Extended Data Fig. 4: MCAO evokes cholesterol metabolic reprogramming in microglia during the chronic stage.
a, b, H&E staining assessing foam cell formation in the infarct area during the chronic stage post-MCAO. Scale bar, 20 µm; n = 3 mice/group. c, Immunofluorescence detection of lipid droplets in microglia using BODIPY and IBA1 co-immunostaining during the acute and chronic stage post-MCAO. Scale bar, 500 µm. The low panel shows a higher-magnification image for detailed visualization. Scale bar, 50 µm. Representative images from one of three independent experiments are shown. d, e, Filipin III (pink pseudo‐color) staining was employed to assess free cholesterol accumulation in microglia during the chronic phase post-MCAO. Scale bar, 20 µm; n = 3 mice/group. f, IBA1 immunostaining and polarized light microscopy (PLM) detecting needle-like cholesterol crystals (CCs) in the infarct area on day 30 and day 90 post-MCAO. Scale bar, 50 µm. Representative images from one of three independent experiments are shown. g, PLM shows birefringent color changes in CCs at days 3, 30, and 90 post-MCAO. Anisotropic crystalline nature confirmed by orientation-dependent interference colors using a first-order λ compensator. Scale bar, 20 µm. Representative images from one of three independent experiments are shown. h, Gene set enrichment analysis (GSEA) revealed changes in the cholesterol metabolism pathway across pairwise comparisons between days 3 vs 30, days 3 vs 90, and days 30 vs 90 post-MCAO. Normalized enrichment scores (NES) and P value for each gene set are displayed. n = 4 mice/group for sham and MCAO-30d groups; n = 3 mice/group for MCAO-3d and MCAO-90d groups. i, KEGG analysis reveals upregulation of cholesterol metabolism-related genes at days 3, 30, and 90 post-MCAO compared to sham controls. NES and P value for each gene set are displayed. n = 4 mice/group for sham and MCAO-30d groups; n = 3 mice/group for MCAO-3d and MCAO-90d groups. Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA (b, e) followed by Dunnett’s multiple comparisons test, a one-sided hypergeometric test, and P-values were adjusted for multiple comparisons to generate q-values (h), or a one-sided hypergeometric test, and P-values were adjusted for multiple comparisons to generate q-values (i).
