The elucidation of molecular mechanism in delayed neruonal death and its control in rat retina

我们探讨了细胞凋亡和Bcl2家族基因在SD大鼠视网膜缺血后迟发性神经元死亡中的作用。升高眼压至130毫米汞柱45min，诱导视网膜缺血。于再灌流后不同时间取组织标本，进行TUNEL染色。短暂性脑缺血后6~48h，神经节细胞层(GCL)和内核层(INL)可见大量TUNEL阳性细胞，缺血后24h达高峰。分别于再灌流后24小时和48小时从缺血区视网膜提取DNA，对侧非缺血区视网膜进行DNA提取并电泳法。琼脂糖凝胶电泳法观察到缺血后24小时和48小时视网膜出现DNA梯状条带，而正常视网膜未见DNA梯状条带。在短暂性脑缺血后不同时间用Bcl2和Bax特异性引物对视网膜进行RT-PCR分析。结果表明，bax基因的表达随着e-…的增加而逐渐增强。短暂性脑缺血后6小时达高峰，24小时达高峰，168小时降至接近基线水平，而短暂性脑缺血后各时间点Bcl2基因表达均无明显变化。利用短暂性脑缺血24小时后的视网膜进行Bax特异性抗体的免疫组织化学研究，并与对侧非缺血视网膜进行比较。用玻璃体内注射Bax反义寡核苷酸(ODN)或随机测序的ODN处理视网膜，检测短暂性脑缺血后TUNEL阳性细胞的数量。再灌流24小时后，缺血后视网膜的GCL和INL有较强的Bax蛋白表达，而非缺血组仅有少量Bax蛋白表达。反义寡核苷酸抑制Bax蛋白表达可减少TUNEL阳性细胞数。我们得出结论，短暂性视网膜缺血引起的部分神经元死亡涉及BAX上调诱导的细胞凋亡的活跃细胞死亡过程。此外，我们观察了神经元过度表达Bcl2的转基因小鼠在短暂性视网膜缺血后的组织学变化，并与野生型小鼠进行了比较。缺血后7d取脑组织切片。用形态计量学方法定量检测过表达Bcl2(NSE73a/ab)的转基因小鼠与野生型小鼠(C57BL/6)的脑缺血损伤。对GCL的细胞数及内网层(IPL)、内网层(INL)和外核层(ONL)的厚度进行了定量。对于每只动物，缺血后眼的这些参数被归一化为完整对侧眼的这些参数，并以百分比表示。转基因小鼠的GCL细胞数比野生型小鼠高约60%。野生型和转基因鼠肺间层厚度分别为33.7±1.8μm和43.8±6.7μm。野生型小鼠GCL细胞数和IPL厚度分别为对照组的64.6±7.5和42.3±0.6%，而转基因小鼠GCL细胞数和IPL厚度分别为81.9±5.6和82.4±19.6。这些结果表明，过度表达Bcl2的视网膜神经元与缺血损伤有关。较少

We evaluated the involvement of apoptosis and contributions of Bcl-2 family genes in delayed neruonal death after retinal ischemia in Sprague-Dawley rat. Retinal ischemia was induced by elevating IOP to 130 mmHg for 45 min. Histological specimens were obtained at various time after reperfusion and examined by TUNEL staining. A significant number of TUNEL positive cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) starting at 6 to 48 hr after transient ischemia and reached a maximum 24 hr after ischemia. DNA was extracted from the-ischemic retina at 24 and 48 hr after reperfusion and the contralateral non-ischemic retina and electrophoresed. DNA laddering was observed on agarose gel electrophoresis in retinas 24 and 48 hr after ischemia but not in normal retina. RT-PCR analysis was carried out with the retina at various time after transient ischemia using specific primers for Bcl-2 and Bax. It demonstrated that Bax gene expression gradually increased as e … More arly as 6 hr, reached a peak at 24 hr, then decreased to near the baseline level at 168 hr, while Bcl-2 gene expression did not show any obvious change at any time point after transient ischemia. Immunohistochemical study using a specific antibody for Bax was performed using retina 24 hr after transient ischemia and fingings were compared with those of the contralateral non-ischemic retina. The number of TUNEL-positive cells after transient ischemia were measured using retinas pretreated by intra-vitreous injection of a Bax antisense oligodeoxynucleotide (ODN) or a randomly sequenced ODN. intense Bax protein immunoreactivity was detected in the GCL and INL of the post-ischemic retina 24 hr after reperfusion, while little immunoreactivity was present in the non-ichemic retina. The inhibition of Bax protein expression by antisense ODNs reduced the number of TUNEL-positive cells. We conclude some of the neuronal death caused by transient retinal ischemia involves an active cell death process of apoptosis induced by the upregulaion of Bax.Furthermore, we examine histological changes after transient retinal ischemia in transgenic mice in which neurons overexpress Bcl-2, in comparison with those of wild-type littermates. Histological sections were obtained 7 days after ischemic insult. Morphometric analysis were performed to quantify the ischemic injury in transgenic mice overexpressing Bcl-2 (NSE73a/ab) vs wild type littermates (C57BL/6). The number of cells in the GCL and the thickness of the inner plexiform layer (IPL), INL, and outer nuclear layer (ONL) were quantified. For each animal, these parameters in the post-ischemic eye were normalized to those in the intact contralateral eye and shown as a percentage. The GCL cell number was approximately 60% greater in transgenic mice than that in wild-type littermates. The IPL thickness was 33.7±1.8 μm in wild-type and 43.8±6.7 μm in transgenic mice. In wild-type mice, ischemia reduced the GCL cell number and IPL thickness to 64.6±7.5, and 42.3±0.6% of the control, respectively, whereas ischemia reduced the GCL cell number and IPL thickness to 81.9±5.6, and 82.4±19.6 in transgenic mice. These results suggest that retinal neurons overexpressing Bcl-2 become torelant to ischemic injury. Less