Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

Takahiro Katayama,Hayato Kobayashi,Yuko Yamasaki-Katayama,Shinichi Kohsaka,Masabumi Minami,Tatsuya Kibayashi,Toshiyuki Okamura,Hiroshi Kimura,Keiko Ohsawa

doi:10.1371/journal.pone.0040813

Abstract

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.

Highlights

Microglia actively survey local brain environments by extending and retracting their processes in various directions in a normal brain [1,2,3]
In the present study using hippocampal slice cultures, we demonstrated that NMDA-induced neuronal injury caused microglial accumulation in the injured areas
Together with the results that there was no difference in the number of Iba1/EdU doublepositive cells between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, the findings suggest that the microglial accumulation in the injured brain areas was due to microglial migration from adjacent and/or remote areas rather than to microglial proliferation at the injured sites

Summary

Introduction

Microglia actively survey local brain environments by extending and retracting their processes in various directions in a normal brain [1,2,3]. It was recently reported that TREM2 expressed on the cell surface of microglia is involved in microglial phagocytosis of injured neurons [7] and that HSP60 is one of the endogenous ligands for TREM2 [8] These studies used dissociated cell cultures of primary microglia or microglia cell lines such as BV-2 and N9 to examine their phagocytic activity, microglia in vivo migrate to and phagocytose injured neurons in the brain parenchyma where neural cells, including neurons and glial cells, and extracellular matrix are densely packed. Using this advantage to examine microglial phagocytosis in living slice cultures, we examined the effects of mitogenactivated protein (MAP) kinase inhibitors on microglial phagocytosis and revealed the crucial role of p38 MAP kinase in microglial phagocytosis of injured neurons

Methods

Results

Conclusion