Blomgren K, McRae A, Elmered A, Bona E, Kawashima S, Saido T C, Ono T, Hagberg H
Institute of Anatomy and Cell Biology, University of Göteborg, Sweden.
Ann N Y Acad Sci. 1997 Oct 15;825:104-19. doi: 10.1111/j.1749-6632.1997.tb48420.x.
Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.
将新生大鼠置于短暂性脑缺氧缺血（HI，单侧颈总动脉闭塞 +7.70%氧气，持续100分钟）环境中，并使其恢复长达14天。发现钙蛋白酶的酪蛋白水解活性在两个半球均至少增加20小时。单纯缺氧暴露会增加钙蛋白酶的活性，在膜相关部分更为明显，可能是通过与细胞成分相互作用，而HI则导致活性丧失，最可能是由于蛋白酶的消耗和损失。连续的组织切片用抗钙蛋白酶抑制蛋白、α - fodrin、α - fodrin的150 kDa降解产物（FBDP，钙蛋白酶蛋白水解的标志物）或微管相关蛋白2（MAP - 2，树突体神经元损伤的标志物）抗体进行染色。脑损伤区域显示MAP - 2明显缺失，这清晰地勾勒出梗死灶。FBDP在同侧的损伤和边缘区域积累，对侧半球也出现不太明显的短暂性FBDP增加，尤其是在白质中。对皮质组织的胞质部分（CF）以及膜和微粒体部分（MMF）进行蛋白质印迹分析，并用抗钙蛋白酶、钙蛋白酶抑制蛋白和α - fodrin的150 kDa降解产物（FBDP）抗体进行染色。在损伤期间，CF中的钙蛋白酶免疫反应性双侧降低（为对照组的62 - 68%），在早期恢复期间仍显著较低，而MMF未显示出显著变化。钙蛋白酶的这种易位与同侧HI半球中FBDP的出现同时发生，从损伤后立即到至少恢复1天，FBDP水平显著升高（为对照组的204 - 292%）。在对侧未受损半球中未发现FBDP有显著变化，尽管两个半球中的钙蛋白酶都发生了易位，这是钙蛋白酶激活的前提条件。这种差异可能与内源性抑制剂钙蛋白酶抑制蛋白的变化有关。发现钙蛋白酶抑制蛋白在同侧半球HI期间及之后不久减少，但对侧半球未减少。HI后钙蛋白酶抑制蛋白的抑制活性也趋于降低，表明钙蛋白酶抑制蛋白的减少可能是广泛激活钙蛋白酶所必需的。m - 钙蛋白酶的mRNA在损伤后48小时在HI半球增加（167%，p < 0.001），此时蛋白质也增加。总之，我们的研究结果表明，钙蛋白酶在HI期间以及HI后再灌注的早期阶段被激活，早于神经元死亡。
