钙蛋白酶同工型在大鼠视网膜缺血再灌注损伤中的作用。

Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina.

作者信息

Sakamoto Y R, Nakajima T R, Fukiage C R, Sakai O R, Yoshida Y R, Azuma M R, Shearer T R

机构信息

Research Laboratories, Senju Pharmaceutical Corporation Limited, Kobe, Japan.

出版信息

Curr Eye Res. 2000 Jul;21(1):571-80.

PMID:11035539

Abstract

PURPOSE

Much evidence has accumulated suggesting that activation of calpain causes neuronal cell death in ischemic brain. However, little is known about the involvement of calpain in retinal cell death in ischemic injury. Thus, the purpose of present study was to investigate the involvement of calpain isoforms (m- and mu-calpain) in ischemia-reperfusion injury in retina from rat.

METHODS

Retinal ischemia was produced by occlusion of the central retinal artery for one hour, and this was followed by reperfusion for seven days. Calpain mRNAs, calpain activities, total calcium content and proteolysis of alpha-spectrin were determined in retina. Effect of a calpain inhibitor SJA6017 was histologically tested in retinal injury after ischemia-reperfusion.

RESULTS

Following retinal ischemia, most of cells in the ganglion cell layer were sloughed off by day 1 after reperfusion, followed by loss of cells in the inner plexiform layer on day 3 and loss of cells in the inner nuclear layer by day 5. These morphologic changes were accompanied by several presumptive biochemical indicators of calpain activation: increased calcium, proteolysis of alpha-spectrin (a sensitive substrate for calpains), decreased caseinolytic activity for both calpains (suggesting calpain activation followed by autolytic degradation), increased mRNA levels for mu-calpain and calpastatin - the endogenous inhibitor of calpains - and decreased mRNA levels for mu-calpain. Moreover, the calpain inhibitor SJA6017 protected the reduction of cell density in the ganglion cell layer after ischemia-reperfusion.

CONCLUSION

These results suggest that calpain isoforms may play an important role in neuronal cell death induced by retinal ischemia-reperfusion injury in rat.

摘要

目的

大量证据表明钙蛋白酶的激活会导致缺血性脑损伤中的神经元细胞死亡。然而，关于钙蛋白酶在缺血性损伤中视网膜细胞死亡的作用却知之甚少。因此，本研究的目的是探讨钙蛋白酶亚型（m-钙蛋白酶和μ-钙蛋白酶）在大鼠视网膜缺血再灌注损伤中的作用。

方法

通过阻塞视网膜中央动脉1小时造成视网膜缺血，随后进行7天的再灌注。测定视网膜中的钙蛋白酶mRNA、钙蛋白酶活性、总钙含量以及α-血影蛋白的蛋白水解情况。在缺血再灌注后的视网膜损伤中，对钙蛋白酶抑制剂SJA6017的作用进行组织学检测。

结果

视网膜缺血后，再灌注1天后神经节细胞层的大多数细胞脱落，3天后内网状层细胞丢失，5天后内核层细胞丢失。这些形态学变化伴随着钙蛋白酶激活的几个推定生化指标：钙增加、α-血影蛋白（钙蛋白酶的敏感底物）的蛋白水解、两种钙蛋白酶的酪蛋白水解活性降低（表明钙蛋白酶激活后发生自溶降解）、μ-钙蛋白酶和钙蛋白酶抑制蛋白（钙蛋白酶的内源性抑制剂）的mRNA水平升高以及μ-钙蛋白酶的mRNA水平降低。此外，钙蛋白酶抑制剂SJA6017可保护缺血再灌注后神经节细胞层细胞密度的降低。