兔眼视网膜下注射N-甲基右旋天冬氨酸诱导视网膜变性的组织病理学研究  

Pathological study of retinal excitotoxicity following subretinal injection of NMDA

作　　者：张纯[1] 徐永胜[1] 王薇[1] 曹安民[1] 

机构地区：[1]北京大学第三医院北京大学眼科中心,100083

出　　处：《中华眼科杂志》2007年第10期922-927,共6页Chinese Journal of Ophthalmology

摘　　要：目的研究视网膜下注射兴奋性氨基酸N-甲基右旋天冬氨酸（NMDA）对神经细胞变性的作用。方法取12只1个月龄有色家兔，视网膜下注射10μl（30mmol／L）NMDA（溶剂为DMEM-F12），形成视网膜隆起，在注射12、24、48h及1周后分别处死家兔，取其视网膜组织进行免疫组织化学检测和电镜观察。应用抗Calretinin、Calbindin、PKCα抗体，分别标记视网膜无长突细胞、水平细胞及视杆双极细胞；采用原位缺口末端标记技术（TUNEL）技术标记凋亡细胞。结果损伤早期（12～24h），实验组视网膜可见散在细胞核固缩浓染的光感受器细胞，并有无长突细胞和神经节细胞的早期严重变性；中期（48h）视网膜各层神经元均出现病理性改变；损伤晚期（1周）视网膜各层细胞数目明显减少。损伤早期，TUNEL技术标记的阳性细胞位于视网膜各层。免疫组织化学和形态学计量资料显示视网膜下注射NMDA后，水平细胞、无长突细胞及神经节细胞数目明显减少，视杆双极细胞数目基本无变化。超微结构观察显示有凋亡、坏死、水肿变性及混合型细胞死亡等多种变性形式。结论视网膜下聚集NMDA时，光感受器细胞、水平细胞、视杆双极细胞、无长突细胞及神经节细胞均表现为视网膜兴奋性毒性反应，与以往体内及体外研究结果显示的仅有内核层神经元死亡情况不同。Objective To study neuronal excitotoxicity induced by subretinal injection of glutamate analogues. Methods We injected 30 mmol/L N-methyl-D-aspartate （NMDA） in 10 μl of DMEM/F12 into subretinal blebs in 24 one-month-old rabbits that were killed 0.5, 1,2 or 7 days after injection. The retinas were studied histopathologically by light and electronic microscope. The horizontal cells, rod bipolar cells and amacrine cells were labeled by antiserum against calbindin, protein kinase Cot, and calretinin, respectively. TUNEL labeling was performed to identify apoptotic cells. Results Compared with vehicle injected control retina, treated retina showed an early and severe morphological changes in both amacrine and ganglion cells and scattered densification in photoreceptor nuclei at acute stage （ 〈 1 d）, all retinal neurons damage at the intermediate phase （2 d）, and a significant loss of cells in all layers of the retina at the chronic phase （7 d）, respectively. Horizontal, amacrine and ganglion cells were reduced after subretinal injection of NMDA. The number of rod bipolar cells had little change. The morphological changes of neuronal death included apoptosis, necrosis, hydropic degeneration and karyorrhectic cell death. TUNEL positive nuclei were significantly （ P 〈 0.01 ） increased in all retinal layers at the acute phases after NMDA administration. Conclusions NMDA administration into sub-retina induces excitotoxicity in photoreceptor, horizontal, rod bipolar, amacrine, and ganglion cells of retina, which is different from the other models of retinal cultures and intravitreal injection that only cause neuronal death in the inner retina.

关 键 词：视网膜变性 N-甲基天冬氨酸 注射 病灶内 

分 类 号：R686[医药卫生—骨科学]