蛇毒神经生长因子对体外培养鼠视网膜神经节细胞的保护作用

目的研究蛇毒神经生长因子（venom nerve growth factor，vNGF）对离体培养的视网膜神经节细胞（retinal ganglion cells，RGCs）的存活和轴突再生的影响。方法采用新生大鼠视网膜神经细胞体外原代培养技术，加入不同浓度的vNGF，通过检测细胞的活力选择一个最佳浓度进行培养，行抗Thy-1．1免疫细胞化学染色，观察RGCs在体外的存活时间,比较实验组和对照组的RGCs个数、胞体直径和轴突长度。结果vNGF的最佳浓度是80μg.L，用该浓度作用于细胞，实验组与对照组细胞存活时间分别为13～14d和7～8d，轴突长度分别为（111.78±10.65）μm和（78.73±8.23）μm。阳性细胞个数分别为（224.23±3.83）个和（182.47±2.79）个。结论vNGF能促进体外视网膜神经细胞和RGCs的存活以及轴突的伸长，有视神经保护作用。     

青光眼视网膜神经节细胞损伤及其保护

青光眼是一种主要的致盲眼病,导致视功能损害的病理基础是视网膜神经节细胞进行性死亡和视神经纤维丧失。视网膜神经节细胞损伤的机理是复杂的,到目前为止还不完全明了,本就其可能的原因及其保护的研究进展作一综述。     

视网膜神经节细胞谷氨酸毒性的防护研究

目前认为谷氨酸兴奋毒性是触发青光眼和／或视网膜缺血性神经节细胞损伤级联反应的最主要因素。大量研究表明,预防性阻断谷氨酸兴奋毒性可以保护视网膜神经节细胞,减少缺血损伤和神经节细胞凋亡。现就近年来抗谷氨酸毒性,保护视网膜神经节细胞的方法和药物作一简要综述。     

青光眼的视网膜神经节细胞损伤及其保护

高眼压一直被认为是引起青光眼视神经损害的重要机制，但是临床发现部分青光眼患者即使眼压得到很好的控制也不能阻止视神经的进一步损害。青光眼造成的视神经萎缩不仅是高眼压导致的视神经受压萎缩，更多的研究结果表明青光眼的视神经改变是一种视神经病变，原因有多种，但均表现为视神经节细胞的死亡。这一视神经节细胞的死亡过程是一种缓慢的凋亡过程，具体可分为两个阶段：第一阶段是缺血、缺氧造成的细胞损害；第二阶段是受损的退变细胞释放有害物质引起基质的改变和损伤。     

褪黑素对大鼠视网膜缺血再灌注损伤后神经节细胞的保护作用

目的:探讨褪黑素(melatonin,MT)对大鼠视网膜缺血再灌注损伤(retina ischemia reperfusion injury,RIR)中神经节细胞(retina ganglion cells,RGC)的防护和修复作用.方法:健康SD大鼠24只随机分为A组和B组.两组均利用前房高眼压灌注法制成左眼RIR模型,A组每日给予溶媒(100mL/L无水乙醇生理盐水)1mL/kg ip,B组每日给予5g/L MT1mL/kgip,直至处死动物.两组按左眼RIR后的存活时间又分为3小组:A1和B1组(损伤后7d),A2和B2组(损伤后14d),A3和B3组(损伤后30d),每组各4只.于处死前7d由双上丘和双外侧膝状体注射30g/L fluorogold(FG)标记双眼RGC,处死日行双眼眼球摘除术,将全视网膜组织铺片置于荧光显微镜下,分鼻上、鼻下、颞上、颞下4个区域作荧光摄影,并输入计算机经图像分析系统计数RGC,计算RGC标识率(即损伤眼RGC数/未损伤眼RGC数)×100%,作统计学分析.结果:A1,A2,A3 3组RGC标识率分别为77.2%±6.4%,65.5%±7.0%,53.9%±4.4%;B1,B2,B3 3组RGC标识率分别为81.3%±9.3%,79.8%±8.4%,80.3%±11.1%.结论:大鼠RIR后MT治疗可提高RGC存活率.     

青光眼视网膜神经节细胞的保护

青光眼特征性的改变是视网膜神经节细胞的死亡。已有实验证实,神经节细胞的死亡是通过细胞凋亡的方式。因而保护神经节细胞免于继续受损已成为青光眼研究的重点。本文从神经营养,谷氨酸及其受体和钙离子,凋亡基因的表达等方面简要介绍其研究进展,提出可能的保护机制。     

视网膜神经节细胞的保护和修复

视网膜神经节细胞(RGCs)的进行性死亡是许多视网膜和视神经疾病发展到最后的必经之路。长期以来一直认为，由于抑制性环境的存在，视神经损伤后不能再生和修复，现在研究证实，在特定的条件下，尽管RGCs的胞体或轴突受损，仍能免于死亡，而且变性的轴突能再生，并能与靶组织建立突触联系。对RGCs的保护和修复的研究进展作一综述。     

黄芪注射液对体外培养视网膜神经节细胞的保护作用

目的 视神经由视网膜神经节细胞(retinal ganglion cells,RGCs)轴突构成,一旦损伤难以再生,视神经保护是治疗视神经疾病的关键,本研究拟评价黄芪注射液的视神经保护作用.方法 体外培养RGCs,采用谷氨酸作为细胞凋亡诱导剂建立RGCs凋亡模型,并用不同浓度黄芪注射液加入细胞培养基进行干预,分别用MTT和流式细胞检测的方法进行细胞存活率及细胞凋亡相关检测.结果 黄芪注射液加入DMEM-F12培养基共培养RGCs 24 h后,MTT检测结果示终浓度为(0.10～0.40)×103g·L-1时黄芪注射液均可促进RGCs存活,其中0.20×103g·L-1、0.30×103g·L-1浓度促生长作用最好(P=0.040、0.005,n=10),谷氨酸浓度为3.2 mmol·L-1时可造成理想的RGCs凋亡模型,0.20×103g·L-1黄芪注射液可促进谷氨酸损伤的RGCs存活(P=0.08,n=10)并减少其细胞凋亡率(P=0.36,n=4).结论 黄芪注射液作为视神经保护中药制剂对RGCs具有一定保护作用,黄芪对RGCs的保护作用很可能跟抑制谷氨酸兴奋性毒性作用有关.     

N—甲基—D—天门冬氨酸和地塞米松对体外培养鼠视网膜神经节细胞凋亡作用观察

目的:研究N-甲基-D-天门冬氨酸(N-methyl-D-aspartate NMDA)及地塞米松对体外培养视网膜神经节细胞(RGCs)的作用。方法:出生1～3天Sprague-Dawley(SD)乳鼠做体外RGCs培养。NMDA按20～500μmol/L加入两组混合培养的RGCs中,24小时后一组用Hochest33258检测凋亡细胞。另一组加0.4%台盼蓝,计数拒染细胞,并算出细胞存活率。地塞米松按1×10~(-4)、1×10~(-5)及1×10~(-6)mol/L分别加入混合培养及纯化培养的RGCs中,对照组不加,24小时后用Hochest33258检测凋亡细胞。结果:混合培养RGCs经不同浓度NMDA处理均可见明显的凋亡细胞形态学改变。对照组则不明显。而且其中细胞的存活率与NMDA浓度呈反相关。不同浓度地塞米松处理混合培养RGCs,经Hochest33258检测均未发现有凋亡形态学改变。而纯化培养RGCs实验组及对照组均有明显凋亡细胞形态学改变。结论:NMDA对体外混合培养RGCs有诱导细胞凋亡的作用;地塞米松则对培养RGCs无诱导细胞凋亡作用。纯化的RGCs生长24小时,不加神经营养因子Neurotrophic Factors(NTFs)有细胞凋亡的形态学改变。眼科学报1999 15:65—69。     

灯盏花素抗叔丁基过氧化氢诱导原代大鼠视神经节细胞凋亡

目的：探讨灯盏花素（BVP）对视神经节细胞的保护作用。方法：实验研究。培养大鼠原代视网膜神经节细胞（PRGCs）,采用0、50、100、200 μmol/L叔丁基过氧化氢（tBHP）诱导细胞氧化损伤建立细胞模型（最后选择100 μmol/L建立模型）,给予不同浓度（0、10、20、50 μmol/L）BVP处理后（最终采用20 μmol/L用于后续研究）,检测神经节细胞的改变,免疫印迹法检测细胞凋亡相关因子的改变。采用t检验进行数据处理。结果：与空白组对照相比,经tBHP处理的PRGCs凋亡明显增加,凋亡相关蛋白Bcl-2（P<0.05）和神经标志蛋白Synaptophysin（P<0.01）的表达明显减少。与模型组相比,各治疗组PRGCs成活率明显增加,细胞凋亡减少,Bcl-2 和Synaptophysin的表达增加（均P<0.05）,而Bcl-2相关X蛋白（Bax）的表达（P>0.05）及Cytochrome C的释放（P>0.05）及Caspase-3的剪切则被抑制（P<0.001）。且BVP治疗作用呈剂量依赖性。结论：BVP可以逆转tBHP对视神经节细胞的损伤,减少视神经节细胞的凋亡。     

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.     

Study on Retinal Ganglion Cell Apoptosis after Explosive Injury of Eyeballs in Rabbits

Purpose: To explore the pathological changes of retinal ganglion cell apoptosis and its relation to the value of glutamate concentration in vitreous body after explosive injury of eyeballs in the rabbits.Method: Similar explosive injury models of eyeballs in 10 adult grey rabbits were made. The rabbits were killed on scheduled time. The retinal tissues of studied eyes and control eyes were obtained for the pathological examination with TUNEL method respectively. The value of glutamate in vitreum of injured eyes was measured and was compared with that of contralateral eyes. Statistical comparison analysis on the experiment data was performed.Result: The value of glutamate in vitreum of injured eyes was significantly higher than that of contralateral eyes in all rabbits in the study. A lot of TUNEL positive cells were observed in the injured eyes. It suggested that apoptosis of the retinal ganglion cells took place.Conclusion: We speculate that apoptosis of the retinal ganglion cells is perhaps among the     

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.     

The Relationship Between Macula Retinal Ganglion Cell Density and Visual Function in the Nonhuman Primate

PurposeLoss of ganglion cell inner plexiform layer (GCIPL) and visual sensitivity in the macula region are known to occur at all stages of glaucoma. While both are dependent on the underlying retinal ganglion cells (RGCs), the relationship between structure and function is modest. We hypothesize that the imprecise relationship is due to a lack of direct correspondence between in vivo measures and RGC counts, as well as the relatively large stimulus size used by standard perimetry, which exceeds spatial summation.MethodsThe relationship between optical coherence tomography (OCT)–derived GCIPL thickness and corresponding inner cell density from retinal flat mounts was determined for four nonhuman primates with varying stages of neuropathy. Normative data for 10-2 threshold using Goldman size I to V stimuli were established for 10 animals, 4 of which were then followed longitudinally with OCT and perimetry. The relationship between GCIPL volume, which incorporated stimulus size after removal of residual thickness, and differential light sensitivity was determined for both experimental glaucoma and healthy eyes.ResultsPeak inner retinal cell density was 63,052 ± 9238 cells/mm² in the healthy eye. Cell density was related to both GCIPL thickness and eccentricity (R² = 0.74, P < .01). For all 10-2 eccentricities, size III stimuli were greater than the critical area (P < 0.01). Based on the structural and histologic relationship, the critical area corresponds to approximately 156 RGCs.ConclusionsThe relationship between cell density and GCIPL thickness is dependent on retinal eccentricity. For 10-2 perimetry, perimetric loss, especially at earlier stages of neuropathy, may best be detected using size II or smaller stimuli.     

Expression of AMPA Receptor Subunit Proteins in Purified Retinal Ganglion Cells

Purpose

To determine whether α-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) receptor (AMPAR) subunit proteins are expressed in cultured retinal ganglion cells (RGCs).

Methods

RGCs were purified from dissociated rat retinal cells (postnatal days 6–8), using a modified two-step panning method and cultured in serum-free medium containing neurotrophic factors and forskolin. Immunohistochemistry was performed on cultured RGCs on days 1, 3, and 7 in vitro (1 DIV, 3 DIV, and 7 DIV) using specific antibodies against AMPAR subunits GluR1 to 4 and microtubule-associated protein (MAP) 2, which is a neuronal marker. Glutamate-induced Ca²⁺ influx was measured with fura-2 acetoxymethyl ester fluorescence.

Results

GluR1 to 4 proteins were expressed in the cell body of RGCs on 1 DIV. RGCs showed strong GluR1 to 4 immunoreactivity in both cell bodies and processes on 3 DIV and 7 DIV, with the gradual spreading of expression and the growth of processes. At all time points examined, GluR2 immunoreactivity was equal to that of the other subunits. Accumulation of intracellular Ca²⁺ levels in RGCs induced by glutamate occurred equally on both 3 DIV and 7 DIV.

Conclusion

All AMPAR subunits are almost equally expressed in cultured RGCs.?Jpn J Ophthalmol 2006;50:217–223 © Japanese Ophthalmological Society 2006     

Retrograde Labeling of Adult Rat Retinal Ganglion Cells with the Flurogold

Purpose: To study the densities and distribution of retinal ganglion cells (RGC) in adultrat retinae with flurogold(FG) labeling retogradely.Methods: FG was injected to the superior colliculi(SC) and dorsal lateral geniculatenuclei (dLGN) in adult rats and the retinae were examined by fluorescence microscopyat various periods of time.Results: FG-labelled RGC were observed in the retina as early as 3 days after applicationof FG. The labelled cells gradually increased in density, reached 95% of the maximalnumber on days 7 and the maximal number on days 30. The density of labelled cellswas higher in the posterior pole than in the peripheral area. The fluorescence intensity inlabelled cells maintained up to 60 days.Conclusion: The FG retrograde labeling method is reliable and effective for quantity ofRGC. Eye Science 2000; 16: 29 - 33.     

青光眼视网膜神经节细胞凋亡的实验研究

目的 研究兔的实验性青光眼视网膜神经节细胞的死亡是否有凋亡参与。方法 前房注入固定的红细胞悬液使免眼压升高,分别在术后７,１４,２１,２８天处死动物,取视网膜组织,ＴＵＮＥＬ标记染色,电镜观察。结果 电镜下可见视网膜神经节细胞死亡特征为典型的凋亡早期特征－核浓染。免疫组ＴＵＮＥＬ法实验组发现神经节细胞凋亡,而正常对照组没有发现。结论 实验性青光眼的视网膜神经节细胞死亡有凋亡参与。这为通过调探凋亡而治疗青光眼的视网膜视神经损伤提供可能。