NMDA诱导青光眼模型的视网膜双极细胞损伤及机制研究

发布时间：2018-04-23 04:00

本文选题：青光眼 + 动物模型　；参考：《武汉大学》2016年博士论文

【摘要】：第一部分NMDA诱导小鼠视网膜兴奋毒性损伤模型的特征目的：研究N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)诱导视网膜兴奋毒性损伤小鼠模型的形态和功能特征。方法：6周龄C57BL/6J小鼠随机分为低、中、高三个剂量组,玻璃体腔内分别注射NMDA10、20、30nmol,建立视网膜兴奋损伤动物模型。我们在整体动物水平通过眼底照相、视网膜电图(electroretinogram, ERG);在组织学水平通过视网膜铺片荧光血管染色、视网膜石蜡切片HE染色、视神经甲苯胺蓝染色、全视网膜铺片Brn-3a免疫荧光染色及RGCs计数观察术后6h、12h、24h、48h、7dRGCs的损伤情况。结果：NMDA干预后24 h,RGCs数量明显减少,IPL厚度变薄。眼底视网膜呈苍白色改变,血管迂曲痉挛。Isolectin IB4荧光染色显示视网膜血管,观察到MDA造模后,24h内视网膜血管结构未见明显损伤。ERG显示a波和b波潜伏期的变化不大,b波振幅下降。光镜下可见RGCs密度逐渐降低；视神经轴索呈退行性变,与NMDA的作用时间呈正相关。RGCs计数显示NMDA诱导的RGC减少主要集中于术后一天,24 h RGCs数量减少83.97%(P0.01)。结论：玻璃体腔内注射NMDA可诱导RGCs的显著减少和视网膜功能损害,与急性青光眼性损伤具有相似性,可为视神经保护研究提供研究条件。检测ERG b波,对于诊断和评估视网膜兴奋损伤具有一定的意义。第二部分NMDA诱导视网膜视杆双极细胞功能性PKCa转运的机制研究目的：观察NMDA诱导的视网膜兴奋毒性损伤小鼠模型中视杆-双极细胞中重要的功能性PKCa转运现象并探讨其机制。方法：6/3周龄C57BL/6J小鼠玻璃体腔内注射NMDA30 nmol,建立视网膜兴奋损伤动物模型。分别在损伤后12 h、24 h、48 h取出视网膜,利用视网膜切片免疫荧光及TUNEL凋亡染色,观察视网膜的分层以及细胞存活情况、PKCa标记的视杆-双极细胞及NMDA受体各亚基在视网膜上的突触表达；利用全细胞膜片钳技术,记录视网膜各类型双极细胞的电生理特性改变；通过单细胞PCR方法,检测视杆-双极细胞中mGluR6-Go-TRPMl信号通路相关组成和调控因子的mRNA水平。结果：观察到NMDA在诱导内层视网膜RGCs急性凋亡的同时,视杆-双极细胞内出现功能性PKCa转运的现象；证实与外丛状层上NR1、NR2B、NR2D亚基的突触表达量明显升高存在空间一致性。利用全细胞膜片钳技术,揭示了NMDA可特异性诱导视杆-双极细胞mGluR6的功能变化,对光反应消失,且损伤具有可逆性。进一步通过单细胞PCR检测,NMDA造模后GDIs的上调和部分GAPs、RGS的下调,共同导致了Gao亚基的失活,TRPM通道开放。结论：玻璃体腔内注射NMDA,可诱导急性的神经节细胞凋亡及双极细胞损伤。通过作用mGluR6-Go-TRPM1这条信号通路,影响视杆双极细胞的生理功能。相关机制研究可对今后治疗人类视杆-双极细胞机制相关的眼病起到指导性作用。
[Abstract]:Part one: the characteristics of NMDA induced retinal excitotoxicity injury model in mice objective: to study the morphological and functional characteristics of the model induced by N-methyl-D-aspartate (NMDA-N-methyl-D-aspartate) -induced retinal excitotoxicity injury in mice. Methods C57BL/6J mice at 6 weeks of age were randomly divided into three groups: low, middle and high dose groups, and intravitreous injection of NMDA1020 + 30nmol into the vitreous cavity to establish the animal model of retinal excitatory injury. At the overall animal level, we take pictures of the fundus, electroretinograms, ERGs; at the histological level, they are stained by fluorescent vessels on the retina, paraffin sections of the retina are stained with HE, and the optic nerves are stained with toluidine blue. Brn-3a immunofluorescence staining and RGCs count were used to observe the injury of RGCs at 6 h, 12 h, 24 h, 48 h and 7 d after operation. Results the number of RGCs in RGCs decreased significantly 24 h after the intervention of 1: NMDA, and the thickness of IPL became thinner. The retinal fundus showed pale white changes, vasospasm. Isolectin IB4 fluorescence staining showed retinal vessels. No obvious damage to retinal vascular structure was observed within 24 hours after MDA modeling. Under the light microscope, the density of RGCs decreased gradually, and the optic axons showed degenerative change, which was positively correlated with the action time of NMDA. RGCs count showed that the decrease of RGC induced by NMDA was mainly focused on the reduction of the number of RGCs at 24 h after operation by 83.97% (P 0.01). Conclusion: intravitreal injection of NMDA can significantly reduce RGCs and damage retinal function, which is similar to that of acute glaucoma injury, and may provide research conditions for the study of optic nerve protection. The detection of ERG b wave is of significance for the diagnosis and evaluation of retinal excitatory injury. The second part is the mechanism of functional PKCa transport in retinal rod bipolar cells induced by NMDA. Objective: to observe the important functional PKCa transport in the rod bipolar cells in the mouse model of retinal excitotoxicity injury induced by NMDA and to explore its mechanism. Methods NMDA30 nmol was injected into the vitreous of 6 / 3 week old C57BL/6J mice to establish the animal model of retinal excitatory injury. The retina was removed at 12 h, 24 h and 48 h after injury. The retinal sections were stained with immunofluorescence and TUNEL apoptosis. The synaptic expression of PKCa labeled optic rod bipolar cells and NMDA receptor subunits on the retina was observed, and the electrophysiological characteristics of various types of bipolar cells in the retina were recorded by whole-cell patch clamp technique. Single cell PCR method was used to detect the relative components of mGluR6-Go-TRPMl signaling pathway and the mRNA level of regulatory factors in rod-bipolar cells. Results: while NMDA induced acute apoptosis of RGCs in the inner layer of retina, functional PKCa transport was observed in rod bipolar cells, which was confirmed to be spatially consistent with the increase of synaptic expression of NR1, NR2BnR2BnR2D subunit in the outer plexiform layer. By using whole-cell patch clamp technique, it was revealed that NMDA could specifically induce the functional changes of mGluR6 in rod and bipolar cells, and the photoreaction disappeared and the damage was reversible. Furthermore, the up-regulation of GDIs and the down-regulation of some GAPs were detected by single cell PCR, which resulted in the opening of inactivated Gao subunit. Conclusion: intravitreal injection of NMDA can induce acute ganglion cell apoptosis and bipolar cell injury. The physiological function of rod bipolar cells is affected by mGluR6-Go-TRPM1 signaling pathway. The study of the related mechanism may play a guiding role in the treatment of ocular diseases related to the mechanism of human optic rod-bipolar cell.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R775;R-332

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