Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat
Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat
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DOI：
10.1016/s0306-4522(02)00376-7
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发表时间：
2002-01-01
期刊：
影响因子：
3.3
通讯作者：
Xu, D
中科院分区：
文献类型：
--
作者：
Davoli, MA;Fourtounis, J;Xu, D
在本研究中，我们评估了大鼠短暂大脑中动脉闭塞引起的局灶性脑缺血后 caspase-3 激活的时间过程以及细胞死亡的演变。再灌注后 3 小时和 6 小时时间点，缺血在纹状体中诱导活性 caspase-3 免疫反应性，但在皮质中则不然。此外，使用一种新的方法来可视化酶活性，DeltaC-APP（由 caspase-3 生成的 APP C 末端裂解产物）被发现与活性 caspase-3 免疫定位到相同的区域。双标记研究证明了这两种蛋白质在细胞水平上的共定位。进一步的双标记实验表明，活性 caspase-3 仅限于仍然存活的神经元细胞，因此对 NeuN 具有免疫反应性。通过末端 dUTP 缺口末端标记 (TUNEL) 进行组织学评估，早在 3 小时就在纹状体的少数细胞中观察到 DNA 碎片，但直到 6 小时才开始出现在皮质中。 DNA 片段化是进行性的，到再灌注后 24 小时，纹状体和皮质的大部分都显示出 TUNEL 阳性细胞。然而，用 TUNEL 双重标记活性 caspase-3 在所有时间点仅显示出极小的共定位。因此，caspase-3 激活似乎是在 DNA 断裂之前发生的事件。作为组织学 TUNEL 数据的证实，细胞死亡酶联免疫吸附测定证明，24 小时缺血也诱导核小体片段的产生。使用一种新的缺血诱导底物裂解生化方法，血影蛋白 P120 片段（一种 α II 血影蛋白（一种环骨架蛋白）的半胱天冬酶特异性裂解产物）通过蛋白质印迹法显示升高。核小体和血影蛋白 P120 的脑浓度与先前通过氯化三苯基四唑染色和梗塞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缺血性细胞死亡之间可能存在关联。 (C) 2002 国际广播组织。由爱思唯尔科学有限公司出版。保留所有权利。
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 cyroskeletal 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. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.