Effects of calpain inhibitor on delayed neuronal death after global forebrain ischemia

短暂前脑缺血诱导沙鼠海马神经元钙蛋白酶激活和胞衬蛋白水解。这种现象先于海马CA 1神经元中的延迟性神经元死亡。我们研究了钙蛋白酶抑制剂对短暂性前脑缺血后迟发性神经元死亡的影响。给沙土鼠经皮注射脂质体包封的选择性钙蛋白酶抑制剂，30分钟后，阻断双侧颈总动脉造成前脑缺血5分钟。在第7天，在用甲酚紫染色的海马切片中检查CA 1神经元损伤。还通过免疫组织化学和免疫印迹检查了钙蛋白酶诱导的胞衬蛋白蛋白水解。此外，为了确保抑制剂被CA 1神经元截留，用FITC标记所述脂质体复合物并给予沙鼠。用激光共聚焦扫描显微镜观察海马脑片的荧光。前脑缺血诱导的选择性CA 1区神经元损伤 ...更多信息 这是通过以剂量依赖性方式给予抑制剂来防止的，但即使以60 mg/kg的剂量也不能完全防止。钙蛋白酶抑制剂能抑制钙蛋白酶诱导的胞衬蛋白水解，且呈剂量依赖性，在60 mg/kg剂量下完全消除。在CA 1神经元中观察到FITC标记的抑制剂的明亮荧光。这些数据表明钙蛋白酶在缺血性迟发性神经元死亡的发展中起重要作用。钙蛋白酶似乎通过降解神经元细胞骨架而产生神经元损伤。我们的数据还显示了钙蛋白酶抑制剂对神经毒性损伤的姑息作用。本实验采用免疫组织化学和免疫印迹方法，观察了内源性钙蛋白酶抑制剂calpain抑制剂calpastatin在前脑缺血时的变化。海马神经元钙蛋白酶抑制素免疫反应在缺血后4小时增强，随后减弱。结果提示钙蛋白酶抑制蛋白可能参与应激反应。少

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CAl neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 minutes later, 5-minute forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CAl neuronal damage was examined in the hippocampal slices stained with cresyl-violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CAl neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CAl neuronal damage induced by forebrain ischemia w … More as prevented by administration of the inhibitor in a dose-dependent manner but not completely even in dose of 60mg/kg. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner and completely abolished in dose of 60mg/kg. Bright fluorescence of the FITC-labeled inhibitor was observed in the CAl neurons. The data show an important role of calpain in development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage. Although the calpain inhibitor does not completely block the ischemic neuronal damage, it offers a new and potent treatment of transient forebrain cerebral ischemia.As an additional experimet, alteration of endogenous calpain inhibitor, calpastatin, in forebrain ischemia was examined by immunohistochemical and immunobloting methods. The immunoreactivity of calpastatin in hippocampal neurons increased 4 hours after the ischemic insult and then decreased. The result suggested that calpastatin may participate in the stress responses. Less