膳食诱导的肥胖型C57BL/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C57BL/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19wk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C57BL/6小鼠视网膜神经节细胞的凋亡过程。     

洛美利嗪对糖尿病早期大鼠视网膜神经节细胞的保护作用

目的： 探讨洛美利嗪(lomerizine,LOM)对糖尿病早期大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡的保护作用及其机制。

方法： SD大鼠随机分为对照组(CON)、糖尿病组(DM)及洛美利嗪组(LOM),每组40只大鼠。DM和LOM组链脲佐菌素(STZ)按60mg/kg一次性腹腔注射诱导糖尿病模型,CON组给予等量无菌柠檬酸钠溶液腹腔注射。LOM组于糖尿病鼠模型成模后,每日予LOM 60mg/kg灌胃,CON组和DM组采用等量生理盐水灌胃。于第4,8,12wk分别行HE、透射电镜、TUNEL检测RGCs的凋亡情况,同时采用激光共聚焦显微镜检测RGCs内钙离子的浓度。

结果：(1)形态学观察：随病程延长,逐渐出现RGCs数量减少、细胞排列紊乱的病理改变。透射电镜下可见DM组RGCs出现不同阶段的凋亡征象,且随病程延长逐渐加重。LOM组与同期DM组对比,在第8,12wk时RGCs凋亡征象减弱。(2)TUNEL检测：CON组大鼠视网膜神经节细胞层未见凋亡细胞。DM组4wk时视网膜神经节细胞层偶见TUNEL 阳性细胞,并随病程延长逐渐增多,凋亡指数与同期CON组比较明显升高,差异有统计学意义(P<0.01)。LOM 组8,12wk时,与同期DM组比较,染色阳性的RGCs明显减少,凋亡指数明显下降,差异显著(P<0.01)。(3)激光共聚焦显微镜钙离子浓度检测：DM组与同期CON组比较：DM组8,12wk 的RGCs内钙离子荧光染色强度明显升高,有显著差异(P<0.01)。LOM组与同期DM组比较：LOM 组8,12wk的RGCs内钙离子荧光染色强度明显下降,差异有统计学意义(P<0.01)。

结论：LOM对糖尿病大鼠早期RGCs的凋亡具有保护作用。     

糖尿病大鼠早期视网膜神经节细胞凋亡机制的探讨

目的：探讨糖尿病大鼠模型早期视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡的机制。

方法：SD大鼠60只,随机分为对照组(CON)及糖尿病组(DM),糖尿病组一次性腹腔注射1% STZ诱发糖尿病鼠模型,两组于第4,8,12wk分别行HE、透射电镜及TUNEL法检测RGCs的凋亡情况,并应用激光共聚焦显微镜检测RGCs内钙离子浓度的变化。

结果：糖尿病组第8wk开始出现RGCs数量减少、细胞排列紊乱的病理改变,第12wk更为明显。糖尿病组透射电镜下可见第4wk时RGCs出现线粒体肿胀; 第8wk时RGCs内肿胀的线粒体更为明显、数目增多,染色质边集于核膜周边,部分细胞体积缩小、细胞器减少; 第12wk时出现RGCs体积变小,甚至出现细胞核断裂。TUNEL阳性RGCs最早于糖尿病组第4wk时出现,随病程延长凋亡的阳性细胞逐渐增多,凋亡指数与同期对照组相比,差异有统计学意义(P<0.01)。糖尿病组第8,12wk时RGCs内钙离子浓度明显高于同期对照组,差异显著(P<0.01); 糖尿病组第8wk与第12wk比较,升高差异亦有统计学意义(P<0.05)。

结论：糖尿病早期出现了视网膜神经节细胞的凋亡,其机制可能与细胞内钙离子浓度升高有关。     

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。     

17-β雌二醇和他莫昔芬对小鼠慢性高眼压模型的作用研究

目的：探讨17-β雌二醇(17-β-estradiol,E2)和他莫昔芬(tamoxifen,TAM)在慢性高眼压小鼠模型中的调节作用。

方法：通过前房注射磁珠堵塞房角构建小鼠慢性高眼压模型。将C57BL/6成年小鼠随机分为对照组、Beads组、E2组和E2+TAM组。采用眼压计监测各组眼内压(intraocular pressure, IOP)的变化; HE染色观察并测量中央视网膜厚度; Brn3a免疫组织化学染色观察并计数存活的视网膜神经节细胞(retinal ganglion cells,RGCs); Western blot法检测视网膜中胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达。

结果：造模后2wk内,Beads组小鼠平均眼压显著高于对照组,而E2组和E2+TAM组眼压均明显低于Beads组,差异均有统计学意义(P<0.05),且E2+TAM组小鼠平均眼压较E2组更低。造模2wk后,Beads组小鼠视网膜RGCs数量较对照组显著减少(P<0.05),而皮下注射E2+TAM可明显抑制该现象。造模2wk后,各组小鼠中央视网膜厚度比较,差异无统计学意义(P>0.05)。Western blot结果显示,Beads组小鼠视网膜GFAP蛋白表达量升高,注射E2和E2+TAM后,GFAP蛋白表达量显著下降(P<0.05)。

结论：皮下注射E2和E2+TAM均能降低慢性高眼压模型小鼠眼内压,增加RGCs存活数量,且降低视网膜GFAP蛋白表达量,表明E2和TAM在青光眼的发生发展过程中具有一定的保护作用。     

发光二极管照射对大鼠高糖视网膜血管内皮细胞的光生物调节作用及其机制

目的：观察发光二极管照射对大鼠高糖视网膜血管内皮细胞中的光生物调节作用及其机制。

方法：大鼠视网膜血管内皮细胞随机分为三组：正常对照组、高糖模型组、高糖模型发光二极管照射组,高糖模型发光二极管组的细胞在造模48h后开始采用发光二极管对培养箱中的细胞进行照射。MTT细胞凋亡实验检测各组细胞凋亡率; 激光共聚焦显微镜观察各组视网膜血管内皮细胞胞内钙离子变化; Western blot 法检测各组磷酸化丝氨酸-苏氨酸激酶(P-AKT)蛋白表达。

结果：正常对照组、高糖模型组、高糖模型发光二极管照射组凋亡率分别为7.54%±2.67%,31.69%±5.74%,21.65%±3.52%(P<0.05)。正常对照组细胞质微弱Ca²⁺荧光染色呈现出绿色的荧光,其荧光像素值为192.65±50.54; 高糖模型组中,细胞质呈现较强烈的绿色荧光,其荧光像素值为710.69±100.38; 发光二极管照射组中,绿色荧光像素值为430.47±80.67,明显高于正常对照组,但明显低于高糖模型组。三组间细胞中内Ca²⁺荧光像素值有差异(P<0.05)。这三组细胞P-AKT蛋白量分别为10.26±2.47、2.35±0.16、7.46±1.64(P<0.05)。

结论：高糖环境抑制苏氨酸激酶通路活性,对大鼠视网膜血管内皮细胞钙稳态产生影响,促使细胞凋亡,低强度的发光二极管照射可激活苏氨酸激酶通路,降低高糖引起的细胞凋亡率。     

糖尿病大鼠早期视网膜形态观察和Bcl-2、Bax及VEGF表达的意义

目的：探讨B-细胞淋巴瘤因子(B-cell lymphoma factor,Bcl-2),Bcl-2相关X蛋白(Bcl2-Associated X protein,Bax)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在早期糖尿病大鼠视网膜上的表达及意义。

方法：用链脲佐菌素(streptozotocin,STZ)腹腔注射(60mg/kg)制作大鼠早期糖尿病模型。于造模后4、8、12wk颈椎脱臼法处死大鼠,取双眼全眼球组织做石蜡切片并做视网膜铺片,通过HE染色观察视网膜各层的形态学和血管分布变化; 取双眼视网膜组织石蜡切片,通过免疫组化法检测Bcl-2、Bax和VEGF在视网膜组织中的表达。行ADP酶视网膜血管染色,观察视网膜血管形态变化。应用激光共聚焦显微镜检测视网膜细胞的形态、细胞中Ca²⁺的荧光强度和分布变化。

结果：糖尿病组造模12wk突破内界膜的内皮细胞核个数呈递增趋势。糖尿病组视网膜中周部和周边部可见无血管区,无血管区面积也明显大于空白对照组,差异有统计学意义(P<0.05)。糖尿病组大鼠VEGF、Bcl-2和Bax光密度值与空白对照组比较,差异均有统计学意义(P<0.05)。糖尿病组大鼠造模4、8、12wk比较,RGCs内钙离子荧光浓度逐渐升高,荧光染色强度比值升高,差异均有统计学意义(P<0.05)。

结论：早期糖尿病大鼠视网膜的Bcl-2和Bax表达非常明显,从而上调 VEGF的表达。Bcl-2、Bax和VEGF是糖尿病视网膜病变中新生血管形成的重要影响因素。     

Elevation of Intracellular Ca<Superscript>2+</Superscript> Concentration Induced by Hypoxia in Retinal Ganglion Cells

Purpose To analyze the mechanism of hypoxia-induced changes of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in retinal ganglion cells (RGCs). Methods Fluo-3 was applied to the cut edge of the optic nerve of 6-week-old rats. The retina was sliced, and the Ca images were captured. A hypoxic condition was created by superfusing the retinal slice with an oxygen/glucose-deprived solution. Results The retrograde staining method filled the RGCs selectively. Fifteen minutes of hypoxic conditions induced an increase in [Ca²⁺]_i in the RGCs (Δ0.13 ± 0.03, n = 23). Application of 60 μM dl-2-amino-5-phosphonovaleric acid partially blocked the hypoxia-induced [Ca²⁺]_i increase in dendrites (Δ0.03 ± 0.02, n = 4, P < 0.05) but not in the somata (Δ0.12 ± 0.02, n = 9). The RGC dendrites showed a further increase in [Ca²⁺]_i after being switched back to an oxygenated solution (Δ0.14 ± 0.04, n = 4). Neither 6-cyano-7-nitroquinoxaline-2,3-dione disodium, dl-threo-β-benzyloxyaspartate, nifedipine, nor bepridil inhibited the hypoxia-induced [Ca²⁺]_i increase. A Ca²⁺-free superfusion prevented the hypoxia-induced [Ca²⁺]_i increase in the somata (Δ0.07 ± 0.02, n = 5, P < 0.05) but not in the dendrites (Δ0.16 ± 0.005, n = 4). Conclusions The mechanism of the hypoxia-induced increase in [Ca²⁺]_i differs between somata and dendrites. The N-methyl-d-aspartate channel of dendrites seems to be the main route of Ca²⁺ influx. Jpn J Ophthalmol 2007;51:175–180 ? Japanese Ophthalmological Society 2007     

大鼠急性高眼压后视网膜神经节细胞自噬和副凋亡的发生

目的：观察急性高眼压后不同时间点大鼠视网膜神经节细胞中自噬及副凋亡的发生,并探讨其机制。

方法：将50只健康成年SD雄性大鼠随机分为正常对照组、急性IOP损伤3d,1、4、8wk组。利用高眼压(elevated intraocular pressure,IOP)前房灌注法建立SD大鼠急性IOP损伤模型,取各组大鼠的视网膜组织,采用免疫荧光染色法检测视网膜微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)的表达; 利用透射电镜(transmission electron microscopy,TEM)检测视网膜神经节细胞(retinal ganglion cells,RGCs)细胞质中自噬体及胞质空泡的产生,验证自噬及副凋亡的发生。

结果：透射电镜观察可见大鼠RGCs细胞质中包裹着电子致密物的双层或多层膜的自噬泡,正常对照组、急性IOP损伤后3d,1、4、8wk组,RGCs细胞质中每50μm²自噬泡数量分别为0.79±0.43、2.14±0.36、2.29±0.47、1.57±0.51、1.21±0.43个,急性IOP损伤后各组大鼠RGCs内每50μm²自噬泡数量均较正常对照组明显增加,差异有统计学意义(P<0.05)。正常对照组视网膜神经节细胞层(ganglion cell layer,GCL)仅见少量LC3阳性表达,LC3阳性细胞百分比15.90%。急性IOP损伤后3d,1、4、8wk组大鼠GCL中LC3阳性细胞百分比均较正常对照组明显增加,差异有统计学意义(P<0.05)。急性IOP损伤后3d,1、4、8wk组大鼠每200μm内RGCs数量较正常对照组明显减少,差异有统计学意义(P<0.05)。急性IOP后3d持续至8wk透射电镜观察可见大量由线粒体和/或内质网肿胀形成的细胞质空泡。

结论：急性IOP损伤后RGCs涉及自噬和副凋亡的激活,各种类型的程序性细胞死亡(programmed cell death,PCD)可作为单一细胞死亡的形式或多种细胞死亡形式共存,参与急性IOP后视网膜神经节细胞的损伤。     

姜黄素对慢性高眼压大鼠视网膜神经节细胞凋亡的影响及机制

目的：探讨姜黄素对慢性高眼压大鼠视网膜神经节细胞(RGCs)凋亡的影响及机制。

方法：将21只SD大鼠随机分为3组,每组7只,高眼压模型组和姜黄素治疗组大鼠通过烧灼巩膜上静脉法建立慢性高眼压模型,假手术组大鼠仅剪开球结膜,不烧灼巩膜上静脉; 姜黄素治疗组给予4mL/kg姜黄素灌胃,假手术组和高眼压模型组给予4mL/kg纯水灌胃,连续3wk。造模后3wk,采用HE染色观察各组大鼠视网膜组织形态病理变化、RGCs数量及神经节细胞层(GCL)厚度; 采用TUNEL染色观察各组大鼠RGCs和视网膜细胞凋亡情况; 采用实时荧光定量PCR、免疫组织化学染色和Western blot法检测各组大鼠视网膜谷氨酰半胱氨酸连接酶调节亚基(GCLM)与血红素加氧酶-1(HO-1)的表达水平。

结果：与假手术组相比,高眼压模型组和姜黄素治疗组大鼠视网膜组织形态紊乱,RGCs数量减少,GCL变薄,RGCs和视网膜细胞凋亡率均升高,GCLM和HO-1表达量均升高; 与高眼压模型组相比,姜黄素治疗组大鼠视网膜组织形态基本正常,RGCs数量增多,GCL增厚,RGCs和视网膜细胞凋亡率均降低,GCLM和HO-1表达量均升高。

结论：姜黄素在慢性高眼压大鼠模型中可通过上调抗氧化基因GCLM与HO-1的表达抑制RGCs凋亡。     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

Tafluprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

Background

To investigate whether tafluprost, which is a prostaglandin-related compound and an anti-glaucoma drug, has a direct anti-apoptotic effect in cultured retinal ganglion cells (RGCs) and rat RGCs in retinas with optic nerve crush (ONC).

Methods

RGC-5 cells were induced to undergo apoptosis by a serum deprivation and by exogenous glutamate. The level of cell death with or without tafluprost was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in intracellular calcium ([Ca²⁺]i) levels were measured with fluo-4 fluorescence. Rat RGCs were degenerated by ONC. After topical instillation of tafluprost for 7 and 14 days, the numbers of retrograde-labeled RGCs were counted. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells.

Results

Tafluprost dose-dependently promoted RGC-5 cell viability with an optimum concentration of 3?μM (p?=?0.006). Tafluprost significantly reduced caspase-3-positive cells and suppressed [Ca⁺²]i evoked by exogenous glutamate. The cGMP-dependent protein kinase inhibitor and KT-5823 partially blocked the rescue effect of tafluprost (p?=?0.002). The survival rate of RGCs significantly increased in eyes treated with tafluprost (p?=?0.01), and the prevalence of TUNEL-positive cells was significantly decreased 14 days after ONC (p?<?0.001).

Conclusions

These data suggest that tafluprost has an anti-apoptotic effect in RGCs.     

高脂诱导肥胖小鼠的视网膜变性及其与氧化应激的关系

目的:探讨高脂饮食建立的C57BL/6小鼠肥胖模型的视网膜变性的超微结构改变及其与氧化应激的关系。方法:高脂饲料喂养19wk后,小鼠分为肥胖抵抗(DIO-R)组和肥胖倾向(DIO)组,同时对照组小鼠给予基础饲料。应用光镜、透射电镜及TUNEL法检测3组小鼠视网膜超微结构的改变及凋亡情况,并应用生化方法检测视网膜的氧化和抗氧化指标。结果:与对照组及DIO-R组比较,DIO组小鼠视网膜及外核层(ONL)变薄(P<0.01);感光细胞出现凋亡及坏死,其外段视盘膜排列紊乱,部分溶解、断裂,神经节细胞体积变小,细胞质分布紊乱,核染色质固缩;TUNEL法检测结果显示,在ONL层可见较多的凋亡细胞,其细胞凋亡率显著升高(P<0.01)。与对照组比较,DIO组小鼠视网膜匀浆丙二醛(MDA)含量明显升高(P<0.01),超氧化物歧化酶(SOD)活力显著下降(P<0.05)。结论:高脂诱导的肥胖可导致C57BL/6小鼠视网膜变性及细胞凋亡,其机制可能与氧化应激有一定关系。     

Current status of emergency treatment of chemical eye burns in workplaces

Chemical eye burns present an avoidable,but frequent,occupational injury with potentially detrimental consequences for the quality of life and occupational rehabilitation of the injured.A periodical review of guidelines is required to assure the optimal emergency management.We reviewed the literature with emphasis on current German guidelines,primarily MEDLINE.If the crucial first-line measure,the injury prevention has failed and an eye burn has been sustained,the immediate and copious rinsing of the eye is the pivotal emergency treatment modality.Whereas the immediacy and sufficiency of the emergency rinsing are largely unanimous,there is an ongoing debate about the benefits and risks of specific rinsing solutions,and regular updates on guidelines and recommendations for the emergency treatment are warranted.The easiest and readily available rinsing solution is tap water,which fulfils the crucial criteria conveniently in most industrialized countries:purity,sterility,and neutral p H.Other rinsing solutions are proposing higher osmolality to stabilize the physiological p H,because of their superior buffering capacity.However,there is no compelling evidence for a substantial benefit,and some reports suggest that there could be unwanted side effects.In combination with the substantially increased expenditure and a more complex handling procedure,currently a general recommendation of any other solution than tap water is not warranted.     

NgR-Rock信号通路在高糖损伤RGC中的作用

目的：探讨NgR-Rock信号通路在高浓度葡萄糖损伤视网膜神经节细胞(retinal ganglion cells,RGCs)中的机制。

方法：实验分4组：对照组、高糖组、SiNgR组(高糖培养基中加入AAV2-siNgR病毒)和SiRNA空白组(高糖培养基中加入阴性核甘酸序列)。在培养的第3d观察细胞生长状态; Western blot检测NgR、Rock及F-actin的表达; MTT法检测细胞活力; 利用F-actin免疫组织化学染色显示细胞形态。

结果：与对照组相比,高糖组及SiRNA空白组细胞体积缩小,突起较少,细胞折光性增强; NgR及Rock的表达明显上调,F-actin表达减少,细胞活力下降(P<0.05); 而SiNgR组与对照组相比NgR、Rock及F-actin的表达,细胞活力无明显改变(P>0.05)。

结论：NgR-Rock信号通路激活可能是导致高糖环境中RGC损伤的重要机制之一。