Apoptosis of cultured microglia by the deprivation of macrophage colony-stimulating factor
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Y Tomozawa1, T Inoue, M Takahashi
1Department of Molecular Pharmacology, Faculty of Pharmaceutical Science, Kyoto University, Japan.
Abstract:
Promotion of microglial proliferation and differentiation by colony-stimulating factors (CSFs) and disappearances of microglia at the late neonatal stage by decreasing of CSFs have been reported. In this study, the effects of the deprivation of macrophage CSF (M-CSF) on enriched microglia in cultures were examined by cytochemical methods including in situ nick-end labeling for DNA fragmentation, and Carrazi's hematoxylin nuclear staining. When M-CSF was deprived from the culture medium: (1) at least 40% of the cells were weakly labeled by nick-end within 3 h and more than 70% of the cells were clearly labeled by 16 h; and (2) nuclear condensation or fragmentation, and formation of apoptotic bodies were observed within 48 h. LeY-positive immunoreactivity, identified as a characteristic of cells undergoing apoptosis, was observed on cells positively labeled by nick-end and condensed nucleus, and ones budding apoptotic bodies. From these results, it is conceivable that microglia undergo apoptosis when M-CSF is deprived from the culture medium and, therefore, require CSFs for their survival. This in vitro phenomenon suggests that one of the mechanisms of microglial disappearance in vivo after synaptogenesis may be due to apoptosis by decreasing level of CSFs.
Insights
Colony-stimulating factors (CSFs) are crucial for microglial survival. Depriving microglia of macrophage colony-stimulating factor (M-CSF) in vitro induces apoptosis, suggesting CSFs are essential for their continued existence.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the immune cells of the central nervous system, are known to proliferate and differentiate in response to colony-stimulating factors (CSFs).
- A decrease in CSFs has been linked to microglial disappearance during the late neonatal stage.
Purpose of the Study:
- To investigate the impact of macrophage colony-stimulating factor (M-CSF) deprivation on cultured microglia.
- To elucidate the role of M-CSF in microglial survival and apoptosis.
Main Methods:
- Primary microglia cultures were established and deprived of M-CSF.
- Cytochemical methods, including in situ nick-end labeling (for DNA fragmentation) and Caracci's hematoxylin nuclear staining, were employed.
- LeY-positive immunoreactivity was assessed as a marker for apoptosis.
Main Results:
- M-CSF deprivation led to significant DNA fragmentation in microglia within 16 hours.
- Nuclear condensation, fragmentation, and apoptotic body formation were observed within 48 hours.
- LeY-positive immunoreactivity correlated with markers of apoptosis, confirming cell death.
Conclusions:
- Microglia undergo apoptosis when M-CSF is withdrawn from the culture medium.
- CSFs, specifically M-CSF, are required for microglial survival.
- This in vitro finding suggests that decreasing CSF levels in vivo may trigger microglial apoptosis after synaptogenesis.