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

短暂性前脑缺血诱导沙土鼠海马区钙蛋白的激活和神经元细胞骨架fodrin的蛋白分解。这种现象发生在海马神经元迟发性死亡之前。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。沙土鼠经静脉注射钙激活酶抑制剂，30min后，结扎双侧颈总动脉造成5min前脑缺血。第7天，用甲酚紫染色观察海马片CA1神经元损伤情况。用免疫组织化学和免疫印迹法检测了钙蛋白酶诱导的Fodrin蛋白降解。此外，为了确保抑制物被Cal神经元捕获，用FITC标记抑制物-脂质体复合体并给予沙土鼠。用激光共聚焦扫描显微镜观察海马片的荧光。前脑缺血致选择性海马神经元损伤的…研究以剂量依赖的方式给予抑制剂，但即使在60 mg/kg的剂量下也不能完全阻止。钙蛋白酶抑制剂能剂量依赖性地抑制钙蛋白酶诱导的Fodrin蛋白降解，在60 mg/kg剂量时完全被完全消除。FITC标记的抑制物在CA1神经元中有较强的荧光。这些数据表明，钙蛋白酶在缺血性迟发性神经元死亡的发生发展中起着重要作用。钙蛋白酶似乎通过降解神经元细胞骨架而造成神经元损伤。我们的数据还显示了Calain抑制剂对神经毒性损伤的缓解作用。虽然钙蛋白酶抑制剂不能完全阻断缺血神经元的损伤，但它为短暂性前脑缺血提供了一种新的有效的治疗方法。作为附加实验，我们用免疫组织化学和免疫印迹的方法检测了内源性钙蛋白酶抑制剂钙蛋白酶抑制剂在前脑缺血中的变化。缺血后4h，海马神经细胞内钙调蛋白的免疫反应增强，然后下降。这一结果提示钙调蛋白可能参与了应激反应。较少

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