Fig. 3: MCAO induces cholesterol metabolic reprogramming in microglia during the chronic stage.
a, BODIPY and IBA1 double immunostaining on frozen sections showing lipid droplet accumulation in microglia during acute and chronic stages post MCAO. Lipid droplets are labelled with BODIPY; IBA1 marks microglia. Colocalization highlights microglia-specific lipid droplet accumulation. Top-right insets show higher magnification of the area inside white dashed rectangles. b, Quantitative data of the BODIPY+ area within IBA1⁺ cells across time points; n = 3 mice per group. c, Principal component analysis (PCA) of lipid species in microglia shows distinct clustering of sham and MCAO groups at days 3, 30 and 90, indicating time-dependent lipidomic shifts. d, Violin plots illustrating alterations in cholesterol esters within microglia, measured by a targeted lipidomics method by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with multiple reaction monitoring during the acute and chronic stages post MCAO. Sham and MCAO-3d, n = 6 mice per group; MCAO-30d and MCAO-90d, n = 5 mice per group. e, Violin plots showing free cholesterol levels in microglia, measured by a targeted lipidomics method by LC–MS/MS with multiple reaction monitoring during the acute and chronic phases post MCAO. Sham and MCAO-3d, n = 6 mice per group; MCAO-30d and MCAO-90d, n = 5 mice per group. f, PLM and IBA1 immunostaining revealing cholesterol crystal (CC) deposition within microglia during acute and chronic phases; insets show higher magnification of the area inside white dashed rectangles. g, Quantification of crystal number per mm2 within IBA1+ cells; n = 3 mice per group. h, Heatmap analysis displaying changes in cholesterol metabolism-associated gene expression in microglia during the acute and chronic stage post MCAO. Sham and MCAO-3d, n = 4 mice per group; MCAO-30d and MCAO-90d, n = 3 mice per group. i, GSEA pathway enrichment analysis showing upregulation of cholesterol metabolism-associated pathways in microglia during the acute and chronic stage post MCAO. Sham and MCAO-3d, n = 4 mice per group; MCAO-30d and MCAO-90d, n = 3 mice per group. Data are mean and s.d. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test (b,g) or a one-sided permutation test, and the resulting P values were adjusted for multiple comparisons (i).
