【摘要】 目的：研究EGb761对视神经夹伤后兔视网膜视神经节细胞(RGC)的保护作用。方法：大白兔24只，右眼为实验组，左眼为对照组，制作视神经夹伤模型。右眼球后注射EGb761(60mg/kg)，左眼球后注射等容量平衡盐液BSS。于伤后4，7，14d取材，应用病理图像分析仪计数RGC;并进行髓鞘碱性蛋白(MBP)的免疫组化染色分析。结果：伤后3～14d，两组RGC 数目均下降，差异有统计学意义(P<0.01);实验组RGC数目明显高于盐水组，差异有统计学意义(P<0.01);伤后两组均有MBP阳性表达，BSS组表现为强阳性;实验组MBP表达呈下降趋势，14d表达呈弱阳性;BSS组表达也随时间有所减弱，但14d时表达仍明显;不同时间点实验组RGC阳性率均低于BSS组，差异均有统计学意义(P<0.01)。结论：EGb761能降低视神经夹伤后MBP的含量，提高RGC的存活数量。
【关键词】 EGb761;视网膜神经节细胞;视神经夹伤;兔;髓鞘碱性蛋白
Preventive effects of extract of ginkgo biloba on retinal ganglion cells after optic nerve crush injury in rabbits
Feng Qiao, YongXia Liu,JinHua Liu,LiPing Jiang, YunCui Cheng
Department of Ophthalmology, Central Hospital of Hengyang, Hengyang 421001, Hunan Province, China;Nanhua University, Hengyang 421001, Hunan Province, China;Eye Center, the 1st Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
AbstractAIM: To observe the protective effect of EGb761 on retinal ganglion cells (RGC )in rabbits with optic nerve crush injury by myelin basic protein (MBP )in rabbits’ retina.METHODS: Twentyfour rabbits were divided into experimental(right eye)and control(left eye) groups. The rabbits’ optic nerves were crushed by minivascular nip. EGb761 60mg/kg were injected into the retrobulbar of right eye and the same volume balanced saline solution (BSS) were injected into the retrobulbar of left eye. 4, 7 and 14 days after injury, the number of the RGC was counted by image analysis system, and the immunohistochemical staining of the MBP was observed.RESULTS: Three to fourteen days after injury, the number of RGC in the experimental group and control group were lower than that in normal by microscope. The differences were significant (P<0.01); the number of RGC in experimental group was higher than that in the control group. The difference was significant (P<0.01); the results of immunohistochemical staining: expression of MBP in both experimental group and control group were gradually decreased after injury. The expression of MBP in experimental group was less apparent than that in BSS group and decreased obviously at 14 days, while the expression of MBP in BSS group was still obvious at 14 days. The positive rates of RGC in different durations of experimental group were lower than those in the BSS group. The differences were significant (P<0.01).CONCLUSION: EGb761 can reduce the content of MBP increase, the survival rate of RGC and may have the protective effect in preventing RGC from degeneration.
KEYWORDS: EGb761; retinal ganglion cell; optic nerve crush injury;rabbit;myelin basic protein
0 引言
视神经损伤是常见的眼外伤，主要造成以视网膜神经节细胞(retinal ganglion cells, RGC)为主的神经元变性死亡[1]。视神经损伤后神经元不易再生，其治疗较困难。银杏叶提取物EGb761的主要活性成分黄酮类和银杏内酯具有清除自由基、抗氧化和影响神经递质释放的特性，在中枢及周围神经损伤的动物模型中均显示出神经保护作用[2]，但目前机制尚不清楚。我们将EGb761用于兔视神经损伤模型，通过观察RGCs数目的变化及髓鞘碱性蛋白(MBP)免疫组织化学的表达，进一步研究EGb761在视神经保护和修复中的作用及机制，探索治疗视神经病变的新途径。
1 材料和方法
1.1 材料
健康成年大耳白兔24只(广西医科大学动物实验中心提供)，雌雄兼用，体质量2.0～2.5kg，眼部检查无异常。EGb761 (上海渌源实业公司)，MBPmAb和DAB试剂(上海长岛生物技术有限公司)。
1.2 方法
每只兔右眼为实验眼，左眼为正常对照眼，于实验前1wk每日右眼球后注射EGb761(60mg/kg)，左眼球后注射等容量平衡盐液BSS，直至处死。戊巴比妥(30mg/kg)耳缘iv麻醉家兔后，仰卧位固定在手术台上，开睑器撑开家兔上下眼睑，沿角巩缘弧形剪开球结膜，分离球后组织，暴露视神经，用微型无创血管钳自球后约3mm处钳夹视神经30s造成视神经损伤。松开血管钳，缝合球结膜，结膜囊里涂四环素眼膏。术后用直接检眼镜检查眼底，防止损伤视网膜供应血管。麻醉苏醒后出现瞳孔散大，直接对光反应消失，间接对光反应存在，眼底无明显出血渗出者记入实验，否则去除。
1.2.1 RGCs计数
分别于伤后3，7，14d取材，每次8只兔。耳缘iv空气10mL，栓塞致死，立即完整摘除眼球，在角巩缘3∶00，9∶00及视神经做缝线标记，多聚甲醛(含5万U肝素)灌注固定24h，乙醇梯度脱水，经视神经纵行剖开眼球，二甲苯透明，常规石蜡包埋视神经和视网膜，切片厚3μm，贴于经多聚赖氨酸处理的载玻片上干燥，染色，光镜下观察由专人进行应用图像分析系统(×400)测量每张标本自视神经两旁1.5mm起，每侧选择3个(共6个)高倍视野，计数RGCs数， 每个密度值由每眼6张切片的平均值作为细胞密度值。
1.2.2 MBP的检测
切片脱蜡至水，PBS缓冲液洗5min×3次;乙醇梯度脱水，抗原修复;PBS缓冲液洗5min×3次，滴加正常血清封闭液，37℃，30min;滴加一抗(MBP单克隆鼠抗)，37℃,1h;PBS缓冲液洗5min×3次;滴加生物素化二抗，20℃，20min;PBS缓冲液洗5min×4次，DAB显色，镜下控制反应时间，蒸馏水洗，苏木素轻度复染，水洗后50℃～60℃热水返兰，盐酸乙醇分化，脱水、透明、中性树胶封片、显微镜下观察。免疫组织化学切片染色结果判断：光镜下细胞质或突起呈黄色或棕黄色者为阳性。
统计学分析：应用SPSS 13.0统计学软件对数据进行统计学分析。各组数据均用±s表示。两组间RGCs数目的比较采用独立样本的t检验，P< 0. 05为差异有统计学意义。
[1] [2] 下一页 |
