Nishiyama T, Nishukawa S, Tamai M
Department of Ophthalmology Tohoku University School of Medicine, Sendai, Japan.
Tohoku J Exp Med. 2000 Aug;191(4):221-32. doi: 10.1620/tjem.191.221.
The role of Müller cells in the preconditioned retinal ischemic injury rat was investigated. In anesthetized Sprague Dawley rats, retinal ischemia for 5 minutes constituted the preconditioning stimulus for the left eye. After 24 hours, both eyes were clamped for 60 minutes. In 30, 60, 90, and 120, minutes and 1 day, 3 days, and 7 days after ischemia, electroretinograms were recorded, and the eyeballs were enucleated. After fixation with 4% paraformaldehyde, the avidin-biotin-peroxidase technique was applied to show glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). Furthermore, for the solubilized retinas, Western blot analysis and enzyme-linked immunosorbent assay were performed to detect GS and GFAP in the extracts. Preconditioning performed 24 hours before ischemia significantly improved the recovery of the a-, and b-waves 1 day after 60 minute ischemia. In the 30, 60, 90, and 120 minutes after ischemia, the recovery of the a-wave only was observed. There was a nonsignificant trend toward greater recovery in the first 120 minutes after 60 minute ischemia, especially in the b-wave. GS immunoreactivity had no significant difference between non-preconditioned and preconditioned groups 30, 60, 90, and 120 minutes after ischemia. In 1 day after ischemia, GS immunoreactivity decreased in both groups. In 3 and 7 days after ischemia, GS immunoreactivity recovered only in the preconditioned group. The retinas at 3 and 7 days after 1 hour of ischemia showed increased GFAP immunoreactivity in the non-preconditioned group. In the preconditioned group, only slight GFAP immunoreactivity was observed. These results suggested that the mechanism of preconditioned retinal ischemia may be related to Müller cells in the retina.
研究了米勒细胞在预处理视网膜缺血性损伤大鼠中的作用。在麻醉的斯普拉格-道利大鼠中,左眼视网膜缺血5分钟作为预处理刺激。24小时后,双眼夹闭60分钟。在缺血后30、60、90和120分钟以及1天、3天和7天,记录视网膜电图,并摘除眼球。用4%多聚甲醛固定后,采用抗生物素蛋白-生物素-过氧化物酶技术显示谷氨酰胺合成酶(GS)和胶质纤维酸性蛋白(GFAP)。此外,对于溶解的视网膜,进行蛋白质印迹分析和酶联免疫吸附测定以检测提取物中的GS和GFAP。缺血前24小时进行的预处理显著改善了60分钟缺血后1天a波和b波的恢复。在缺血后30、60、90和120分钟,仅观察到a波的恢复。在60分钟缺血后的前120分钟,尤其是b波,有恢复程度更高的不显著趋势。缺血后30、60、90和120分钟,未预处理组和预处理组之间的GS免疫反应性无显著差异。缺血后1天,两组的GS免疫反应性均降低。缺血后3天和7天,仅预处理组的GS免疫反应性恢复。缺血1小时后3天和7天的视网膜显示,未预处理组的GFAP免疫反应性增加。在预处理组中,仅观察到轻微的GFAP免疫反应性。这些结果表明,预处理视网膜缺血的机制可能与视网膜中的米勒细胞有关。