Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y
Department of Cell Biology and Neuroanatomy, Iwate Medical University School of Medicine, Japan.
J Neurosci. 1995 Feb;15(2):1001-11. doi: 10.1523/JNEUROSCI.15-02-01001.1995.
The CA1 pyramidal neurons in the hippocampus are selectively vulnerable to transient ischemic damage. In experimental animals, the CA1 pyramidal neurons undergo cell death several days after brief forebrain ischemia. It remains, however, unknown whether this delayed neuronal death is necrosis or apoptosis. To investigate the degenerating processes of the CA1 pyramidal neurons in gerbil hippocampus after brief ischemia, lysosomal and nuclear alterations in the cells were examined using immunocytochemistry, in situ nick-end labeling, and Southern blotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L, representative lysosomal cysteine proteinases, increased in the CA1 pyramidal neurons 3 d after ischemic insult, which showed cell shrinkage. By morphometric analysis, the volume density of cathepsin B-positive lysosomes markedly increased 3 d after ischemic insult, while that of autophagic vacuole-like structures also increased at this stage, suggesting that cathepsin B-immunopositive lysosomes increasing in the neurons after ischemic insult are mostly autolysosomes. Nuclei of the CA1 neurons were nick-end labeled by biotinylated dUTP mediated by terminal deoxytransferase 3 and 4 d after ischemic insult, but not in the prior stages. Simultaneously, dense chromatin masses appeared in nuclei of the neurons. By Southern blotting, laddering of DNA occurred only in CA1 hippocampal tissues obtained 4 d after ischemic insult. Confocal laser scanning microscopy demonstrated that the fragmented DNA in the CA1 pyramidal layer was phagocytosed by microglial cells. The results suggest that delayed death of the CA1 pyramidal neurons after brief ischemia is not necrotic but apoptotic.
海马体中的CA1锥体神经元对短暂性缺血损伤具有选择性易损性。在实验动物中，短暂性前脑缺血几天后，CA1锥体神经元会发生细胞死亡。然而，这种延迟性神经元死亡是坏死还是凋亡仍不清楚。为了研究沙鼠海马体中CA1锥体神经元在短暂缺血后的退变过程，采用免疫细胞化学、原位缺口末端标记和Southern印迹法检测了细胞中的溶酶体和细胞核变化。通过光学显微镜和电子显微镜观察，缺血损伤后3天，CA1锥体神经元中组织蛋白酶B、H和L（代表性的溶酶体半胱氨酸蛋白酶）的免疫反应性增加，此时细胞出现萎缩。通过形态计量分析，缺血损伤后3天，组织蛋白酶B阳性溶酶体的体积密度显著增加，而自噬泡样结构的体积密度在这个阶段也增加，这表明缺血损伤后神经元中增加的组织蛋白酶B免疫阳性溶酶体大多是自溶酶体。缺血损伤后3天和4天，CA1神经元的细胞核被末端脱氧转移酶介导的生物素化dUTP进行缺口末端标记，但在之前阶段未出现这种情况。同时，神经元细胞核中出现致密染色质团块。通过Southern印迹法检测，只有在缺血损伤后4天获得的CA1海马组织中出现了DNA梯状条带。共聚焦激光扫描显微镜显示，CA1锥体层中破碎的DNA被小胶质细胞吞噬。结果表明，短暂缺血后CA1锥体神经元的延迟死亡不是坏死性的，而是凋亡性的。
