Single-Cell RNA Sequencing Reveals Microglial Heterogeneity and Functional States After Cerebral Ischemia-Reperfusion Injury.

Background: Ischemic stroke remains a leading cause of mortality and disability worldwide. Microglia, the resident immune cells of the central nervous system, perform critical roles in immune surveillance, debris clearance, and tissue repair. Analyzing the heterogeneity, activation trajectories, and metabolic states of microglia in ischemic stroke is essential for discovering therapeutic targets. Methods: We performed single-cell RNA sequencing (scRNA-seq) on brains from ischemic and control rats and identified transcriptionally distinct microglial subpopulations. We conducted differential expression, pathway enrichment, pseudotime analysis, regulatory network inference, and cell-cell communication mapping to characterize functional states, key transcription factors, and intercellular crosstalk underlying microglial responses following cerebral ischemia-reperfusion injury. We used Real-Time Quantitative PCR (RT-qPCR), Western blot, and immunohistochemistry to measure relative RNA and protein expression levels. Results: We identified seven microglial subclusters with distinct transcriptional signatures. Ischemia-associated clusters exhibited strong activation of inflammatory pathways, increased glycolysis and lipid metabolism, and suppressed TCA cycle and oxidative phosphorylation (OXPHOS), reflecting a shift toward proinflammatory, energy-demanding phenotypes. Pseudotime analysis revealed transitions from homeostatic to pathological states, highlighting potential therapeutic windows. Regulatory network analysis identified activating transcription factor 3 (ATF3) as a central regulator controlling the expression of cholesterol 25-hydroxylase (CH25H) and secreted phosphoprotein 1 (SPP1). Notably, ATF3 overexpression enhanced CH25H expression and selectively increased proinflammatory cytokine production, linking metabolic reprogramming to neuroinflammatory responses. Cell-cell communication analysis further revealed extensive remodeling of interactions with astrocytes, endothelial cells, and fibroblasts, potentially amplifying postischemic neuroinflammation. Conclusion: Our study demonstrates that cerebral ischemia induces transcriptionally and functionally distinct microglial subpopulations, characterized by metabolic reprogramming and proinflammatory activation. ATF3 serves as a central regulator linking CH25H-mediated metabolic changes to cytokine-driven neuroinflammation, highlighting potential therapeutic targets for modulating microglial responses and mitigating ischemia-induced brain injury.