Related Experiment Video
Updated: Aug 17, 2025
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Delayed microglial activation associated with the resolution of neuroinflammation in a mouse model of sublethal
Atsuyoshi Shimada1, Makiko Murata1, Sayaka Aoyagi2
1Pathology Research Team, Faculty of Health Sciences, Kyorin University, 5-4-1 Shimorenjaku, Mitaka, Tokyo 181-8612, Japan.
Abstract:
Systemic inflammation affects brain functions. In our previous study in which lipopolysaccharide (LPS) was injected intraperitoneally into mice at sublethal doses, choroid plexus macrophages produced interleukin-1β and stimulated neighboring stromal cells. Activated stromal cells stimulate choroid plexus epithelial cells, and then choroid plexus epithelium-derived cytokines enter the brain parenchyma and stimulate astrocytes. Stimulated astrocytes then produce cytokines such as CCL11, CXCL10 and G-CSF and change the brain parenchymal microenvironment. However, the effects of an altered brain microenvironment on other brain cells remain to be determined. In the present study, we hypothesized that microglia are activated in response to astrocyte-induced changes in the brain microenvironment. Using the brains of mice treated with intraperitoneal LPS injection, Luminex multiplex cytokine immunoassays revealed increased hippocampal concentrations of CCL11, CXCL10 and G-CSF at 48 h after systemic LPS challenge. The concentrations of all cytokines examined returned to control levels at 72 h after LPS injection, which indicated a resolution of the neuroinflammation. Immunohistochemistry revealed that microglia were hypertrophied in mice at 48 h after systemic LPS challenge. Following isolation of microglial cells from the brain using magnetic-activated cell sorting, gene expression assays were performed with real-time reverse transcriptase-polymerase chain reaction. Isolated microglial cells exhibited much higher gene expression of the receptors for CCL11, CXCL10 and G-CSF than other brain cells. Microglial cells isolated from the brains of mice at 48 h after systemic LPS challenge exhibited the M2-like phenotype. In conclusion, microglial hypertrophy occurs following astrocytic reactions in a mouse model of sublethal endotoxemia-induced systemic inflammation, and hypertrophic microglia are polarized toward the M2-like phenotype and involved in the resolution of neuroinflammation.
Insights
Systemic inflammation triggers astrocyte activation, leading to microglial M2 polarization and neuroinflammation resolution. This study reveals how microglia respond to altered brain environments post-LPS challenge.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Systemic inflammation impacts brain function.
- Previous work showed choroid plexus macrophages and astrocytes respond to lipopolysaccharide (LPS).
- The effect of astrocyte-derived cytokines on other brain cells, particularly microglia, was unknown.
Purpose of the Study:
- To investigate microglial activation in response to astrocyte-induced brain microenvironment changes.
- To determine the phenotype and role of microglia during systemic inflammation.
Main Methods:
- Mice were injected with sublethal doses of lipopolysaccharide (LPS).
- Luminex assays measured hippocampal cytokine levels (CCL11, CXCL10, G-CSF).
- Immunohistochemistry assessed microglial morphology, and gene expression analysis (RT-PCR) was performed on isolated microglia.
Main Results:
- Hippocampal CCL11, CXCL10, and G-CSF levels increased at 48 hours post-LPS, returning to baseline by 72 hours.
- Microglia showed hypertrophy and an M2-like phenotype 48 hours after LPS injection.
- Isolated microglia expressed higher levels of receptors for CCL11, CXCL10, and G-CSF.
Conclusions:
- Microglial hypertrophy and M2 polarization follow astrocytic responses in a mouse model of endotoxemia.
- Activated microglia play a role in resolving neuroinflammation induced by systemic inflammation.
