Number of ganglion cells in glaucoma eyes compared with threshold visual field tests in the same persons

PURPOSE: To compare the number of retinal ganglion cells (RGCs) topographically mapped with specific visual field threshold test data in the same eyes among glaucoma patients. METHODS: Seventeen eyes of 13 persons with well-documented glaucoma histories and Humphrey threshold visual field tests (San Leandro, CA) were obtained from eye banks. RGC number was estimated by histologic counts of retinal sections and by counts of remaining axons in the optic nerves. The locations of the retinal samples corresponded to specific test points in the visual field. The data for glaucoma patients were compared with 17 eyes of 17 persons who were group matched for age, had no ocular history, and had normal eyes by histologic examination. RESULTS: The mean RGC loss for the entire retina averaged 10.2%, indicating that many eyes had early glaucoma damage. RGC body loss averaged 35.7% in eyes with corrected pattern SD probability less than 0.5%. When upper to lower retina RGC counts were compared with their corresponding visual field data within each eye, a 5-dB loss in sensitivity was associated with 25% RGC loss. For individual points that were abnormal at a probability less than 0.5%, the mean RGC loss was 29%. In control eyes, the loss of RGCs with age was estimated as 7205 cells per year in persons between 55 and 95 years of age. In optic nerves from glaucoma subjects, smaller axons were significantly more likely to be present than larger axons (R2 = 0.78, P<0.001). CONCLUSIONS: At least 25% to 35% RGC loss is associated with statistical abnormalities in automated visual field testing. In addition, these data corroborate previous findings that RGCs with larger diameter axons preferentially die in glaucoma.     

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.     

Neuroprotective effect of topically applied brimonidine tartrate 0.2% in endothelin-1-induced optic nerve ischaemia model

BACKGROUND: To investigate the neuroprotective effects of topically applied brimonidine tartrate 0.2% (BMD), an alpha(2)-receptor agonist, on the retinal ganglion cell (RGC) layer and inner nuclear layer (INL) of rabbit retina in endothelin-1 (ET-1)-induced optic nerve (ON) ischaemia model. METHODS: Osmotic minipumps were surgically implanted into one eye of 16 New Zealand Albino rabbits to deliver ET-1 at the constant rate of 0.5 microL/h for 2 weeks. Eyes were divided into four groups. ET-1 was given with (Group 3) and without topical BMD therapy (Group 1). Groups 2 and 4 were taken as controls. Rabbits were sacrificed at day 14. Morphological alterations, total cell number and proportion of cells undergoing apoptosis in INL and RGC layer were assessed by histopathological analysis to determine the survival of the cells of the INL and RGC layer. RESULTS: Endothelin-1 led to severe reduction of cells in both the RGC layer and INL in Group 1 (P < 0.05). In Group 3, the total cell number and the proportion of cells undergoing apoptosis in the RGC layer were comparable with the control group (Group 4), whereas the former was found to be higher and the latter was found to be lower than those recorded for Group 1. However, the total cell number in the INL was found to be lower in Group 3 compared with that of Group 4, despite topical BMD therapy (P < 0.05). CONCLUSIONS: Topically applied BMD seems to be neuroprotective and antiapoptotic in the ET-1-induced ON ischaemia model, especially for RGCs. BMD might be used as an adjuvant agent for its neuroprotective effects in hypoxic-ischaemic conditions such as diabetic retinopathy, normotensive glaucoma and other retinal vascular occlusive conditions which require further investigations.     

Heat Shock Protein 72 Protects Retinal Ganglion Cells in Rat Model of Acute Glaucoma

Purpose: To investigate whether the induction of heat shock protein (HSP)72 by heat stress (HS) or zinc (Zn2 ) administration can increase survival of retinal ganglion cells (RGC)in rat model of acute experimental glaucoma. Methods: Acute glaucoma model was made by intracameral irrigation with BSS at 102 mmHg for two hours in right eyes of male Wistar rats. Glaucoma model rats were treated with HS once a week (six rats) or intraperitoneal injection of zinc sulfate (24.6 mg/kg) every two weeks (six rats), and were referred to as HS group and zinc group, respectively. Untreated model rats served as damage group (six rats). In control groups, querc-etin (400 mg/kg) was intraperitoneally injected to inhibit the induction of heat shock proteins 6 hours before HS or zinc administration, and were referred to as HS que group (six rats) and zinc que group (six rats), respectively. Subsequent to 16 days of IOP elevation, the rats were sacrificed. Eyes were quickly enucleated, and the retinas were dissected. RGC were labeled with Nissl staining and counted under microscope. Results: The average RGC density in normal Wistar rats was (2504±181) cells/mm2. In damage group, it decreased to (2015±111) cells/mm2. The RGC densities at 1,2, and 3 mm from the center of the optic nerve head were (2716±215), (2496±168), and (2317±171) cells/mm2, respectively, for normal rats and (2211±133), (1969±154), and (1872±68) cells/mm2, respectively, for damage group. The latter was significantly lower at all locations compared with the former (P=0.027 for each, Mann-Whitney test). The average RGC densities were (2207±200) cells/mm2 for HS group, (2272±155) cells/mm2 for zinc group, (1964±188) cells/mm2 for HS que group, (2051 ±214) cells/mm2 for zinc que group and (2015±111) cells/mm2for damage group. There were significant differences in density of labeled RGCs among the five groups (P=0.040, Kruskal-Wallis test). Both HS and zinc group had higher RGC densities than damage group (P =0.036 between HS and damage group,P=0.019 between zinc and damage group,Mann-Whitney test). There was no significant difference in RGC densitiy between control groups and damage group (P=0.260 between HS que and damage group,P=0.748 between zinc que and damage group, Mann-Whitney test). Conclusions: The results demonstrated that the induction of HSP72 in RGCs by HS or zinc administration plays an important role in the survival of RGCs in rat model of acute glaucoma. A novel therapeutic approach to glaucoma through an enhanced induction of endogenous HSP72 could be possible. Eye Science 2005;21:163-168.     

高良姜素对青光眼大鼠视网膜神经节细胞的保护作用

目的 研究高良姜素对青光眼大鼠视网膜神经节细胞(RGC)的保护作用。方法 取48只SD大鼠随机分为对照组、对照+高良姜素组、高眼压组和高眼压+高良姜素组,每组12只(12眼)。对照+高良姜素组和高眼压+高良姜素组大鼠接受高良姜素滴眼液治疗,对照组和高眼压组大鼠则接受相同剂量二甲基亚砜溶剂滴眼。于造模后第0～28天监测大鼠眼压,并于造模后第28天处死大鼠,收集各组大鼠右眼球,并采用HE染色观察大鼠视网膜形态变化,采用免疫组织化学染色分析各组大鼠视网膜中GFAP、Toll样受体4(TLR4)和NOD样受体3(NLRP3)表达。从4只4 d龄大鼠视网膜中分离RGC,分为空白组、光气组和CoCl₂+高良姜素组。光气组和光气+高良姜素组RGC与200μmol·L^-1 CoCl₂一起孵育48 h,光气+高良姜素组RGC中加入20μmol·L^-1高良姜素干预48 h。收集各组细胞,采用流式细胞术和Western blot检测RGC的凋亡率及相关蛋白的相对表达情况,并采用ELISA检测空白组、光气组和光气+高良姜素...     

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.     

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.     

Neuroprotection in glaucoma - Is there a future role?

In glaucoma, the major cause of global irreversible blindness, there is an urgent need for treatment modalities that directly target the RGCs. The discovery of an alternative therapeutic approach, independent of IOP reduction, is highly sought after, due to the indirect nature and limited effectiveness of IOP lowering therapy in preventing RGC loss. Several mechanisms have been implicated in initiating the apoptotic cascade in glaucomatous retinopathy and numerous drugs have been shown to be neuroprotective in animal models of glaucoma. These mechanisms and their potential treatment include excitotoxicity, protein misfolding, mitochondrial dysfunction, oxidative stress, inflammation and neurotrophin deprivation. All of these mechanisms ultimately lead to programmed cell death with loss of RGCs. In this article we summarize the mechanisms involved in glaucomatous disease, highlight the rationale for neuroprotection in glaucoma management and review current potential neuroprotective strategies targeting RGCs from the laboratory to the clinic.     

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.     

Neuroprotective effect of systemic and/or intravitreal rosuvastatin administration in rat glaucoma model

AIM: To evaluate the neuroprotective effect of rosuvastatin, in a rat experimental glaucoma model. METHODS: Ocular hypertension was induced in right eyes of Long-Evans rats (n=30) by cauterization of three episcleral veins. Left eyes were defined as controls. Rats were divided into five groups: oral rosuvastatin, intravitreal rosuvastatin, oral+intravitreal rosuvastatin, intravitreal sham and glaucoma without intervention. Rats were sacrificed at day 14. Retinal ganglion cell (RGC) number was assessed by histopathological analysis. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) staining and the expression of glial fibrillary acidic protein (GFAP) in RGC layer was also examined. RESULTS: A significant intraocular pressure (IOP) elevation was seen (P=0.002). Elevated IOP resulted in a significant decrease in number of RGCs in group 5 (70.33±8.2 cells/mm²) when compared with controls (92.50±13.72 cells/mm²; P=0.03). The RGC number in group 1 (92.4±7.3 cells/mm²) was significantly higher than group 5 (P=0.03). The numbers of RGC in groups 2, 3 (57.3±8.2 cells/mm², 60.5±12.9 cells/mm²) were comparable with that of group 5 (P=0.18 and P=0.31). The apoptosis rates with TUNEL staining were also parallel to RGC number. Animals with experimentally induced glaucoma showed an increase in retinal GFAP immunoreactivity. CONCLUSION: Decrease in RGC loss and apoptosis suggest the neuroprotective potential of oral rosuvastatin treatment in a rat model of ocular hypertension. However intravitreal rosuvastatin showed a contrary effect and further studies are required.     

热休克蛋白72（HSP72）对大鼠青光眼模型视网膜神经节细胞和视神经的保护作用

目的　探讨热休克蛋白72（heat shock protein 72,HSP72）对大鼠青光眼模型视网膜神经节细胞（retinal ganglial cells,RGCs）和视神经的保护作用。方法　选取Wistar大鼠46只，采用随机数字表法将所有大鼠分为正常对照组（6只）和实验组（40只），实验组40只大鼠中8只不作处理（实验对照组），在制作青光眼模型后2 d，给予16只大鼠热休克反应处理（热休克反应组），16只大鼠硫酸锌腹腔注射（硫酸锌注射组）。观察及比较治疗前后各组大鼠眼压、RGCs中HSP72抗体含量、RGCs平均密度。结果　激光后3 d、7 d、14 d、28 d，各组大鼠右眼眼压均较激光前有所上升，且各组间差异均无统计学意义（均为P>0.05）。正常对照组大鼠不同时间点HSP72抗体在RGCs中无表达，激光后3 d实验组各组大鼠RGCs中HSP72抗体含量缓慢上升，7 d、14 d达高峰，此后逐渐下降至接近正常水平。激光后，实验组各组大鼠RGCs中HSP72抗体含量显著高于正常对照组，且热休克反应组RGCs中HSP72抗体含量均高于实验对照组，低于硫酸锌注射组，差异均有统计学意义（均为P<0.05）。激光后3 d、7 d、14 d、28 d实验组各组大鼠RGCs平均密度均明显低于正常对照组，且热休克反应组RGCs平均密度显著高于实验对照组和硫酸锌注射组，差异均有统计学意义（均为P<0.05）。结论　热休克蛋白反应可在青光眼模型大鼠RGCs中诱导HSP72的生成，且对青光眼性RGCs和视神经具有保护作用。     

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.     

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.     

Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma

PURPOSE: The aim of this pilot study was to evaluate the potential neuroprotective effect of an intravitreal injection of erythropoietin (EPO) on retinal ganglion cell (RGC) preservation in an episcleral vessel cautery-induced rat model of glaucoma. METHODS: The animals were randomly assigned into an unoperated control group (n = 11) and three experimental groups: episcleral vessel cautery only (EVC: n = 4), episcleral vessel cautery with intravitreal normal saline injection (EVC-NS; n = 5), and episcleral vessel cautery with intravitreal EPO treatment (EVC-EPO; n = 9). The intravitreal injections were limited to 5 mul containing either normal saline alone or 200 ng of EPO in normal saline administered immediately after the cautery procedure. RGCs were labeled retrogradely by FluoroGold neuron tracer 5 to 7 days prior to the collection of eyes at day 21 and counted in whole flat-mounted retinas with fluorescence microscopy. RESULTS: Compared to the RGC counts in retinal specimens from unoperated control rats (12,619 +/- 310), the corresponding RGC counts were significantly decreased in both the EVC (9116 +/- 273; p < 0.005) and EVC-NS (9489 +/- 293; p < 0.005) groups but not significantly decreased in the EVC-EPO (11,212 +/- 414; p = 0.051) treated retinas. CONCLUSIONS: A single intravitreal 200 ng dose of EPO appears to have a protective effect on RGC viability in an in vivo rat model of glaucoma. Further experimental studies are needed to confirm these preliminary results and to optimize the appropriate dose and frequency of EPO delivery in animal models of glaucoma.     

Topically administered timolol and dorzolamide reduce intraocular pressure and protect retinal ganglion cells in a rat experimental glaucoma model

AIMS: This study sought to elucidate the effects of timolol and dorzolamide on intraocular pressure (IOP) and retinal ganglion cell (RGC) death in an experimental model of glaucoma in rat. METHODS: Mild elevation of IOP was induced in rats by intracameral injection of India ink and subsequent laser trabecular photocoagulation. IOP was measured before the surgical procedures and weekly thereafter. Timolol (0.5%), timolol XE (0.5%), dorzolamide (1%), and artificial tears (vehicle) were topically applied daily. Retinal sections were prepared for histology to determine RGC number. RESULTS: Timolol, timolol XE, and dorzolamide induced a significant reduction in IOP (p<0.05) and counteracted the reduction in RGC number that occurred in vehicle treated glaucomatous eyes (p<0.05). The coefficient of correlation between RGC number and IOP was significant in the dorzolamide treated group (r = -0.908, p<0.005), but not in other groups (p>0.05). CONCLUSIONS: Both timolol formulation and dorzolamide reduced IOP and protected RGCs in a rat model of experimental glaucoma. It cannot be ruled out that timolol might protect RGCs by additional mechanisms other than simply lowering of IOP.     

Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma

PURPOSE: To characterize the early reaction of retinal ganglion cells (RGCs) in a rat model of glaucoma using in vivo imaging and to examine the involvement of retinal microglia in glaucomatous neuropathy. METHODS: Glaucoma was induced in adult female Sprague-Dawley rats by cauterizing two episcleral veins, which resulted in a 1.6-fold increase in intraocular pressure (IOP). Retinal ganglion cells were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10ASP) and monitored in vivo after elevation of IOP using fluorescence microscopy imaging. The number of RGCs was quantified on retinal flatmounts. Dying RGCs were surrounded by activated microglia that became visible after taking up the fluorescent debris. Immunocytochemistry was conducted to characterize further the ganglion cells and microglia. RESULTS: Cauterizing two of the four episcleral veins resulted in a consistent increase of IOP to 25.3 +/- 2.0 mm Hg, as measured with a handheld tonometer. IOP remained high for at least 3 months in glaucomatous eyes. The earliest sign of RGC death was detected in anesthetized animals 20 hours after induction of glaucoma. RGCs continued to decrease in number over time, with 40% of RGCs having degenerated after 2.5 months. Fundoscopic examination of the optic nerve head revealed cupping 2 months after induction of glaucoma. In addition, microglia were detected on retinal flatmounts as early as 72 hours after induction. Activated microglia and RGCs were also identified immunocytochemically, with an antibody against ionized calcium-binding adaptor molecule (Iba)-1 and an antibody specific to the 200-kDa subunit of the neurofilament protein, respectively. CONCLUSIONS: Occlusion of episcleral veins is a reproducible method that mimics human glaucoma, with chronically elevated IOP-induced RGC loss. This study shows that in vivo imaging permits the detection of ganglion cells in the living animal in the early stages of the disease and highlights the importance of in vivo imaging in understanding ophthalmic disorders such as glaucoma. Secondly, activation of intraretinal microglia coincides with degeneration of RGCs in glaucoma.