石斛散对人视网膜神经细胞凋亡的影响

目的探讨石斛散治疗视网膜变性类疾病方面的作用及机制，为临床治疗同类疾病提供药理学依据。方法以人视网膜神经细胞为研究对象，用AnnexinV＋PI为染料标记的凋亡试剂盒，利用流式细胞仪分析药物干预后不同组别中各类凋亡细胞及活细胞的比例。结果正常情况下，对照组活细胞占61．32％，石斛散组为73．8％，两组比较有显著统计学差异。用1mmol／L的谷氨酸干预细胞2h后，活细胞比例降为44．23％，而晚期凋亡细胞和死亡细胞分别增为24．90％和21．63％。预先给予石斛散干预的中药组，活细胞明显多于对照组，其他各类细胞少于对照组。结论石斛散具有抵抗符氨酸损伤，延缓视网膜神经细胞凋亡过程的作用。     

石斛散对体外培养人视网膜细胞的作用

目的 观察中药方剂石斛散及单味药对人视网膜细胞的增生以及谷氨酸损伤的保护作用,探讨其在视网膜细胞损伤或变性类疾病方面的作用及机理,为临床治疗同类疾病提供依据.方法 体外培养人视网膜细胞,通过四甲基偶氮唑盐实验观察石斛散复方以及各单味药对视网膜细胞增生的影响.另用Rhodamine123标记培养的视网膜细胞,通过激光共聚焦扫描显微镜动态观察和记录不同药物作用下细胞的线粒体膜电位(mito-chondrial membrane potential,MMP)变化.结果 体外培养人视网膜细胞贴壁、伸展以及生长良好.四甲基偶氮唑盐实验:石斛散组1.58 g·L-1和0.50 g·L-1以及石斛组5.00 g·L-1对人视网膜细胞活性分别增加21.8%、14.2%和13.4%.MMP实验:石斛散轻度升高MMP,增加约13.5%,同时它可以抵抗谷氨酸损伤细胞后引起的MMP下降,由降低46.4%减少为16.2%.结论 石斛散能够影响人视网膜细胞线粒体功能、促进细胞活性、抵抗谷氨酸对人视网膜细胞损伤作用.可作为临床治疗视网膜变性类疾病的一种选择.     

视网膜缺血再灌注损伤中谷氨酸的兴奋毒性及其机制

视网膜缺血再灌注(retinal ischemia reperfusion,RIR)损伤是多因素综合作用的结果,兴奋性氨基酸学说是其机制之一。谷氨酸本是一种主要的神经兴奋递质,行使重要的生理功能,而RIR过程中,细胞间质内谷氨酸浓度明显升高,过度激活谷氨酸受体,引起兴奋毒性,其机制主要有钙离子超载、过多一氧化氮合成、细胞内氧自由基升高及细胞凋亡等方面。     

膳食诱导的肥胖型 C57 BL／6小鼠视网膜神经节细胞凋亡的机制

目的：探讨高脂饮食诱导的C57 BL/6肥胖小鼠视网膜神经节细胞（ RGCs）凋亡的机制。 方法：高脂饲料喂养19 wk后,小鼠分为肥胖抵抗（ DIO-R）组和肥胖倾向（ DIO）组,同时对照组（ CON）小鼠给予基础饲料。 TUNEL法检测各组小鼠RGCs的凋亡情况,并应用激光共聚焦显微镜检测RGCs内钙离子的浓度。 结果：TUNEL法凋亡检测结果显示,DIO组小鼠视网膜神经节细胞层可见较多黄色着染的凋亡细胞,其凋亡指数为（6．7±1．2）％,显著高于对照组和DIO-R组（P＜0．01, P＜0．05）;对照组和DIO-R组间比较无显著差异（ P＞0.05）。激光共聚焦结果显示,与对照组和DIO-R组比较,DIO组小鼠视网膜神经节细胞内Ca2＋荧光染色明显增强,其荧光染色强度比值显著升高（均P＜0．01）;对照组和DIO-R组视网膜神经节细胞内Ca2＋荧光染色强度无明显差异（P＞0．05）。 结论：细胞内钙离子超载可能介导了肥胖型C57 BL/6小鼠视网膜神经节细胞的凋亡过程。     

视网膜色素上皮细胞体外凋亡过程中钙离子平衡与caspase-3基因表达

目的:研究钙通道拮抗剂-维拉帕米(verapamil,Ver)诱导视网膜色素上皮(retinal pigment epithelium,RPE)细胞凋亡过程中钙离子及凋亡基因caspase-3变化。方法:应用80mg/L的Ver分别作用健康人眼RPE细胞12,24及48h诱导凋亡,设立对照组。逆转录聚合酶链反应(RT-PCR)检测凋亡基因caspase-3的表达,采用Fluo-3/AM负载技术,MetaFluo4.5/coolsnapfx/IX70细胞内钙离子荧光成像系统测定每组20个RPE细胞钙荧光值,并计算RPE细胞内钙浓度([Ca2+]i)。结果:对照组RPE细胞Ca2+荧光分布胞核最强,胞质次之。Ver作用12,24及48h后,细胞内[Ca2+]i明显降低(P<0.01)。对照组RPE细胞可见caspase-3的mRNA有少量的表达。Ver作用12h后,可见caspase-3的mRNA有较高的表达,与对照组比较,具有显著性差异(P<0.01)。随着Ver作用时间的延长,caspase-3的mRNA表达逐渐增强,在48h时有所下降。结论:Caspase-3基因表达上调及RPE细胞内钙离子稳态失调可能在Ver诱导RPE细胞凋亡中起关键作用。     

bcl-XL抗视网膜光感受器细胞凋亡作用的实验研究

目的评价抗凋亡基因bcl-XL对视网膜光感受器细胞的抗凋亡作用.方法首先建立谷氨酸损伤的SD大鼠视网膜光感受器细胞凋亡模型.将体外培养的光感受器细胞分为A组(正常对照组)、B组(谷氨酸组)及C组(rAd-gfp-bcl-XL转染+谷氨酸组);其中C组在加入谷氨酸前48 h用滴度为6.5×l012 pfu/mL的重组腺病毒rAd-gfp-bcl-XL转染光感受器细胞;荧光显微镜下观察光感受器细胞中的绿色荧光蛋白表达情况;用免疫组化法分析rAd-gfp-bcl-XL转染细胞和未转染细胞Bcl-XL蛋白水平;DNA琼脂糖凝胶电泳以评判3个组神经元凋亡发生与否及凋亡程度;采用Hoechst33258染色做正常核和凋亡核的形态学检测.结果免疫组化检测结果表明转染细胞与未转染细胞的Bcl-XL蛋白表达水平有差异,DNA电泳分析发现B组呈典型的DNA"梯度"条带,而A组和C组几乎无DNA"梯度"条带,核形态学检测结果亦证实转染组较未转染组的核有明显差异.结论重组腺病毒介导转染的bcl-XL对体外培养的视网膜光感受器细胞有抗凋亡作用,提高视网膜光感受器细胞bcl-XL的表达水平可能为视网膜变性疾病提供潜在有效的治疗方法.     

维拉帕米诱导人视网膜色素上皮细胞凋亡及细胞内钙变化

目的:探讨钙通道拮抗剂—维拉帕米(verapamil,Ver)对体外培养的人视网膜色素上皮(humanretinalpigmentepithelium,hRPE)细胞凋亡诱导作用及凋亡过程中细胞内钙浓度[Ca2 ]i的变化。方法:应用80mg/L的Ver作用体外培养hRPE细胞12,24及48h,采用吖叮橙(AO)荧光染色法、透射电镜、流式细胞术观察hRPE细胞凋亡;Fluo-3/AM负载技术,观察凋亡过程中细胞[Ca2 ]i的变化。结果:一定浓度Ver可诱导hRPE细胞凋亡,荧光显微镜及电镜观察hRPE细胞具有早期凋亡特征:核荧光呈黄绿色,电镜下核染色质浓染、边集。流式细胞术显示,Ver作用后的hRPE细胞凋亡百分率增加(F=12.3415,P<0.05)。Ver作用的hRPE细胞内钙浓度([Ca2 ]i)呈下降趋势(F=23.607,P<0.01)。结论Ver能诱导体外培养的hRPE细胞凋亡,细胞内[Ca2 ]i浓度的变化可能是诱导hRPE凋亡的机制之一。     

bcl-X_L抗视网膜光感受器细胞凋亡作用的实验研究

目的　评价抗凋亡基因bcl XL 对视网膜光感受器细胞的抗凋亡作用。方法　首先建立谷氨酸损伤的SD大鼠视网膜光感受器细胞凋亡模型。将体外培养的光感受器细胞分为A组 (正常对照组 )、B组 (谷氨酸组 )及C组 (rAd gfp bcl XL 转染 +谷氨酸组 ) ;其中C组在加入谷氨酸前 4 8h用滴度为 6 5×l0 12 pfu/mL的重组腺病毒rAd gfp bcl XL 转染光感受器细胞 ;荧光显微镜下观察光感受器细胞中的绿色荧光蛋白表达情况 ;用免疫组化法分析rAd gfp bcl XL 转染细胞和未转染细胞Bcl XL蛋白水平 ;DNA琼脂糖凝胶电泳以评判 3个组神经元凋亡发生与否及凋亡程度 ;采用Hoechst332 5 8染色做正常核和凋亡核的形态学检测。结果　免疫组化检测结果表明转染细胞与未转染细胞的Bcl XL 蛋白表达水平有差异 ,DNA电泳分析发现B组呈典型的DNA“梯度”条带 ,而A组和C组几乎无DNA“梯度”条带 ,核形态学检测结果亦证实转染组较未转染组的核有明显差异。结论重组腺病毒介导转染的bcl XL 对体外培养的视网膜光感受器细胞有抗凋亡作用 ,提高视网膜光感受器细胞bcl XL 的表达水平可能为视网膜变性疾病提供潜在有效的治疗方法。     

米诺环素对压力作用下体外培养神经细胞凋亡的抑制

目的在开放式压力控制培养系统作用下体外培养大鼠视网膜神经细胞（retinal neurons，BNs），观察米诺环素对其活性及凋亡的影响，并进一步探讨其对受损RNs保护的可能机制。方法采用出生0～3d的Sprague Dawley（SD）大鼠视网膜神经细胞体外混合培养，制备RNs加压培养模型。通过细胞形态学观察、四唑盐（MTT）比色法测定细胞活力、吖啶橙／溴化乙锭（AO／EB）染色法检测细胞的凋亡率来观察米诺环素对上述损伤细胞的保护及治疗作用，以及应用免疫细胞化学染色法，观察诱导型一氧化氮合酶（iNos）和半胱天冬酶-3（caspase-3）表达的改变。结果加压培养后，在倒置显微镜下RNs与对照组相比形态改变较明显，细胞活力降低，大量细胞发生凋亡（占53．93％），而米诺环素治疗组（20μmol／L）细胞则形态改善，活力显著增高，凋亡数目减少（占17．29％），差异具有显著性（P〈0．05）。免疫细胞化学染色法示米诺环素治疗组细胞内iNOs和caspase-3表达较加压损伤组减少。结论一定剂量的米诺环素在体外可有效抑制压力引起的大鼠视网膜神经细胞损伤及凋亡，抑制iNOs和caspase-3的表达可能是其潜在作用机制。     

缺氧培养下大鼠视网膜祖细胞与脑神经干细胞内钙离子浓度差异的研究

目的:比较缺氧培养条件下新生大鼠视网膜祖细胞(retinal progenitor cells,RPCs)与脑神经干细胞(neural stemcells,NSCs)钙离子浓度的差异。方法:分离新生大鼠RPCs及大脑皮质NSCs,进行无血清体外培养;采用荧光免疫细胞化学的方法进行干细胞鉴定;缺氧培养后,光镜观察细胞形态,采用钙离子荧光探针结合激光共聚焦显微镜测定细胞内钙离子荧光强度。结果:缺氧培养后,大脑皮质NSCs较早出现肿胀、伸出突起等变化,随缺氧时间延长,两种细胞内钙离子荧光强度逐渐增强,缺氧12h后RPCs内钙离子荧光强度小于脑皮质NSCs(P<0.05),差异具有统计学意义。结论:缺氧培养12hRPCs内钙离子浓度低于大脑皮质NSCs。     

Susceptibilities to and mechanisms of excitotoxic cell death of adult mouse inner retinal neurons in dissociated culture

PURPOSE: To explore the susceptibilities of adult retinal neurons in dissociated culture to treatments with excitotoxic agonists and the mechanisms of the resultant retinal cell death. METHODS: C57B6 mice were used. Retinas were removed, dissociated, plated on a polylysine/laminin substrate, and maintained in vitro for 5 to 7 days. Excitotoxic agonists (glutamate, N-methyl-D-aspartate [NMDA], or kainic acid [KA]) were added for 30 minutes or 24 hours, sometimes in the presence of modified extracellular ion concentrations or potential blocking agents. The next day, cells were fixed and immunocytochemically stained to identify ganglion and amacrine cells. Surviving cells were counted. RESULTS: Ganglion cells from adult mouse retinas were much less susceptible to excitotoxic death than those prepared from neonatal retinas. Adult amacrine cells were killed by KA, NMDA, or glutamate. Experiments with selective blockers demonstrated that KA killed through AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, whereas NMDA and glutamate exerted toxicity through a combination of AMPA and NMDA receptors. The KA-induced death of amacrine cells was not mediated by chloride ions. Removal of extracellular sodium, however, completely prevented the amacrine cell death, and removal of extracellular calcium prevented approximately 70% of the death. The path of calcium entry was investigated. Experiments with selective blockers indicated that the lethal calcium entry was via reverse operation of a sodium-calcium exchanger. CONCLUSIONS: There is a profound developmental regulation in the sensitivity of retina ganglion cells to excitotoxic insults. Excessive intracellular sodium and calcium are the proximal causes of amacrine cell death. The pathologic calcium entry is dependent on the sodium overload, which then drives a sodium-calcium exchanger to take up calcium.     

谷氨酸对视网膜Muller细胞超微结构和谷氨酰胺合成酶表达的影响

目的探讨谷氨酸对大鼠视网膜Muller细胞超微结构和谷氨酰胺合成酶（GS）表达的影响。方法取5～7d清洁级Wistar乳鼠视网膜,在含质量分数10%新生牛血清的Dulbecco改良Eagle培养基中传代培养大鼠视网膜Muller细胞。第3代Muller细胞培养基中加入终浓度分别为10、20、50、100、200μmol/L的谷氨酸作用10min。正常对照组Muller细胞培养基中未加入谷氨酸。通过免疫细胞化学法检测GS的表达鉴定视网膜Muller细胞。透射电镜观察各组Muller细胞超微结构的改变。采用Westernblot法半定量检测GS的表达。结果培养的Muller细胞中,GS阳性细胞达95%以上。透射电镜下正常对照组Muller细胞超微结构正常,谷氨酸10、20、50μmol/L组Mller细胞超微结构无明显改变。100μmol/L谷氨酸组可见Muller细胞线粒体肿胀和染色质的凝集、边聚。在200μmol/L谷氨酸组可见Muller细胞空泡样变性和凋亡小体。Westernblot结果显示,10、20、50μmol/L组GS在Muller细胞中表达较正常对照组升高,差异均有统计学意义（q=29.32、q=75.54、q=48.36、q=130.20,P〈0.01）,50μmol/L谷氨酸组GS的表达达高峰,100μmol/L谷氨酸组表达开始下降,200μmol/L谷氨酸组的表达低于正常对照组（q=46.67,P〈0.01）。结论谷氨酸浓度影响视网膜Muller细胞中GS的表达,谷氨酸浓度超过50μmol/L可引起视网膜Muller细胞超微结构的改变。     

Glutamate regulates the viability of retinal cells in culture

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.     

维拉帕米对兔眼挫伤后视网膜谷氨酸含量的影响

目的观察探讨Ca^2＋通道阻滞剂维拉帕米对兔眼挫伤后视网膜谷氨酸含量的影响及其在跟挫伤中的作用。方法60只兔（60眼）建立右眼挫伤模型,治疗组每日球结膜下注射维拉帕米,挫伤组每日球结膜下注射生理盐水,分别于30min、6h、1d、3d、7d、14d取材,高压液相色谱仪定量检测视网膜谷氨酸水平。另5只兔10眼为正常对照。结果兔眼挫伤后视网膜谷氨酸含量高于正常,并在6h、7d形成两个高峰。维拉帕米治疗可明显抑制6h高峰,并使7d高峰的峰值降低。结论提示维拉帕米可降低眼桦伤后视网膜谷铕西夸7k平。     

视网膜缺血-再灌注损伤干预治疗新进展

视网膜缺血-再灌注损伤(RIRI)是眼科常见的病理生理损伤性眼病,常发生于视网膜动静脉阻塞、糖尿病视网膜病变、急性闭角型青光眼等与缺血相关的眼病.表现为缺血性患眼在血液再灌注后,细胞功能发生代谢性障碍,视网膜组织结构损伤,视功能下降.缺血-再灌注损伤是由多种因素共同作用的结果,目前公认的假说主要包括氧自由基的损伤、细胞内的钙超载、白细胞介素介导作用和细胞凋亡等.但目前对于RIRI的保护及治疗研究有限,本文就国内外有关RIRI的干预治疗新进展综述如下.     

米诺环素在L-谷氨酸诱导的视网膜神经节细胞损伤中的保护作用及分子机制

目的 探讨米诺环素在L-谷氨酸诱导的视网膜神经节细胞(RGC)毒性中的保护作用和分子机制.方法 实验研究.原代小鼠RGCs体外培养24 h后,随机分为3组:对照组,L-谷氨酸组(100 μmol/L、500 μmol/L、1 mmol/L和2 mmol/L)及L-谷氨酸+米诺环素组(30 μmol),观察不同浓度L-谷氨酸对RGC的存活率与轴突生长的损伤作用及米诺环素的保护作用.体内实验,将雌性B6小鼠随机分为实验组(30只)和对照组(30只).两组小鼠腹腔内分别注射米诺环素(实验组,60 mg/kg)或生理盐水(对照组),每天1次,连续7 d.第2天时,两组小鼠玻璃体腔内注射2μl L-谷氨酸(2 mmol/L),诱导RGC损伤.免疫组化染色分析β-Ⅲ-tubulin阳性细胞数目变化及视网膜神经胶质酸性蛋白(GFAP)表达情况,Real-time PCR和免疫印迹法分别检测小鼠视网膜组织中干扰素γ(IFN-γ)、白细胞介素(IL-1)、肿瘤坏死因子α(TNF-α)、GFAP与波形蛋白(Vimentin)的mRNA及蛋白表达水平.结果 体外实验显示,与对照组相比,L-谷氨酸降低RGC的存活率,与剂量和干预时间呈负相关.同时L-谷氨酸可明显抑制RGC轴突的生长,RGC轴突长度>2BL、1～2 BL、<1 BL占总细胞数比例分别从50.38%、7.83%和3.72%降至31.43%、5.05%和1.29%.而米诺环素能明显减轻L-谷氨酸对RGC的毒性作用,改善RGC轴突生长,各组细胞比例回升至51.00%、8.10%和2.43%,谷氨酸与对照组相比、米诺环素组与谷氨酸相比,差异有统计学意义(F=18.87,P<0.01).体内实验结果显示,与对照组相比,L-谷氨酸组小鼠RGC数目显著减少(45.00±10.21和68.50±2.86),而米诺环素治疗后可明显改善L-谷氨酸诱导的RGC损伤,RGC数目恢复至62.00±11.65,(F=7.6,P<0.01).谷氨酸处理后视网膜组织中GFAP的表达水平明显增高,而米诺环素明显降低视网膜组织中GFAP的表达.同时,L-谷氨酸显著提高小鼠视网膜组织中炎症相关因子IFN-γ、IL-1、TNF-α及胶质细胞相关蛋白Vimentin和GFAP的基因及蛋白表达水平,而米诺环素可显著抑制这些因子的表达.结论 L-谷氨酸损伤可诱导RGC凋亡、抑制RGC轴突生长,并上调炎症因子及视网膜相关胶质蛋白的基因与蛋白表达水平,米诺环素对L-谷氨酸所导致的视网膜神经节细胞损伤具有明显的保护作用.     

青光眼视神经保护研究进展

青光眼是以视网膜神经节细胞进行性死亡为特征的一类疾病,其病理学机制包括慢性缺血、氧自由基损害、谷氨酸兴奋性毒素导致神经变性、轴突运输障碍、神经营养因子缺乏、电活动消失等。临床上主要通过降眼压治疗青光眼,但往往有一部分患者控制眼压后仍不能有效减少视网膜神经节细胞的死亡。因此,合理的青光眼治疗应包括视神经保护,本文就近几年青光眼视神经保护治疗的研究进展作一综述。     

Retinal Ganglion Cell Dysfunction Induced by Hypoxia and Glutamate: Potential Neuroprotective Effects of β-Blockers

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2–50 μM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 μM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 μM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.     

Protective effect of arachidonic acid on glutamate neurotoxicity in rat retinal ganglion cells

PURPOSE: Low concentrations of excitotoxic agents such as glutamate decrease survival of retinal ganglion cells (RGCs) and may be an important cause of RGC death in a variety of retinal diseases. Arachidonic acid (AA), an intercellular messenger in the central nervous system, has been reported to have multiple effects on glutamate receptors, including an inhibitory effect on non-N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to test the hypothesis that AA could protect RGCs from glutamate neurotoxicity. METHODS: RGCs were purified from the rat retina on postnatal days 7 and 8 by a modified two-step panning method. Survival of RGCs after exposure to glutamate, with or without AA treatment, was measured after 3 days in culture. To visualize calcium signals, RGCs were loaded with a calcium indicator dye, fluo-3 acetoxymethyl ester, and the fluorescence was measured by laser scanning confocal microscopy. Electrophysiological effects of AA on non-NMDA ionotropic receptors were examined by using whole-cell patch clamp configurations. RESULTS: Incubation of RGCs with 25 microM glutamate caused 60% loss of RGCs. This glutamate neurotoxicity was significantly ameliorated by low concentrations of AA. Concentrations of AA above 10 microM were toxic to RGCs. Calcium imaging showed that glutamate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA) and kainate-induced intracellular calcium accumulation in these cells was reduced by AA. Electrophysiological recordings revealed that currents mediated by non-NMDA ionotropic receptors were inhibited by AA in a dose-dependent manner. CONCLUSIONS: Low concentrations of AA can reduce glutamate neurotoxicity to RGCs by the inhibition of non-NMDA ionotropic receptors. These results suggest that endogenous or exogenous AA may be used to protect RGCs from glutamate neurotoxicity and that AA may be one potential treatment for RGC loss in a variety of eye diseases, including glaucoma.     

Detection of Glutamate and γ-aminobutyric Acid in Vitreous of Patients with Proliferative Diabetic Retinopathy

Purpose: To study the levels of glutamate and γ-aminobutyric acid (GABA) in vitreous of patients with proliferative diabetic retinopathy(PDR) and to investigate their roles in retinal ischemia.Method: Vitreous samples were collected from 25 patients (27 eyes) with PDR and 14patients ( 14 eyes) with idiopathic macular hole. Glutamate and GABA detection were performed by high-performance liquid chromatography (HPLC).Results: Patients with PDR had significantly higher concentrations of glutamate and GABA than the control group. The glutamate level has a significantly positive correlation with GABA level.Conclusion: Detection of glutamate and GABA in vitreous provides biochemical support for the mechanism and treatment of ischemic retinal damage in patients with PDR.