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The study of apoptosis and contributions of Bcl-2 family genes in delayed neruonal death after retinal ischemia

视网膜缺血后迟发性神经元死亡中Bcl-2家族基因凋亡及贡献的研究

基本信息

批准号：
09671796
负责人：
KASHII Satoshi
金额：
$ 2.18万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671796/
关键词：
delayed neruonal death Apoptosis TUNEL positive cell DNA fragmentation Bax Bcl-2 Glutamate Nitric oxide 虚血性網膜細胞死 TUNE陽性(DNA断片化)細胞 bal-2遺伝子虚血網膜遅発性神経細胞死 DNAの断片化 N-メチル-D-アスパラギン酸(NMDA)

项目摘要

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 post-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 … More as early 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 findings 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 upregulation 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 mum in wild-type and 43.8*6.7 mum 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

我们评估了Bcl-2家族基因在Sprague-Dawley大鼠视网膜缺血后延迟性神经元死亡中的凋亡参与和贡献。将眼压升高至130 mmHg 45 min，诱导视网膜缺血。再灌注后各时间取组织学标本，TUNEL染色检查。瞬时缺血后6 ~ 48小时，在神经节细胞层（GCL）和内核层（INL）中观察到大量TUNEL阳性细胞，缺血后24小时达到最大值。再灌注后24和48小时提取缺血后视网膜DNA，对侧非缺血视网膜进行电泳。琼脂糖凝胶电泳在缺血后24和48小时观察到DNA阶梯，而在正常视网膜则没有。利用Bcl-2和Bax特异性引物对短暂性缺血后不同时间的视网膜进行RT-PCR分析。结果表明，Bax基因表达在缺血后6小时逐渐升高，24小时达到峰值，168小时降至接近基线水平，而Bcl-2基因表达在缺血后各时间点均无明显变化。短暂性缺血24小时后，用Bax特异性抗体对视网膜进行免疫组化研究，并与对侧非缺血视网膜的结果进行比较。用玻璃体内注射Bax反义寡脱氧核苷酸（ODN）或随机测序的ODN预处理的视网膜，测量短暂缺血后tunel阳性细胞的数量。再灌注24小时后，缺血视网膜的GCL和INL中检测到强烈的Bax蛋白免疫反应性，而非缺血视网膜的免疫反应性不明显。反义odn对Bax蛋白表达的抑制减少了tunel阳性细胞的数量。我们认为，短暂性视网膜缺血引起的神经元死亡与Bax上调诱导的细胞凋亡的主动死亡过程有关。此外，我们研究了神经元过表达Bcl-2的转基因小鼠与野生型小鼠相比，在短暂性视网膜缺血后的组织学变化。缺血损伤后7天取组织学切片。形态学分析量化过表达Bcl-2 （NSE73a/ab）转基因小鼠与野生型同窝小鼠（C57BL/6）的缺血性损伤。定量测定GCL细胞数及内丛状层（IPL）、内丛状层（INL）、外核层（ONL）厚度。对于每只动物，缺血后眼的这些参数与完整对侧眼的参数归一化，并以百分比显示。转基因小鼠的GCL细胞数量比野生型小鼠高约60%。野生型和转基因小鼠的IPL厚度分别为33.7*1.8 mum和43.8*6.7 mum。野生型小鼠GCL细胞数量和IPL厚度分别为对照组的64.6*7.5和42.3* 0.6%，转基因小鼠GCL细胞数量和IPL厚度分别为81.9*5.6和82.4*19.6。这些结果提示过表达Bcl-2的视网膜神经元与缺血性损伤相关。少