神经生长因子对脱髓鞘性视神经炎小鼠视网膜神经组织保护作用的实验研究

Protective effects of nerve growth factor against retinal ganglion cell damage in demyelinated optic neuritis in mice

目的 研究神经生长因子（NGF）对脱髓鞘性视神经炎小鼠视网膜神经组织的保护作用.方法 实验研究.50只C57BL/6小鼠按随机数字表法分为对照组（10只）、BSS组（20只）和NGF组（20只）,取右眼为实验眼.后两组应用MOG35-55多肽加完全弗氏佐剂皮下注射建立实验性视神经炎小鼠模型,每天对各组小鼠进行称重及神经功能评分.NGF组和BSS组分别于免疫后的第4天和第10天,对右眼进行玻璃体腔注射3μg/μl NGF或2 μl BSS.分别于免疫当天、免疫后的第7天和免疫后的第14天,对每组小鼠进行闪光视觉诱发电位（f-VEP）和闪光视网膜电图（f-ERG）检查;采用HE染色、LFB染色、Bielschowsky银染分别评估视神经炎性细胞浸润、髓鞘脱失、轴突病理改变;使用TUNEL法检测视网膜神经节细胞凋亡并计算凋亡指数.对两组实验数据采用t检验进行统计学分析.结果 NGF组与BSS组小鼠发病时间和临床评分比较,差异均无统计学意义（t=-1.844,P=0.079;t=-2.012,P=0.059）.在不同时间点,NGF组和BSS组的f-VEP差异均无统计学意义（P＞0.05）.在免疫后的第14天,NGF组f-ERG b波潜伏期较BSS组缩短,振幅较BSS组增大,两组差异有统计学意义（t=5.909,P=0.000;t=3.602,P=0.043）.LFB染色示,NGF组和BSS组视神经的脱髓鞘面积占横截面比例分别为（31.50±8.72）%、（29.91±10.00）%,差异无统计学意义（t=0.298,P=0.709）.TUNEL检测结果示,NGF组小鼠视网膜神经节细胞凋亡指数[（15.18±3.36）%]低于BSS组[（34.14±3.83）%],差异有统计学意义（t=11.790,P=0.000）.结论 NGF可以促进脱髓鞘性视神经炎小鼠视网膜神经节细胞的存活,对其视网膜神经组织可能具有一定的保护作用.

Objective To investigate the protective effects of nerve growth factor （NGF） against retinal ganglion cell （RGC） damage in demyelinated optic neuritis in mice. Methods Experimental study. Fifty C57BL/6 mice were randomly divided into 3 groups： the NGF-treated group （NGF）, the balanced salt solution group （BSS）, and the control group （CON）. The right eye was used as the experimental eye. Optic neuritis and experimental autoimmune encephalomyelitis （EAE） were induced in the mice by subcutaneously injecting MOG35-55 peptide and complete Freund＇s adjuvant （CFA） in the NGF and BSS groups. NGF （3 μg/2 μl） was intravitrealy injected into the right eyes of the NGF group and BSS （2 μl） was intravitreally injected into the right eyes of the BSS group on days 4 and 10 post-immunization. Flash visual evoked potential （f-VEP） and flash electroretinogram （f-ERG）responses were recorded on days 0, 7 and 14 in all groups. After examination by visual electrophysiology, histological evaluation was performed. Consecutive sections of optic nerve were stained with hymatoxylin-eosin, Luxol fast blue and Bielschowsky silver impregnation to assess inflammation, demyelination, and axonal pathology. Apoptosis of RGC was measured by the TUNEL technique. The t-test was used to analyze the data of NGF and BSS groups. Results The clinical score and weight in the NGF group and BSS group did not change （t=-1.844, P=0.079;t=-2.012, P=0.059）. At all times, the results of the f-VEP in the NGF group were not significantly different from f-VEP in the BSS group （P〉0.05）. The ERG amplitude and latency of the b-wave were significantly different from the BSS group at day 14 after immunization （t=5.909, P=0.000; t=3.602,P=0.043）. LFB staining showed that the areas of demyelination in the NGF and BSS groups were （31.50±8.72）% and （29.91±10.00）%, respectively; there was no singnificant difference between the two groups （t=0.298, P=0.709）. At the same time, TUNEL assay showed the apoptotic index of RGC [（15.18±3.36）%] in the NGF group was significantly lower than that in the BSS group [（34.14±3.83）%）]（t=11.790, P=0.000）. Conclusion NGF has no significant effect on clinical score or degree of demyelination in mice. It can not decrease the incidence of disease, but treatment with NGF shows a protective effect on the survival and function of RGC.