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Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
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Single-Cell RNA Sequencing Reveals Microglial Heterogeneity and Functional States After Cerebral Ischemia-Reperfusion
Yushi Tang1,2, Qi Zhang3, Yewei Qu1
1Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China.
Journal of Inflammation Research
|December 10, 2025
Summary
Microglia in ischemic stroke show distinct activation states and metabolic shifts. Activating transcription factor 3 (ATF3) links metabolic changes to neuroinflammation, offering therapeutic targets for brain injury.
Area of Science:
- Neuroscience
- Immunology
- Metabolic Biology
Background:
- Ischemic stroke is a major global cause of death and disability.
- Microglia, the brain's immune cells, are crucial for CNS immune surveillance, debris clearance, and repair.
- Understanding microglial heterogeneity and metabolic states in stroke is key for therapeutic development.
Purpose of the Study:
- To analyze microglial heterogeneity, activation trajectories, and metabolic reprogramming in ischemic stroke.
- To identify key transcription factors and intercellular communication networks involved in microglial responses to cerebral ischemia-reperfusion injury.
- To uncover potential therapeutic targets for mitigating stroke-induced brain damage.
Main Methods:
- Single-cell RNA sequencing (scRNA-seq) on rat brains post-ischemia.
- Differential gene expression, pathway enrichment, and pseudotime analyses.
- Regulatory network inference, cell-cell communication mapping, RT-qPCR, Western blot, and immunohistochemistry.
Main Results:
- Seven distinct microglial subclusters were identified, with ischemia-associated clusters showing inflammatory pathway activation and altered metabolism (increased glycolysis/lipid, decreased OXPHOS).
- Pseudotime analysis indicated transitions from homeostatic to pathological states, revealing potential therapeutic windows.
- Activating transcription factor 3 (ATF3) was identified as a key regulator linking metabolic changes (via CH25H) to neuroinflammation and cytokine production.
Conclusions:
- Cerebral ischemia induces distinct microglial subpopulations with metabolic reprogramming and proinflammatory activation.
- ATF3 plays a central role in connecting metabolic shifts to cytokine-driven neuroinflammation.
- Targeting microglial responses, particularly ATF3-mediated pathways, offers a promising strategy for treating ischemic stroke.
