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Updated: May 5, 2026
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
Activated microglia in a rat stroke model express NG2 proteoglycan in peri-infarct tissue through the involvement of
Kana Sugimoto1, Ryutaro Nishioka, Airi Ikeda
1Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan.
Abstract:
We investigated activated microglia in ischemic brain lesions from rats that had been subjected to transient middle cerebral artery occlusion. Activated microglia expressing NG2 chondroitin sulfate proteoglycan (NG2) were found only in the narrow zone (demarcation zone) that demarcated the peri-infarct tissue and ischemic core. NG2(-) activated microglia were abundantly distributed in the peri-infarct tissue outside the demarcation zone. NG2(+) microglia but not NG2(-) microglia expressed both CD68 and a triggering receptor expressed on myeloid cells 2 (TREM-2), suggesting that NG2(+) microglia eliminated apoptotic neurons. In fact, NG2(+) microglia often attached to degenerating neurons and sometimes internalized NeuN(+) or neurofilament protein(+) material. Kinetic studies using quantitative real-time RT-PCR revealed that expression of transforming growth factor-β1 (TGF-β1) was most evident in the ischemic core; with this marker produced mainly by macrophages located in this region. TGF-β receptor mRNA expression peaked at 3 days post reperfusion (dpr) in the peri-infarct tissue, including the demarcation zone. Primary cultured rat microglia also expressed the receptor mRNA. In response to TGF-β1, primary microglia enhanced the expression of NG2 protein and TREM-2 mRNA as well as migratory activity. A TGF-β1 inhibitor, SB525334, abolished these effects. The present results suggest that TGF-β1 produced in the ischemic core diffused toward the peri-infarct tissue, driving activated microglial cells to eliminate degenerating neurons. Appropriate control of NG2(+) microglia in the demarcation zone might be a novel target for the suppression of secondary neurodegeneration in the peri-infarct tissue.
Insights
Transforming growth factor-β1 (TGF-β1) drives NG2(+) microglia to clear degenerating neurons in stroke. Targeting these cells in the demarcation zone may prevent further neurodegeneration.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Ischemic stroke triggers complex inflammatory responses in the brain.
- Microglia, the brain's resident immune cells, play critical roles in lesion progression and repair.
- Understanding microglial subtypes and their functions is crucial for developing effective stroke therapies.
Purpose of the Study:
- To investigate the role of specific activated microglial populations in ischemic brain lesions.
- To elucidate the involvement of NG2 chondroitin sulfate proteoglycan (NG2) and transforming growth factor-β1 (TGF-β1) in microglial-mediated neurodegeneration after stroke.
Main Methods:
- Transient middle cerebral artery occlusion model in rats.
- Immunohistochemistry to identify microglial markers (NG2, CD68, TREM-2).
- Quantitative real-time RT-PCR for gene expression analysis (TGF-β1, TGF-β receptor).
- In vitro studies with primary microglia treated with TGF-β1 and an inhibitor.
Main Results:
- NG2(+) microglia were localized to the demarcation zone, expressing CD68 and TREM-2, and were observed engulfing apoptotic neurons.
- TGF-β1 expression was highest in the ischemic core, produced by macrophages.
- TGF-β1 signaling upregulated NG2, TREM-2, and migration in primary microglia, effects blocked by an inhibitor.
Conclusions:
- TGF-β1 from the ischemic core promotes NG2(+) microglial activation and phagocytosis of degenerating neurons in the peri-infarct area.
- NG2(+) microglia in the demarcation zone are key players in clearing neuronal debris.
- Targeting NG2(+) microglia presents a potential therapeutic strategy to mitigate secondary neurodegeneration post-stroke.
