SPATIAL CHARACTERIZATION OF MYELOID CELL HETEROGENEITY IN ISCHEMIC STROKE UNDER NORMOLIPIDEMIC AND HYPERLIPIDEMIC CONDITIONS

Ischemic stroke is a major cause of disability and mortality and is closely linked to atherosclerosis-driven vascular dysfunction. This study aimed to characterize the phenotypic and spatial heterogeneity of myeloid cells, particularly macrophages and microglia, in experimental ischemic stroke and to assess the impact of hyperlipidemia on these immune responses. Transient middle cerebral artery occlusion was induced in young male C57BL/6 mice fed either a standard diet or a high-fat diet, with naïve mice used as controls. Spatial transcriptomics was performed on frozen brain sections using the CosMx platform with a 960-gene panel. After quality control, cell types were identified using InSituType with the Allen Brain Mouse Atlas as reference, followed by unsupervised clustering of myeloid populations based on gene expression and spatial distribution. Marked differences in cellular composition were observed between naïve and ischemic brains, with neuronal enrichment in naïve and contralateral regions and pronounced immune infiltration, astrogliosis and microglial activation in the ipsilateral hemisphere. Microglia displayed homeostatic profiles in naïve and contralateral tissue, whereas reactive, phagocytic and proliferative phenotypes predominated in the ischemic core and peri-infarct regions, with distinct spatially defined subpopulations. Comparisons between diets revealed largely similar spatial patterns, although a microglial and vascular subset showed diet-associated differential gene expression. Spatial interaction analysis highlighted robust communication between vascular cells, infiltrating immune cells, activated microglia and astrocytes. These findings demonstrate pronounced spatial and functional heterogeneity of myeloid cells after stroke and suggest that hyperlipidemia subtly modulates microglial transcriptional states, providing insights into immune mechanisms relevant for therapeutic targeting.