Abstract WP316: Single-Cell RNA Sequencing Reveals a Massive Alteration in the Molecular Landscape of Microglia After Ischemic Stroke

Abstract

Introduction: Ischemic stroke, a prevalent and often devastating disease, remains poorly treated. Neuroinflammation is a key element of stroke pathophysiology and represents a promising therapeutic target. Among the central components driving neuroinflammation, microglia - the primary resident immune cells within the brain parenchyma - are key players in mediating the immune response in the stroke brain. However, how microglia respond to stroke is incompletely understood. In this study, we took advantage of a mouse genetic tool (ie, Tmem119-CreERT2), sorted microglia, and performed single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of permanent stroke. Moreover, as stroke exhibits sexual dimorphism, we included both male and female mice. Methods: Young male and female Ai6/TMEM119-CreERT2;Ai6 (Tmem119-Ai6) mice with microglia labeled with ZsGreen underwent permanent middle cerebral artery occlusion (pMCAO) or sham surgery. On day 3 after stroke, ZsGreen+ cells (microglia) in the brain were isolated by FACS, and used for scRNA-seq analysis. Results: First, we confirmed that in Tmem119-Ai6 mice, expression of fluorescent ZsGreen was almost exclusively in microglia in the brain, with minimal presence in blood cells. Our unbiased scRNA-seq analysis of ZsGreen+ cells revealed 13 distinct microglial clusters after stroke. Importantly, 4 major stroke-related microglial clusters were identified. One cluster shared a gene signature with the reported disease-associated microglia (DAM) subset. The other 3 clusters were characterized by an interferon (IFN) response signature, a highly proliferative state, and a macrophage-like profile, respectively. RNA in situ hybridization analysis showed that cells from these clusters mainly resided within the stroke boundaries. Notably, our scRNA-seq data suggest no major sexual dimorphism in the microglial response to permanent stroke. Conclusions: This is the first scRNA-seq study with a focus on microglia in the context of sex and stroke. We propose that 4 microglial subsets may be deemed as common stroke-associated microglia. Future work will be designed to elucidate the functional significance of these subsets and their potential implications for stroke therapy.