Analysis of the molecular mechanism of delayed neuronal death in hippocampus following transient ischemia

本研究采用蒙古沙土鼠短暂性同侧脑缺血模型，研究了迟发性神经元死亡的分子机制，并着重研究了一氧化氮(NO)诱导细胞凋亡的作用。在海马CA1区锥体神经元中，短暂性脑缺血后6h在消息水平表达诱导型一氧化氮合酶(INOS)，在蛋白水平表达iNOS。在海马钙层检测到硝基酪氨酸的形成，表明细胞毒性氧化剂过氧亚硝酸盐(0N00)是由N0与超氧化物(O2)相互作用产生的。腹腔注射Aminoguanid320 mg/kg可抑制海马CA1区锥体神经元DNA的断裂和ONOO的产生。亚低温(34ﾟC)可抑制大鼠海马CA1区锥体神经元bcl2的表达和诱导型一氧化氮合酶的诱导。它还可以改善同一区域的迟发性神经元死亡。此外，短暂性脑缺血后20~30min，海马CA1层可检测到iNOS基因的重要诱导物之一--核因子-kB的激活。这些结果清楚地表明，iNOS在短暂性脑缺血再灌流后不久的海马区锥体神经元中的表达参与了凋亡诱导机制的上游，提示iNOS产生的N0和0N00可能具有不同的功能。在短暂性脑缺血再灌流后，由于兴奋性氨基酸的神经毒性，核因子-kB可能上调了锥体神经元iNOS基因的转录，而核因子-kB又被钙信号激活。此外，短暂性脑缺血后，海马CA1区锥体细胞表达FasL。FasL的表达可能也参与了细胞凋亡诱导机制的上游，也可能在中枢神经系统的免疫豁免中发挥重要作用。

This study examined the molecular mechanism of delayed neuronal death using a mongolian gerbil, model for transient isohemia, and focused particularly on the induction of apoptosis by nitric oxide (NO). In the hippocampal CAl pyramidal neurons, inducible NO synthase (iNOS) was induced at the message level at 6 hours, and at the protein level at 24 hours following transient ischemia. Nitrotyrosine formation was detected in the hippocampal CAl layer, indicating that peroxynitrite (0N00), a cytotoxic oxidant, was produced from N0 by the interaction with superoxide (0_2). Aminoguanidine 320 mg/kg, injected intra-peritoneally, suppressed fragmentation of DNA, as well as production of ONOO in the hippocampal CAl pyramidal neurons. Intraischemic mild hypothermia (34ﾟC) inhibited both the decrease of bcl-2 and induction of iNOS in the hippocampal CAl pyramidal neurons. It also ameliorated delayed neuronal death in the same region. Furthermore, activation of NF-_KB, one of the important inducers of the iNOS gene, was detected in the hippocampal CAl layer at 20 to 30 min after transient ischemia. These results clearly showed that iNOS expressed in the hippocampal CAl pyramidal neurons soon after transient ischemia and reperfusion was involved upstream of the apoptosis-induction mechanism, indicating that N0 and 0N00 produced by iNOS may have various functions. Transcription of the iNOS gene in the pyramidal neurons seems to be up-regulated by NF-_KB, which is activated by calcium-signaling due to excitatory amino acid neurotoxicity after transient ischemia and reperfusion. In addition, FasL was expressed in the hippocampal CAl pyramidal neurons after transient ischemia. The expression of FasL may also be involved upstream of the apoptosis-induction mechanism, or it may play a significant role in immune privilege for the central nervous system.