大鼠视网膜光损伤模型中小胶质细胞的迁移及活化

摘要

目的 探讨大鼠视网膜光损伤模型中小胶质细胞的迁移、活化及其与光感受器凋亡的关系.方法 将SD大鼠分为光照组(n=78)和正常对照组(n=15),光照组大鼠在自制的光损伤箱中接受强度为2500 lux的宽谱蓝光照射24 h,建立光损伤模型.在光照结束后2 h、6 h、1天、3天、7天和14天时(每一时间点,n=13),用原位末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)染色观察两组大鼠视网膜细胞凋亡情况;用免疫荧光染色观察视网膜OX42( + )小胶质细胞的形态改变和迁移活动,并对视网膜外层的TUNEL( + )细胞和OX42( + )细胞分别计数并绘制时间-数量曲线;用透射电镜观察进入光感受器层的小胶质细胞的吞噬行为;用real-time PCR法定量分析光照后视网膜胶质源性神经毒性物质IL-1β mRNA的表达变化.结果 光照结束后2 h视网膜外核层(out nuclear layer,ONL)即可见TUNEL( + )细胞,1天后达高峰,3天后逐渐减少;光照结束后6 h视网膜ONL开始出现少量OX42( + )细胞,逐渐增多并于3天后达高峰,7天后渐消失,其形态转变为肥大细胞体的激活型.从时间-数量曲线可见OX42( + )细胞的迁移高峰落后并紧随凋亡高峰;强光照射上调了视网膜IL-1β mRNA的表达,其随时间变化的趋势同小胶质细胞的激活和迁移趋势基本一致;透射电镜显示进入ONL的小胶质细胞吞噬了光感受器的外节膜盘.结论 视网膜光损伤模型中光感受器的凋亡诱导小胶质细胞向ONL的迁移、活化及吞噬行为,并伴有视网膜IL-1β表达水平的升高,小胶质细胞的活化可能在加速光感受器变性过程中起重要作用.%Objective To investigate the relationship between the migration and activation of retinal microglia and the apoptosis of photoreceptors in SD rats with retinal photic injury. Methods SD rats in illumination group ( n = 78) and control group ( n = 15) accepted dark adaptation prior to light exposure at 2 500 lux and each for 24 hours. At the time points of 2 hours,6 hours, 1 day, 3 days, 7 days and 14 days after light exposure, TdT-mediated dUTP nick end labeling (TUNEL) assay and mouse-antirat OX42 antibody were used to detect DNA fragmentation and label retinal microglia cells. The number of TUNEL(+) and OX42(+) cells in the outer retina were counted and the time-number curve were established. Electron micrographic image was used to observe the phagocytization of microglia. Real-time PCR was used to evaluate the retinal IL-1β mRNA level. Results TUNEL(+) cells were noted in the outer nuclear layer (ONL) as early as 2 hours after light exposure, and their presence were noticeably increased to reach the peak on the 1st day,but declined on the 3rd day. In contrast,OX42(+) cells assumed as a more ameboid configuration were seen in the ONL from 6 hours after light exposure,and then reached the peak on the lst day and gradually disappeared on the 7th day, so as the expression of retinal IL- 1 β mRNA.Electron micrography showed that microglia migrating into the ONL phagocytized the ROS (rod out segment) disc of photoreceptors. Conclusions The activation and migration of retinal microglia, as well as the expression of microglia-derived toxic factor (IL-1β), coincides with photoreceptor apopotosis,suggesting activated microglia play a major role in the further degeneration of photoreceptors after the exposure to intense light.