视神经损伤诱导小胶质细胞表型变化与视网膜神经节细胞死亡的关系  

Correlation of changes in retinal microglia phenotype with ganglion cell death in mice after optic nerve injury

作　　者：游添敬 杨元星 何军材 阿洛丹 马翔[1] 徐海伟 YOU Tianjing;YANG Yuanxing;HE Juncai;A Luodan;MA Xiang;XU Haiwei(Department of Ophthalmology,the First Affiliated Hospital of Dalian Medical University,Dalian,Liaoning Province,116014;Department of Ophthalmology,Chongqing Key Lab of Visual Damage and Regeneration&Restoration,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]大连医科大学附属第一医院眼科,辽宁大连116014 [2]陆军军医大学(第三军医大学)第一附属医院眼科,视觉损伤与再生修复重庆市重点实验室,重庆400038

出　　处：《陆军军医大学学报》2024年第17期1934-1942,共9页Journal of Army Medical University

基　　金：国家重点研发计划(2021YFA1101203)。

摘　　要：目的探究视神经损伤后视网膜小胶质细胞表型改变与视网膜神经节细胞(retinal ganglion cells,RGCs)死亡的关系。方法将6~8周龄雄性C57BL/6J小鼠按照随机数字表法分为造模后1、3、7、14 d损伤组和Sham组,每组4只。对损伤组进行视神经钳夹(optic nerve crush,ONC),对Sham组行相同的手术操作但不夹持视神经;结合闪光视觉诱发电位(flash visual evoked potential,fVEP)和免疫荧光染色评估视神经损伤对小鼠视觉功能和RGCs数量的影响;RT-qPCR和免疫荧光染色检测视神经损伤对视网膜小胶质细胞表型变化的影响。结果fVEP结果显示,与Sham组相比,损伤组的视觉传导功能随时间进展逐渐下降(P<0.01)。免疫荧光染色结果显示RGCs数量减少主要发生在损伤后7 d内(P<0.01);同时视网膜小胶质细胞的数量在损伤后7 d达到峰值(P<0.01)。RT-qPCR显示与Sham组相比,ONC后7 d视网膜疾病相关型小胶质细胞(disease-associated microglia,DAM)及干扰素反应型小胶质细胞(interferon-responsive microglia,IRM)特征基因表达均显著增加(P<0.01)。免疫荧光染色提示DAM的数量在ONC后3 d达到峰值(P<0.01),但随着时间的进展比例逐渐降低(P<0.05);而IRM的数量及比例在ONC后7 d达到峰值(P<0.01)。相关性分析提示IRM的数量与神经节细胞死亡强烈相关(P<0.01)。结论视神经损伤后视网膜小胶质细胞由早期的DAM型向IRM型转化可能是RGCs死亡的重要原因。Objective To explore the relationship between phenotypic changes of retinal microglia and retinal ganglion cells(RGCs)death after optic nerve injury.Methods Male C57BL/6J mice(6 to 8 weeks old)were randomly divided into 1-,3-,7-,and 14-day injury groups and sham operation group,with 4 mice in each group.The eyes in the injured groups were inflicted with optic nerve crush(ONC),while the eyes of the sham operation group were treated with the same operation procedure but without optic nerve clamp.Flash visual evoked potential(fVEP)and immunofluorescence staining were employed to evaluate the impact of optic nerve injury on visual function and number of RGCs.RT-qPCR and immunofluorescence staining were applied to detect the effecy of optic nerve injury on phenotypic changes in retinal microglia.Results fVEP results showed that the visual conduction of the injured eye was gradually decreased over time when compared with that of the sham group(P<0.01).Immunofluorescence staining revealed that the number of RGCs was lost mainly within 7 d after injury(P<0.01).At the same time,the number of retinal microglia reached its peak at 7 d after injury(P<0.01).RT-qPCR indicated that the expression of disease-associated microglia(DAM)and interferon-responsive microglia(IRM)specific genes were significantly increased when compared with the sham group at 7 d after ONC(P<0.01).Immunofluorescence staining displayed that the number of DAM peaked at 3 d after ONC(P<0.01),but the proportion was decreased gradually with the progress of time(P<0.05).The number and proportion of IRM peaked 7 d after ONC(P<0.01).Correlation analysis suggested that the number of IRM was strongly correlated with the loss of ganglion cells(P<0.01).Conclusion The conversion of retinal microglia from DAM type to IRM type after optic nerve injury may be an important cause of ganglion cell loss.

关 键 词：视神经损伤 视网膜 小胶质细胞 表型重塑 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R339.146[医药卫生—基础医学] R774.6