糖尿病大鼠早期视网膜小胶质细胞活化与神经节细胞损害的关系

目的　观察糖尿病早期视网膜小胶质细胞的活化特征与视网膜神经节细胞（RGC）损害的关系。方法　20只成年雄性Sprague-Dawley（SD）大鼠,采用链脲佐菌霉素腹腔注射方法制作糖尿病动物模型,分为糖尿病1、3个月组及相应正常对照组,每组5只大鼠。对所有大鼠行上丘定位注射逆行标记RGC,分别用免疫组织化学法标记视网膜铺片、冰冻切片小胶质细胞和RGC,共聚焦显微镜下观察小胶质细胞细胞形态及分布特征。结果　糖尿病组视网膜铺片小胶质细胞胞体增粗,形态不规则。与对照组相比,糖尿病3个月组RGC层发生吞噬的小胶质细胞密度显著增加（t=3.83,P＜0.01）。与对照组相比,糖尿病大鼠1、3月个组RGC层小胶质细胞平均密度均显著增加（t=2.71,4.22;P＜0.05）;糖尿病大鼠3个月组RGC层小胶质细胞平均密度较糖尿病1个月组显著增加（t=7.45,P＜0.0001）。糖尿病早期小胶质细胞与RGC数量之间存在相关关系（r=0.9,P＜0.05）。结论　糖尿病早期小胶质细胞活化与RGC损伤关系密切。     

视神经损伤后发生非折叠蛋白反应的超微证据

目的:探索视神经损伤后视网膜节细胞(retinal ganglion cell,RGC)及其纤维渐进性死亡机制。方法:利用包埋前与包埋后免疫金细胞化学标记技术结合电镜观察研究大鼠(n=15)视神经钳夹损伤后RGC与视神经干少突胶质细胞内的非折叠蛋白反应(unfolding protein response,UPR)。结果:视神经钳夹后,需肌醇酶1(inositol requiring enzyme 1,IRE1)在RGC与少突胶质细胞内胶体金标记数目增多,在钳夹0.5d后RGC内即有显著地增加(18.4±5.1～30.4±7.2个,P<0.05),随损伤时间延长,增多更为明显,在钳夹3d后达高峰(48.5±9.7个),IRE1在视神经干上的少突胶质细胞内增多时程变化与在RGC内相似。IRE1在RGC与少突胶质细胞内胶体金标记颗粒分布部位距离ER管腔距离增加为特征,在钳夹0.5d内神经干少突胶质细胞以及视网膜RGC内均表现非常明显的距离增加。结论:视神经钳夹导致损伤细胞触发UPR,UPR可能参与视神经损伤后RGC及其纤维渐进性死亡机制。     

早期糖尿病大鼠视网膜神经节细胞逆行轴浆流损害的研究

目的 观察早期糖尿病大鼠视网膜神经节细胞（RGC）逆行轴浆流速是否受损，并判断损伤细胞的类型。 方法 Sprague-Dawley(SD)成年雄性大鼠6只, 腹腔注射链脲佐菌霉素（STZ）形成糖尿病模型，4周时行荧光金（FG）上丘定位逆行标记。分别于FG注射后12、72h行视网膜铺片，在荧光显微镜下拍照后做尼氏染色，统计FG标记的不同大小RGC比例。另6只正常SD成年雄性大鼠作为对照。 结果 FG注射12h后，实验组RGC总数与对照组无差别，但小直径RGC比例明显低于对照组；72h后，实验组RGC数比对照组明显减少，小细胞减少尤其明显。 结论 糖尿病早期大鼠RGC逆行轴浆流速受到影响，小直径RGC容易受损。 (中华眼底病杂志, 2006, 22: 4-6)     

银杏叶制剂EGb 761对外伤后视网膜神经节细胞的保护作用

目的 探讨银杏叶提取物制剂EGb 761对大鼠视神经损伤后视网膜神经节细胞(RGC)存活的影响.方法 大鼠48只经荧光金逆行标记后制备视神经损伤的动物模型,随机分为治疗组和对照组,两组分别给予EGb 761 150 mg/kg·d和生理盐水灌胃.在视神经损伤后4d、7d及14d观察视网膜铺片,进行RGC计数.结果 大鼠视神经损伤后4d、7d及14d,RGC数量持续减少,但治疗组均高于对照组,各时间点的差异均有统计学意义(依次为P＜0.05,P＜0.01,P＜0.01).结论 EGb761可促进视神经损伤后RGC的存活,对RGC有保护作用.     

银杏叶提取物对幼年大鼠大视网膜神经节细胞保护作用的研究

目的 观察银杏叶提取物（EGb761）对培养幼年大鼠大视网膜神经节细胞（RGC）的保护作用;建立用荧光金逆行上丘标记鉴定体外培养大鼠RGC的方法。方法 12只鼠龄20 d Sprague-Dawley大鼠用荧光金行双侧上丘注射,标记RGC。6 d后摘除眼球,其中一只眼球行视网膜铺片,荧光显微镜下观察标记情况。另一只眼球摘除后分离视网膜,制成细胞悬液,倒置荧光显微镜下观察荧光着染的RGC形态。将细胞悬液分为对照组及浓度分别为0.03%、0.10%、0.30%、1.00%、3.00%的EGb761组,接种后3 h,1、3、5、7 d锥虫蓝染料排斥 法检测RGC的存活情况,同时记录细胞存活率。结果 逆行上丘标记后观察到RGC标记良好,视网膜细胞悬液中大RGC特征明显,离体后迅速死亡,荧光消失。锥虫蓝染料排斥法观察到在视网膜细胞悬液中,这种大RGC死亡迅速。加入不同浓度的EGb761后大RGC存活率明显增加,不同时间点与对照组比较大RGC存活率均有统计学意义（P＜0.01）,且呈明显剂量依赖关系（P＜0.01）。结论 EGb761对体外培养的RGC具有明显的保护作用;荧光金逆行上丘标记鉴定体外培养的RGC方法可行。     

肌苷毫微粒对成年大鼠视网膜节细胞的保护作用

目的 研究载有肌苷的毫微粒对视神经切断后视网膜节细胞(RGC)存活的影响。方法 制备肌苷毫微粒，体外测定理化性质。将等体积的肌苷毫微粒、空载毫微粒或生理盐水溶液分别注入成年大鼠左眼内，对照组未经任何治疗。1d后于眶内切断所有动物左侧视神经，术后7d取左视网膜，计数荧光金逆行标记的存活RGC。结果 肌苷毫微粒形态规整，具有缓释特点。同对照相比，肌苷毫微粒能显著提高存活RGC的密度，而空载体和生理盐水无此作用；空载毫微粒与生理盐水、对照之间以及空载毫微粒和肌苷毫微粒两组间RGC密度均无显著差异。结论 注入眼球的肌苷毫微粒至少在7d内能有效缓释肌苷，进而对轴突损伤RGC发挥显著的神经保护作用。     

实验动物视网膜神经节细胞的定量研究方法

尼氏染色、逆行标记和免疫标记是目前对视网膜神经节细胞(RGCs)进行定量研究的基本方法。尼氏染色是用碱性染料与神经元细胞内的尼氏体结合,使RGCs染色,并行计数,记数时需要排除非神经元的干扰。逆行标记是将荧光染液注射或贴附到上丘、外侧膝状体或视神经,染液通过神经元细胞轴突内的逆行轴浆运输,向视网膜神经节细胞的胞体内移行,并使其染色。免疫标记是使用特异性的Brn-3b或者Thy-1抗体与视网膜神经节细胞上的抗原结合标记,再行染色和计数。本文综述尼氏染色,逆行标记及免疫标记3种方法对RGCs进行定量计数的研究进展。     

荧光金逆行标记检测CNTF基因眼内转移对视神经损伤大鼠的影响

目的通过荧光金逆行标记的方法,检测腺病毒(adenovirus,Ad)介导的睫状神经营养因子(ciliary neurotrophic factor,CNTF)对视神经钳夹伤大鼠视网膜神经节细胞(retinal ganglion cell,RGC)数目的影响。方法采用钳夹视神经法制作大鼠视神经损伤模型,夹伤后向大鼠伤眼内注射Ad-CNTF,在取材前7d进行荧光金逆行标记RGC,于伤后28d,对大鼠的视网膜铺片进行荧光金标记的RGC记数。结果伤后28d,单纯损伤组、PBS组、Ad-LacZ组视网膜标记RGC数均较低;CNTF处理组视网膜标记RGC数为每视野(12.11±2.29)个(n=9),高于前3组,且差异非常显著(P<0.01)。Ad-CNTF组视网膜标记RGC数为每视野(20.93±2.67)个(n=8),在各组标记RGC数中最多,也好于CNTF组,2组间差异非常显著(P<0.01)。结论视神经损伤后眼内单次注射Ad-CNTF,可明显增加钳夹伤后28d大鼠视网膜的标记RGC数。与单次注射CNTF相比,对损伤的RGC具有更加显著的神经保护作用。     

青光眼模型视神经损伤的评估方法

对青光眼动物模型视神经损伤的评估有利于青光眼发病机制和治疗方法的研究.目前主要从视网膜神经节细胞(retinal ganglion cell,RGC)计数、视网膜内层厚度测定和球后视神经轴突计数三方面进行评估.几种评估方法各有优缺点.RGC的凋亡直接反映视神经损伤的程度,视网膜全铺片染色计数RGC方法操作较简单,但视网膜铺片各层细胞易于重叠;逆行荧光染料标记需进行颅内手术且染料可能泄露到邻近小胶质细胞,导致不准确的计数;视网膜厚度测量不能直接反映视神经损伤的程度;视神经轴突计数难度相对较高;体视学方法可直接计数RGC,但需要熟悉相关体视学公式及方法,目前在该领域的报道较少.     

地西泮对大鼠视神经切断后视网膜神经节细胞保护作用的研究

目的 探讨地西泮对成年大鼠视神经切断后视网膜神经节细胞(retinal ganglion cells,RGC)的保护作用及机制.方法 取雌性SD大鼠36只,随机平均分为2组,麻醉后于眶内距视神经根部1.5 mm处切断左侧视神经,眶侧残端留置浸有荧光金的明胶海绵以逆行标记RGC.术前30 min及术后每天腹腔注射地西泮(地西泮组)和生理盐水(对照组),分别手术后2 d、7 d及14 d各处死6只大鼠.根据上述实验结果,将另外24只大鼠平均随机分为2组,每天腹腔注射γ-氨基丁酸A型(GABAA)受体阻断剂荷苞牡丹碱(荷苞牡丹碱组)和荷苞牡丹碱+地西泮(联合应用组)以探讨地西泮的神经保护机制,2组在动物存活2 d及7 d后分别处死6只大鼠.动物处死后视网膜平铺,计数每只动物荧光金标记的存活RGC并得出存活RGC的平均密度.比较各组RGC密度.结果 地西泮组视神经切断后7 d RGC平均密度(1 730±75)mm-2显著高于同一时间点对照组RGC密度(1 095±94)mm-2.在此时间点,联合应用组RGC密度(1 120±63)mm-2明显低于地西泮组,而荷苞牡丹碱组与对照组RGC密度差异无统计学意义(P>0.05),说明地西泮对RGC的保护作用可被荷苞牡丹碱拮抗.结论 地西泮可通过激活GABAA受体的途径在大鼠视神经切断后7 d促进RGC的存活.     

Distribution and size of ganglion cells in the retinae of large Amazon rodents

The topographical distribution of density and soma size of the retinal ganglion cells were studied in three species of hystricomorph rodents. Flat-mounted retinae were stained by the Nissl method and the ganglion cells counted on a matrix covering the whole retinae. Soma size was determined for samples at different retinal regions. The agouti, a diurnal rodent, shows a well-developed visual streak, reaching a peak density of 6250 ganglion cells/mm2. The total number of ganglion cells ranged from 477,427-548,205 in eight retinae. The ganglion-cell-size histogram of the visual streak region exhibits a marked shift towards smaller values when compared to retinal periphery. Upper and lower regions differ in both cell density and cell size. The crepuscular capybara shows a less-developed visual streak with a peak ganglion cell density of 2250/mm2. The shift towards small-sized cells in the visual streak is less marked. Total ganglion cell population is 368,840. In the nocturnal paca, the upper half of the fundus oculi includes a tapetum lucidum. The retina of this species shows the least-developed visual streak of this group, with the lowest peak ganglion cell density reaching 925/mm2. The total ganglion cell number (230,804) is also smaller than in the two other species. Soma-size spectra of this species are characterized by the presence, in the lower hemi-retina, of very large perikarya comparable in size to the cat's alpha ganglion cells.     

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.     

Retinal ganglion cells resistant to advanced glaucoma: a postmortem study of human retinas with the carbocyanine dye DiI

PURPOSE: The present study was conducted to examine whether the morphology of the retinal ganglion cells is altered in advanced glaucoma. Perikaryal, axonal, and dendritic alterations were monitored in glaucoma-resistant retinal ganglion cells by postvitam application of the fluorescent dye DiI. METHODS: The retinas of four amaurotic glaucomatous eyes and four normal eyes enucleated after death were used in this study. The retinas were freed from surrounding tissue, prepared as flatmounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. The retinal ganglion cells were labeled by introducing crystals of the fluorescent carbocyanine dye DiI, into the optic fiber layer. This dye diffuses along membranes of ganglion cell axons, completely labeling them and their cell bodies and dendrites. Further characterization of the retinas and optic nerves included hematoxylin-eosin and van Gieson histochemical staining as well as immunohistochemistry against glial fibrillary acidic protein. RESULTS: Because of the advanced stage of the disease, the retinas were almost completely depleted of ganglion cells, which had degenerated and therefore could not be stained. The few remaining ganglion cells were considered to be resistant to glaucoma. They showed drastic morphologic alterations, such as abnormal axonal beading, the cell bodies were normal in size but had irregular silhouettes or swellings, and there were fewer dendritic bifurcations. The size of the dendritic trees was smaller, implicating pruning of smaller dendritic branches. Glial cells were also detected immunocytochemically indicating their involvement in the pathologic course of glaucoma. CONCLUSIONS: The data suggest that the few ganglion cells that survive the elevated intraocular pressure associated with loss of visual function display morphologic changes that are manifested both on the cell body and on their intraretinal processes, including axons and dendrites.     

Ganglion cell distribution in the retina of the mouse

The distribution of ganglion cells in the mouse retina was studied with the use of Nissl criteria for distinguishing cell types in the ganglion cell layer. Retrograde filling with horseradish peroxidase (HRP) from the optic fiber tract helped to validate Nissl criteria and served to identify displaced ganglion cells. We estimated a total of 117,000 nonvascular cells in the ganglion cell layer; of these, 70,000 were probably ganglion cells, and 47,000 could not be classified. The density of the presumed ganglion cells was highest-more than 8000 cells/mm2-just temporal to the optic disk, and lowest-less than 2000 cells/mm2-in the most dorsal retina. The retinal region with highest ganglion cell density was slightly elongated in a nasotemporal direction. About 2% of all HRP-filled ganglion cells had their cell bodies in the inner nuclear layer. These displaced cells differed in topographical distribution from the normally positioned ganglion cells: although occurring throughout the retina, they were more common along the retinal periphery. Measurements of ganglion cell areas showed a tendency toward larger size with eccentricity. At no retinal location did cell-size histograms reveal clearly separate size classes.     

褪黑素通过Müller细胞保护糖尿病视网膜神经节细胞的机制

目的　探讨褪黑素对糖尿病视网膜Müller细胞的影响及其对视网膜神经节细胞的保护作用。方法　SD大鼠54只随机分为对照组、糖尿病组和褪黑素治疗组，糖尿病组和褪黑素治疗组制造糖尿病模型。造模后，对照组和糖尿病组大鼠腹腔注射体积分数10%乙醇溶液，褪黑素治疗组大鼠腹腔注射褪黑素溶液（10 mg·kg^-1）。3个月后，试剂盒检测视网膜中丙二醛（malondialdehyde，MDA）和还原型谷胱甘肽（reduced glutathione，GSH）的含量，Western blot检测视网膜中神经胶质酸性蛋白（glial fibrillary acidic protein，GFAP）及谷氨酰胺合成酶（glutamine synthetase，GS）的表达变化，免疫组织化学染色观察GFAP阳性染色的空间分布并进行定量分析，高效液相色谱检测视网膜中谷氨酸含量变化，HE染色计数视网膜神经节细胞密度。结果　对照组及褪黑素治疗组GFAP免疫阳性染色主要局限于视网膜神经纤维层，而糖尿病组GFAP阳性染色几乎贯穿视网膜全层。与对照组相比，糖尿病组视网膜MDA含量增加而GSH含量减少，GFAP表达增加而GS表达减少，谷氨酸含量增加，视网膜神经节细胞密度明显下降（均为P<0.01）。褪黑素治疗组与对照组各指标相比差异均无统计学意义（均为P>0.05）。结论　褪黑素能抑制糖尿病视网膜的氧化应激反应，恢复视网膜Müller细胞功能酶GS的含量，减少视网膜内谷氨酸堆积，保护视网膜神经节细胞。     

A key role for calpains in retinal ganglion cell death

PURPOSE: The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death. METHODS: Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates. RESULTS: In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death. CONCLUSIONS: These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.     

腺病毒载体介导的色素上皮衍生因子对大鼠视网膜缺血再灌注损伤的保护作用

目的:观察重组腺病毒介导的色素上皮衍生因子(Ad-PEDF)对大鼠视网膜缺血再灌注损伤的保护作用及机制。方法:选用健康大鼠96只,随机分为正常组、缺血再灌注组、缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组,以前房加压的方法制备大鼠视网膜缺血再灌注模型,缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组分别玻璃体腔注射Ad-CMV或Ad-PEDF1μL(滴度3.8×109/PFU),每组按照时间点12,24,72,168h,为4亚组,光学显微镜观察视网膜组织切片情况,并测量视网膜内层厚度及神经节细胞层神经节细胞数量。以TUNEL方法观察大鼠视网膜神经节细胞凋亡情况。结果:Ad-PEDF组视网膜内层厚度均超过缺血组及缺血+Ad-CMV组,Ad-PEDF组神经节细胞数目多于缺血组及Ad-CMV组,Ad-PEDF组视网膜神经节细胞凋亡细胞少于缺血组及Ad-CMV组,凋亡程度减轻,上述差异均具有显著性(P<0.05)。结论:腺病毒介导的色素上皮衍生因子玻璃体腔注射能够恢复大鼠视网膜缺血再灌注损伤所致的视网膜内层厚度降低,神经节细胞密度减少,具有保护作用。     

Pathological changes in human retinal ganglion cells associated with diabetic and hypertensive retinopathy.

BACKGROUND: To examine whether systemic diseases like diabetes and arterial hypertension, which frequently cause retinopathies leading to blindness effect the morphology of retinal ganglion cells (RGC). METHODS: Histological retina material with a history of being untreated, or laser-coagulated (LC) diabetic retinopathy (DR), or arterial hypertensive retinopathy (AHR) was used. The RGC were labeled by introducing crystals of the fluorescent carbocyanine dye DiI into the nerve fiber layer, which contains ganglion cell axons. RESULTS: The typical silhouettes of both major types of RGC, parasol and midget cells, were identified. The axons in DR and AHR retinas showed morphology changes such as irregular swelling and beading. Dendritic field sizes were significantly reduced in RGC of both the hypertonic and diabetic retinas. A significant reduction in branching frequency was evident in both the diabetic and hypertonic retinas, in both the midget and the parasol cells. In LC retinas, both parasol and midget RGC were observed within the LC spots, although their numbers were dramatically decreased compared with normal retinas. CONCLUSIONS: The data suggest that diabetes and arterial hypertonia have similar effects on the morphology of RGC, in addition to causing microvascular alterations and bleeding. Therefore, therapeutic measures and prognostic outcomes in diabetic and hypertensive retinopathy should also consider regressive changes in retinal neurons.     

Pathological changes in human retinal ganglion cells associated with diabetic and hypertensive retinopathy

Background To examine whether systemic diseases like diabetes and arterial hypertension, which frequently cause retinopathies leading to blindness effect the morphology of retinal ganglion cells (RGC). Methods Histological retina material with a history of being untreated, or laser-coagulated (LC) diabetic retinopathy (DR), or arterial hypertensive retinopathy (AHR) was used. The RGC were labeled by introducing crystals of the fluorescent carbocyanine dye DiI into the nerve fiber layer, which contains ganglion cell axons. Results The typical silhouettes of both major types of RGC, parasol and midget cells, were identified. The axons in DR and AHR retinas showed morphology changes such as irregular swelling and beading. Dendritic field sizes were significantly reduced in RGC of both the hypertonic and diabetic retinas. A significant reduction in branching frequency was evident in both the diabetic and hypertonic retinas, in both the midget and the parasol cells. In LC retinas, both parasol and midget RGC were observed within the LC spots, although their numbers were dramatically decreased compared with normal retinas. Conclusions The data suggest that diabetes and arterial hypertonia have similar effects on the morphology of RGC, in addition to causing microvascular alterations and bleeding. Therefore, therapeutic measures and prognostic outcomes in diabetic and hypertensive retinopathy should also consider regressive changes in retinal neurons.