作者:解正高, 吴星伟, 庄朝荣, 陈放, 王正, 王雅坤, 华欣
【摘要】 目的: 探讨腹腔注射银杏叶提取物(Ginkgo biboba extract,EGb 761)对豚鼠视神经横断伤后视网膜神经节细胞(retinal ganglion cell, RGC)形态及功能的保护作用。方法: 75只白化豚鼠随机等分为正常对照组、假手术组、模型组、生理盐水组和EGb 761组。分离暴露豚鼠的右眼视神经并于球后1.0 mm处进行横断造模,正常对照组不作任何处理,假手术组仅分离暴露视神经。生理盐水组和EGb 761组分别于实验开始前1周每日腹腔注射1次相应体积生理盐水和EGb 761(100 mg/kg),术后继续给药4周。术后第4天,各组随机处死3只豚鼠,采用脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick endlabeling, TUNEL)法检测RGC凋亡;分别于术后第14和28天进行图形视网膜电图(pattern electoretinogram, PERG)检查;摘取眼球作组织病理学检查并计数视网膜垂直经线RGC数目。结果: 术后第4天,正常对照组、假手术组和EGb 761组均未见TUNEL阳性RGC,模型组和生理盐水组均见有TUNEL阳性RGC。术后第14和28天,模型组和生理盐水组RGC数目均少于正常对照组和假手术组(P<0.05),EGb 761组少于正常对照组和假手术组(P<0.05),但显著多于模型组和生理盐水组(P<0.05);术后第14和28天,EGb 761组PERG的N95振幅较模型组和生理盐水组高(P<0.05),与模型组和假手术组相近(P>0.05)。RGC数目与N95振幅呈正相关(r=0.859, P=0.001 5)。结论: 腹腔注射银杏叶提取物EGb 761能抑制豚鼠视神经横断后的RGC凋亡,对RGC的结构和功能具有保护作用。
【关键词】 银杏叶提取物; 视神经损伤; 视网膜神经节细胞; 图形视网膜电图; 神经保护; 豚鼠
Objective: To investigate the effects of Ginkgo biloba extract (EGb 761) on the morphology and function of retinal ganglion cells (RGC) in guinea pigs with optic nerve transection.
Methods: Seventyfive albino guinea pigs were randomly divided into five groups: normal control group, shamoperated group, untreated group, normal saline group and EGb 761 group. No operation was performed in the normal control group. Optic nerve was merely exposed in the shamoperated group, but transected at 1.0 mm from posterior pole of the eye ball in the untreated, normal saline and EGb 761 groups. Guinea pigs in the EGb 761 group or the normal saline group received daily intraperitoneal injection of EGb 761 (100 mg/kg) or corresponding volume of normal saline from 7 days before experiment to 28 days after experiment. Three guinea pigs in each group were sacrificed for apoptosis assay (TUNEL method) of RGC. Pattern electoretinograms (PERGs) were recorded 14 and 28 days after transection, respectively. At the end of the examination, six guinea pigs were killed for histological examination and RGC count.
Results: No TUNELpositive cells were observed in the normal control, shamoperated and EGb 761 groups, but there were TUNELpositive cells in the untreated group and the normal saline group. The numbers of RGCs in the untreated and normal saline groups were less than those in the normal control and shamoperated groups at 14 days or 28 days (P<0.05). Although the number of RGCs in the EGb 761 group was less than those in the normal control and shamoperated groups (P<0.05), it was more than those in the untreated and normal saline groups (P<0.05). N95 amplitude in EGb 761 group was higher than those in the untreated and normal saline groups (P<0.05) and close to those in the normal control and shamoperated groups (P>0.05) at 14 days or 28 days. The number of RGCs was positive correlated to N95 amplitude (r=0.859, P=0.001 5).
Conclusion: EGb 761 can inhibit the apoptosis of RGCs in guinea pigs after optic nerve transection, thus protect the morphology and function of RGCs.
Keywords: Ginkgo biloba extract; optic nerve lesion; retinal ganglion cell; pattern electroretinogram; neuroprotective effect; guinea pigs
青光眼是一种威胁视神经视觉功能,主要与眼压升高有关的眼病,也是目前我国第二大致盲眼病。虽然青光眼的具体发病机制尚不清楚,但目前一致认为视网膜神经节细胞(retinal ganglion cell, RGC)凋亡为青光眼神经损害的中心环节。研究证明银杏叶提取物(Ginkgo biloba extract,EGb 761)具有抗氧化[1]、抗缺血[2]、保护线粒体[3]和抑制一氧化氮合酶活性[4]等广泛的生物学效应。因此,我们采用横断豚鼠球后视神经的方法建立RGC损伤模型观察EGb 761对RGC结构的保护作用,并采用图形视网膜电图(pattern electroretinogram, PERG)评价EGb 761对RGC功能的保护作用。
1 材料和方法
1.1 实验材料
1.1.1 实验动物 75只雌性白化豚鼠,体质量190~210 g,购于中国科学院上海实验动物中心,许可证号为SCXK(沪)2007005。
1.1.2 实验药物及试剂 EGb 761,17.5 mg/支(5 mL),批号为9941209C,德国威玛舒培博士大药厂产品;速眠新Ⅱ注射液,又名846合剂,1 mg/mL,军事医学科学院军事兽医研究所产品,批号为(2004)005013;陆醒宁注射液,1 mg/mL,军事医学科学院军事兽医研究所产品,批号为(2006)006011;甲基纤维素,上海建华精细生物制品有限公司产品;脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick endlabeling, TUNEL)检测试剂盒,美国Promega公司产品;碘化丙啶(propidium iodide,PI),美国Sigma公司产品。
1.1.3 实验设备 手术显微镜(SM2000J,上海轶德医疗设备有限公司);PERG检查仪(EP1000,日本Tomey公司);荧光显微镜(Axioplan 2,德国Zeiss公司);光学显微镜(Olympus BX50,日本Olympus公司)。
1.2 实验方法
1.2.1 实验动物分组 豚鼠使用严格遵循视觉与眼科研究协会(Association for Research in Vision and Ophthalmology, ARVO)有关视觉和眼科研究动物使用条款。所有豚鼠均在12 h明/12 h暗,温度为22~25 ℃,湿度为55%~60%的环境中饲养。采用计算机随机数字法将75只豚鼠分为5组:正常对照组、假手术组、模型组、模型+生理盐水组(生理盐水组)、模型+EGb 761组(EGb 761组),每组15只。每只鼠的右眼为实验眼,左眼为用于计算RGC存活指数的对照眼。
1.2.2 视神经横断方法 按照Nakazawa等[5]报道的方法进行视神经横断,建立豚鼠视神经横断模型。臀部注射速眠新Ⅱ注射液0.6 mL/kg麻醉后,在手术显微镜下剪开外眦角;自12∶00处沿角膜缘逆时针呈180°打开球结膜并向后钝性分离暴露视神经,钝性分离视神经鞘表面的血管后于球后1.0 mm处横断球后视神经。分别缝合球结膜和外眦角,涂金霉素眼膏。手术结束后立即在臀部注射陆醒宁(1.2 mL/kg)催醒。术中尽可能钝性分离血管以不影响眼球的血液供应,同时尽可能减少对视神经的牵拉。
1.2.3 各组处理方法 正常对照组:不给任何处理;假手术组:按造模方法暴露视神经,但不横断;模型组:按上述视神经横断方法横断视神经;生理盐水组:于实验前7 d腹腔注射生理盐水,每日1次,体积参照EGb 761的剂量,视神经横断后继续注射28 d;EGb 761组:于实验前7 d腹腔注射EGb 761,剂量为100 mg/kg[6],每日1次,持续至视神经横断后继续注射28 d。视神经横断后第4天,各组随机处死3只豚鼠,摘取眼球作石蜡切片,进行RGC凋亡的TUNEL标记检测。分别于视神经横断后14 d和28 d进行PERG检查,检查后每组处死6只豚鼠作视网膜组织病理学检查,并作RGC计数。
1.2.4 TUNEL检测RGC凋亡 按TUNEL检测试剂盒说明书提供的方法进行石蜡切片和常规脱蜡。用蛋白酶K孵育后滴加TUNEL反应混合液,最后用PI衬染细胞核。经上述处理后,在荧光显微镜下进行观察并采用AxioVision软件进行同步图像采集,视网膜组织中凋亡的RGC细胞核呈黄色荧光着色。
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