Namura S, Zhu J, Fink K, Endres M, Srinivasan A, Tomaselli K J, Yuan J, Moskowitz M A
Stroke and Neurovascular Regulation, Neurosurgical Service, Departments of Surgery and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
J Neurosci. 1998 May 15;18(10):3659-68. doi: 10.1523/JNEUROSCI.18-10-03659.1998.
We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.
我们通过免疫组织化学、免疫印迹以及对荧光底物N-苄氧羰基-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-7-氨基-4-三氟甲基香豆素（zDEVD-afc）的切割，研究了成年小鼠大脑中，在通过将细丝插入颈动脉造成短暂性（2小时）大脑中动脉闭塞后，半胱天冬酶-3（CPP32）的表达、激活及细胞定位情况。免疫反应性半胱天冬酶-3p32而非其切割产物半胱天冬酶-3p20在全脑神经元中组成性表达，在梨状皮质内的神经元胞体中最为显著。再灌注后0至3小时，脑匀浆中的半胱天冬酶样酶活性升高，并在30至60分钟内达到峰值。再灌注时及1至12小时后的免疫印迹显示，半胱天冬酶-3p20免疫反应性在大脑中动脉区域内的神经元胞体中变得显著。再灌注后6至24小时检测到DNA梯状条带（琼脂糖凝胶）和末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记（TUNEL）染色细胞。在12至24小时时，免疫反应性p20在TUNEL阳性细胞中可见，这一发现也在出生后第5天的凋亡小鼠小脑颗粒细胞中观察到。总之，这些观察结果表明存在缺血性损伤的时间依赖性演变，其特征为半胱天冬酶样酶激活与再灌注时或再灌注前开始的免疫反应性产物（半胱天冬酶-3p20）相关增加之间密切对应，数小时后接着出现凋亡的形态学和生化特征。
