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

本研究以蒙古沙鼠为模型，研究了延迟性神经元死亡的分子机制，并特别关注一氧化氮（NO）对细胞凋亡的诱导。在海马CAl锥体神经元中，诱导型NO合成酶（iNOS）在短暂缺血后6小时的信息水平和24小时的蛋白水平上被诱导。在海马CAl层检测到硝基酪氨酸的形成，表明N0与超氧化物（0_2）相互作用产生细胞毒性氧化剂过氧亚硝酸盐（0N00）。氨基胍320 mg/kg腹腔注射，抑制海马CAl锥体神经元DNA断裂和ONOO的产生。缺血亚低温（34‰）可抑制海马锥体神经元中bcl-2的减少和iNOS的诱导。它还改善了同一区域的延迟神经元死亡。此外，短暂性缺血后20 ~ 30 min海马CAl层检测到iNOS基因重要诱导剂之一NF-_KB的活化。这些结果清楚地表明，海马CAl锥体神经元在短暂缺血再灌注后不久表达的iNOS参与了细胞凋亡诱导机制的上游，表明iNOS产生的N0和0N00可能具有多种功能。锥体神经元中iNOS基因的转录似乎被NF-_KB上调，NF-_KB在短暂缺血再灌注后由于兴奋性氨基酸神经毒性而被钙信号激活。短暂性缺血后海马胼胝体锥体神经元中有FasL表达。FasL的表达也可能参与凋亡诱导机制的上游，或者在中枢神经系统的免疫特权中发挥重要作用。

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.