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Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Lipopolysaccharide extends the lifespan of mouse primary-cultured microglia
Yoko S Kaneko1, Akira Nakashima, Keiji Mori
1Department of Physiology, Fujita Health University School of Medicine, Kutsukake-cho, Toyoake, Japan.
Abstract:
Microglial activation has been implicated in the recognition and phagocytic removal of degenerating neurons; however, this process must be tightly regulated in the central nervous system, because prolonged activation could damage normal neurons. We report that mouse primary-cultured microglia, which are destined to die within a few days under ordinary culture conditions, can live for more than 1 month when kept activated by lipopolysaccharide (LPS) treatment. Primary-cultured microglia treated with sublethal doses of LPS remained viable, without any measurable increase in apoptotic or necrotic cell death. LPS-treated microglia had an arborescent shape, with enlarged somata and thickened cell bodies. Although the amount of intracellular ATP in these microglia was reduced by 2 h after the start of LPS treatment, this had no effect on the viability of the cells. LPS treatment of microglia increased the antiapoptotic factor Bcl-xL protein level at day 1, although the level of the proapoptotic Bcl-associated X-protein was unaffected. Furthermore, the level of microtubule-associated light chain 3, a marker protein for autophagy, decreased at 3 h after exposure to LPS. These data show that the optimal dose of LPS suppresses the induction of both apoptosis and autophagy in primary-cultured microglia, allowing the cells to stay alive for more than 1 month. Because long-lived microglia may play critical roles in the exacerbation of neurodegeneration, our findings suggest that inducing a resting stage in active microglia could be a new and promising strategy to inhibit the deterioration of neurodegenerative disease.
Insights
Lipopolysaccharide (LPS) treatment keeps primary microglia alive for over a month by suppressing apoptosis and autophagy. This finding suggests targeting microglial activation could treat neurodegenerative diseases.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglial activation is crucial for clearing degenerating neurons but requires tight regulation to prevent damage to healthy neurons.
- Prolonged microglial activation can exacerbate neurodegeneration.
Purpose of the Study:
- To investigate the effects of lipopolysaccharide (LPS) on primary microglia survival and cell death pathways.
- To determine if modulating microglial activation can influence neurodegenerative disease progression.
Main Methods:
- Primary mouse microglia were cultured and treated with sublethal doses of LPS.
- Cell viability, apoptosis, necrosis, ATP levels, and protein expression (Bcl-xL, Bcl-associated X-protein, microtubule-associated light chain 3) were assessed.
Main Results:
- LPS treatment significantly extended primary microglia survival to over 1 month without increasing cell death.
- LPS-induced microglia exhibited an arborescent morphology and reduced intracellular ATP levels initially, without compromising viability.
- LPS upregulated the anti-apoptotic factor Bcl-xL and downregulated autophagy marker LC3.
Conclusions:
- Optimal LPS doses inhibit both apoptosis and autophagy in primary microglia, promoting long-term survival.
- Long-lived microglia may contribute to neurodegeneration, suggesting that inducing a resting state in activated microglia is a potential therapeutic strategy for neurodegenerative diseases.
