Fig. 7

Pan-probiotic (PP) treatment decreases microglia density and macrophage infiltration at 3 days post-injury. (a) PP treatment significantly reduced microglial density (Iba-1 + cells, red) in male mice compared to vehicle (VH)-treated males (***p < 0.001, **p < 0.01) at 3 days post-injury (dpi). (b) The percentage of the area occupied by Iba-1 + cells also exhibits a noteworthy decrease in PP-treated male mice compared to their VH-treated counterparts and the VH-treated females compared with VH-treated males. (c, d, e, f) Representative images depict Iba-1 + cells in the injured cortex of mice treated with VH and PP. (g) Skeletonized microglial structures with color-coded branches illustrating morphological complexity using the Neurolucida 360 tracing software. (h) Higher magnification of individual microglia demonstrating branch intersections and total process length. (i) An image displays concentric circles (red) drawn at a 2 μm distance for Sholl analysis. (c1, d1, e1, f1) Representative confocal microscopy images of Iba-1 + staining in PP and VH brains at 40X magnification. The Sholl analysis reveals significant differences in the number of intersections from the distance of the cell body of Iba-1 + cells (j) and the total average length of the branch from the distance of the cell body (k) in the injured cortex of males treated with PP compared to VH, indicating reduced activation and more ramified microglia in PP-treated males. No differences were observed in female mice (l, m). (n) PP treatment reduced macrophage infiltration (F4/80 + cells, green) in male mice (**p < 0.01) but not in females. (n1-4) Representative images illustrate F4/80 + cells in the injured cortex of PP- and VH-treated male and female mice. (o–q) Co-Localization of Iba-1 + microglia with TGFβ, Arg1, and TNFα. PP treatment increased TGFβ + microglia in males (*p < 0.05) and decreased TNFα + microglia in both sexes (*p < 0.05). No significant differences were observed in Arg1 + microglia. Iba-1 + microglia (green), DAPI (blue), and markers TGFβ (o1–o4), Arg1 (p1–p4), and TNFα (q1–q4) in the cortex of male (o1–q2) and female (o3–q4) mice treated with VH or PP. The data represents n = 4 mice per group, and statistical significance was determined using two-way ANOVA with post-hoc Tukey’s multiple comparison test. For the Sholl analysis, the area under the curve was calculated, and an unpaired t-test using the mean, SEM, and n was utilized (*p < 0.05, **p < 0.01, ***p < 0.001). mRNA expression in red, and nuclei in blue. Scale bars represent 50 μm in c-f and 20 μm in c1, d1, e1, f1, o1-4, p1-4, and q1-4