Davoli M A, Fourtounis J, Tam J, Xanthoudakis S, Nicholson D, Robertson G S, Ng G Y K, Xu D
Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorval, QC, Canada H9R 4P8.
Neuroscience. 2002;115(1):125-36. doi: 10.1016/s0306-4522(02)00376-7.
In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.
在本研究中，我们评估了大鼠短暂性大脑中动脉闭塞所致局灶性脑缺血后caspase-3激活的时间进程以及细胞死亡的演变过程。再灌注后3小时和6小时时，缺血诱导纹状体而非皮质出现活性caspase-3免疫反应性。此外，采用一种可视化酶活性的新方法，发现由caspase-3产生的APP的C末端裂解产物deltaC-APP与活性caspase-3免疫定位到相同区域。双标记研究在细胞水平证实了这两种蛋白的共定位。进一步的双标记实验显示，活性caspase-3局限于仍存活且因此对NeuN呈免疫反应性的神经元细胞。通过末端脱氧尿苷三磷酸缺口末端标记法（TUNEL）进行组织学评估，早在3小时时就在纹状体的少数细胞中观察到DNA片段化，但直到6小时才开始出现在皮质中。DNA片段化是渐进性的，再灌注后24小时时，纹状体和皮质的大部分区域均显示TUNEL阳性细胞。然而，活性caspase-3与TUNEL的双标记在所有时间点均仅显示极少的共定位。因此，caspase-3激活似乎是在DNA片段化之前发生的事件。作为对组织学TUNEL数据的证实，24小时缺血还诱导了核小体片段的产生，细胞死亡酶联免疫吸附测定法证明了这一点。采用一种新的缺血诱导底物裂解生化方法，通过蛋白质印迹法显示细胞骨架蛋白αII血影蛋白的caspase特异性裂解产物血影蛋白P120片段升高。核小体和血影蛋白P120的脑内浓度与先前通过氯化三苯基四氮唑染色和梗死体积计算评估的损伤程度相关。总之，我们的研究结果表明caspase-3激活与大脑中动脉闭塞性脑损伤后的缺血性细胞死亡之间可能存在关联。
