Sasaki C, Kitagawa H, Zhang W R, Warita H, Sakai K, Abe K
Department of Neurology, Okayama University Medical School, Japan.
Neurol Res. 2000 Mar;22(2):223-8. doi: 10.1080/01616412.2000.11741065.
Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 1. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats.
尽管凋亡途径在缺血性神经元损伤中发挥重要作用，但凋亡酶级联反应的确切机制尚未得到充分研究。在大鼠永久性大脑中动脉（MCA）闭塞模型中，检测了细胞色素c和半胱天冬酶-3的免疫组织化学染色，以及末端脱氧核苷酸转移酶（TdT）介导的dUTP生物素缺口末端标记法（TUNEL）的组织化学染色。MCA闭塞后3小时，缺血半暗带神经元中细胞色素c被强烈诱导，同一区域的半胱天冬酶-3从3小时开始诱导，8小时达到峰值。MCA区域的神经元细胞在延迟时间出现TUNEL阳性，24小时达到峰值。因此，细胞色素c的诱导峰值先于半胱天冬酶-3的诱导峰值，这两个峰值也先于DNA片段化峰值。蛋白质印迹分析显示细胞色素c在线粒体中的胞质表达。本研究表明：1. 缺血3小时后，细胞色素c主要在皮质神经元中从线粒体快速释放到细胞质中。2. 随后依次出现半胱天冬酶-3和TUNEL的峰值。这些时间特征表明大鼠永久性MCA闭塞后神经元死亡中凋亡途径的一系列级联激活。
