Extended Data Fig. 2: MCAO induces chronic neuroinflammation and microglial cholesterol metabolic reprogramming in female mice.
a, IBA1 immunostaining showing altered microglial morphology in the lesion area at days 3 and 90 post-MCAO compared to sham controls in female mice. Scale bar, 20 µm; n = 3 mice/group; Representative images from one of two independent experiments are shown. b, Heatmap depicting upregulation of inflammatory genes in microglia isolated at days 3 and 90 post-MCAO compared to sham controls in female mice. n = 4 mice for the sham group; n = 3 mice per MCAO group. c, Gene set enrichment analysis (GSEA) identifying enrichment of inflammatory and immune-related pathways. Normalized enrichment scores (NES) and P-value for each gene set are displayed. sham, n = 4 mice/group; MCAO group, n = 3 mice/group. RES: Running enrichment score, RLM: Ranked list metric. d, e, Immunofluorescence detection of lipid droplets in microglia using BODIPY and IBA1 co-staining at 3 and 90 days post-MCAO in female mice, highlighting lipid accumulation over time. Scale bar, 20 µm; n = 3 mice/group; f, g, Polarized light microscopy (PLM) assessing cholesterol crystals at 3 and 90 days post-MCAO in female mice, demonstrating cholesterol crystal accumulation during the chronic phase. Crystal birefringence was confirmed by orientation-dependent interference colors under PLM with a first-order λ compensator. Main image: scale bar, 20 µm; Higher-magnification view: scale bar, 10 μm. n = 3 mice/group; ns: no significance; Representative images from one of three independent experiments are shown. h, Heatmap illustrating differential expression of cholesterol metabolism-associated genes in microglia at 3 and 90 days post-MCAO in female mice, revealing metabolic reprogramming. n = 4 mice for the sham group; n = 3 mice per MCAO group. Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA (a, e, g) followed by Dunnett’s multiple comparisons test, or a one-sided permutation test, and the resulting P-values were adjusted for multiple comparisons©.
