Abstract 103: Acute Ischemia Induces Spatially and Transcriptionally Distinct Microglial Subclusters

Abstract

Introduction: Damage in the ischemic core and penumbra after stroke affects patient prognosis. Microglia immediately respond to ischemic insult after stroke. However, the microglial heterogeneity and the underlying mechanisms remain unclear. In this study, we sought to uncover stroke-associated microglia and identify their heterogenous features. Methods: We performed single-cell RNA-sequencing and spatial transcriptomics on middle cerebral artery occlusion (MCAO) mice to determine stroke-associated microglial clusters. The expression of marker genes and the localization of different microglial subclusters were verified through RNAscope and immunofluorescence. GSVA was employed to reveal functional characteristics of microglial subclusters. IPA was used to explore upstream regulators of microglial clusters, which was confirmed by immunofluorescence, RT-qPCR, and targeted metabolomics. Finally, the infarct size and neurological deficits were evaluated in MCAO mice after pharmacological manipulation of specific microglial subcluster. Results: We discovered stroke-associated microglial subclusters in MCAO mice. We also identified novel marker genes of these microglial subclusters and defined these cells as ischemic core-associated (ICAM) and ischemic penumbra-associated (IPAM) microglia, according to their spatial distribution. ICAM, induced by damage-associated molecular patterns, are fueled by glycolysis, and exhibit increased pro-inflammatory cytokines and chemokines production. BACH1 is a key transcription factor driving ICAM generation. In contrast, glucocorticoids enriched in the penumbra triggers IPAM formation, which are powered by citrate cycle and oxidative phosphorylation and are characterized by moderate pro-inflammatory responses, inflammation-alleviating metabolic features, and myelinotrophic properties. Conclusions: ICAM induce excessive neuroinflammation, aggravating brain injury, whereas IPAM could be essential for the homeostasis and survival of cells in the penumbra. Our findings provide a biological basis for targeting specific microglial subtypes as a potential therapeutic strategy for ischemic stroke.