Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.
Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.
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DOI：
10.1371/journal.pone.0040813
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Minami M
中科院分区：
文献类型：
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作者：
Katayama T;Kobayashi H;Okamura T;Yamasaki-Katayama Y;Kibayashi T;Kimura H;Ohsawa K;Kohsaka S;Minami M
在这项研究中，小胶质细胞的迁移和吞噬作用进行了检查，在小鼠器官型海马脑片培养，这是处理与N-甲基-D-天冬氨酸（NMDA）选择性损伤神经元细胞。通过增强的绿色荧光蛋白的表达使小胶质细胞可视化。每日观察显示锥体细胞层中的小胶质细胞积聚，其在NMDA处理后5至6天达到峰值。延时成像显示，小胶质细胞迁移到锥体细胞层从邻近和/或偏远地区。在锥体细胞层和放射层中，对照和NMDA处理的切片之间的增殖小胶质细胞的数量没有差异，这表明损伤区域中的小胶质细胞积累主要是由于小胶质细胞迁移，而不是增殖。延时成像还显示，碘化丙啶（PI）显示的受损神经元在被小胶质细胞包围后立即消失。每日观察显示PI荧光强度逐渐减弱，并且这种减弱通过用氯膦酸盐（一种小胶质细胞毒素）预处理而被抑制。这些发现表明，积累的小胶质细胞吞噬损伤的神经元，PI荧光可能是一个有用的指示小胶质细胞的吞噬。利用这一优势，以检查小胶质细胞的吞噬作用，在活切片培养，我们调查了小胶质细胞的积累和吞噬作用的丝裂原活化蛋白（MAP）激酶的参与。p38 MAP激酶抑制剂SB 203580可抑制PI荧光的衰减，而MAP激酶/细胞外信号调节激酶抑制剂PD 98059或c-Jun N-末端激酶抑制剂SP 600125则无此作用。另一方面，损伤区域中的小胶质细胞积累不受任何这些抑制剂的抑制。这些数据表明，p38 MAP激酶在小胶质细胞吞噬损伤神经元中起着重要作用。
In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.