Fig. 4

Pharmacological inhibition of sEH reduced proinflammatory microglia/macrophage activation, neuronal damage, and MMP-9 activity and attenuated BBB permeability after ICH. a The experimental design scheme used to study the effect of sEH inhibition by AUDA. ELISA enzyme-linked immunosorbent assay, EB Evans blue, MMP-9 matrix metalloproteinase-9, WB Western blot. b Iba1 staining and quantitative data at 1 day post-ICH. The inset is a representative activated microglia/macrophage at higher magnification. Low magnification of a cresyl violet-stained brain section of a core hemorrhagic region at 0.24 mm from the bregma (top left). The white box indicates the location of representative images. c Double-labeling shows colocalization of CD16/32 (classic activation marker) and Iba1 in the perihematomal area at 1 day. Arrowheads indicate the cell of colocalization. The inset images represent higher magnification of the colocalized regions in the corresponding images. Nuclei were stained with DAPI (blue). The bar graph shows the degree of Iba1 and CD16/32 colocalization in gray pixel intensity. d FJB staining and quantitative data at 1 day. The inset is a representative FJB-positive cell at higher magnification. e Western blot analysis of c-CP and f MPO staining and quantitative data at 1 day. The inset is a representative MPO-positive cell at higher magnification. g Representative gelatin zymography and quantitative data of MMP-9 activity and f hemoglobin levels at 1 day. h Quantification of brain Evans blue leakage at 1 day. i Quantification of hemoglobin contents at 1 day. The white box in the cresyl violet-stained image in b indicates the location of representative images for Iba1, FJB, and MPO stainings. The number of Iba1-, FJB-, and MPO-positive cells is expressed as the mean number per field of view (0.8 mm²). Values are mean ± S.E.M.; ^## P < 0.01, and ^### P < 0.001 vs. sham group; *P < 0.05, **P < 0.01, and ***P < 0.01 vs. vehicle group; ^† P < 0.05 vs. 1 μM AUDA group (n = 5 mice/group for Iba1, FJB staining, and hemoglobin assay and n = 6 mice/group for MPO staining, Student’s t test; n = 5 mice/group for CD16/32 and Iba1 double staining, n = 6 mice/group for Western blot and Evans blue quantification, and n = 7 mice/group for zymography, one-way ANOVA)