Fig. 1 

H-I, in the presence or absence of metformin, increases microglia numbers in the cortex and striatum, but not the SEZ. A Experimental timeline. Mice received H-I on P8 and metformin administration from P9-P14. Immunohistochemistry (IHC) was performed on P9, P12, P14 and P21. B Representative image of microglia (Iba1+, red) and proliferating microglia (Iba1 + (red) EdU + (white) double-positive cells) at P12 in the cortex in Sham mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. C Representative image of microglia (Iba1+, red) and proliferating microglia (Iba1 + (red) EdU + (white) double-positive cells) at P12 in the cortex in Met-treated mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. D Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the cortex after H-I (×20 magnification). Proliferating microglia are depicted by yellow arrows. Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the cortex after H-I (×20 magnification). Proliferating microglia are depicted by yellow arrows. E Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the cortex after H-I + Met treatment (×20 magnification). Proliferating microglia are depicted by yellow arrows. F Quantification of the number of microglia (Iba1 + cells) in the cortex across time points and treatment groups expressed as fold change. Microglia were significantly increased after H-I at P12 relative to Sham (1.00 ± 0.05-fold change of Iba1 + cells in Sham mice vs. 2.36 ± 0.38 in H-I-injured mice, p = 0.0063). This trend was observed after HI + Met (1.20 ± 0.24-fold change of Iba1 + cells in Met-treated mice vs. 2.13 ± 0.09-fold change of Iba1 + cells in H-I + Met-treated mice, p = 0.073). Average number of Iba1 + cells/unit area: Sham = 22.69 ± 0.97; Met = 27.88 ± 5.57; H-I = 58.13 ± 4.00, H-I + Met = 50.59 ± 1.77. G Quantification of percent proliferating microglia (Iba1 + EdU + /Iba1 +) in the cortex at P12 across treatment groups. A significant increase was observed after H-I (7.50 ± 2.86% Iba1 + EdU + cells/unit area in Sham vs. 45.98 ± 11.45% Iba1 + EdU + cells/unit area in H-I, p = 0.0064) and Met treatment did not prevent this expansion (45.98 ± 11.45% Iba1 + EdU + cells/unit area in H-I vs. 32.38 ± 2.07% Iba1 + EdU + cells/unit area in H-I + Met, p = 0.37). There was no significant difference between Met-treated mice and H-I + Met-treated mice (8.38 ± 4.13% Iba1 + EdU + cells/unit area in Met vs. 32.38 ± 2.07% Iba1 + EdU + cells/unit area in H-I + Met-treated mice, p = 0.056). H Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the striatum in Sham mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. I Representative image of microglia (Iba1+, red) and proliferating microglia (Iba1 + (red) EdU + (white) double-positive cells) at P12 in the striatum in Met-treated mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. J Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the striatum after H-I (×20 magnification). Proliferating microglia are depicted by yellow arrows. K Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the striatum after H-I + Met treatment (×20 magnification). Proliferating microglia are depicted by yellow arrows. L Quantification of the number of microglia (Iba1 +) in the striatum across time points and groups expressed as fold change. Microglia were significantly increased after H-I at P12 relative to Sham (1.00 ± 0.15-fold change of Iba1 + cells in Sham mice vs. 2.70 ± 0.10 in H-I-injured mice, p = 0.032). There was no significant difference in Iba1 + cells in Met-treated mice compared to H-I + Met (1.86 ± 0.31-fold change of Iba1 + cells in Met-treated mice vs. 2.95 ± 0.27-fold change of Iba1 + cells in H-I + Met-treated mice, p = 0.29). Average number of Iba1 + cells/unit area: Sham = 8.25 ± 1.11; Met = 14.97 ± 2.71; H-I = 58.13 ± 4.00, H-I + Met = 27.83 ± 2.13. M Quantification of proliferating microglia (Iba1 + EdU + /Iba1 +) in the striatum at P12 across groups. Significant increases were observed between Sham and H-I (7.99 ± 2.41% Iba1 + EdU + cells/unit area in Sham vs. 33.58 ± 7.02% Iba1 + EdU + cells/unit area in H-I, p = 0.0085) and Met-treated mice vs. H-I + Met-treated mice (6.12 ± 1.16% Iba1 + EdU + cells/unit area in Met vs. 47.19 ± 2.64% Iba1 + EdU + cells/unit area in H-I + Met, p = 0.15). Quantification of proliferating microglia (Iba1 + EdU + /Iba1 +) in the striatum at P12 across groups. Significant increases were observed between Sham (7.99 ± 2.41% Iba1 + EdU + cells/unit area) vs. H-I (33.58 ± 7.02% Iba1 + EdU + cells/unit area) (p = 0.009) and Sham (7.99 ± 2.41% Iba1 + EdU + cells/unit area) vs. H-I + Met (47.19 ± 2.64% Iba1 + EdU + cells/unit area) (p = 0.006). N Representative image of microglia (Iba1+) and proliferating microglia (Iba1 + EdU +) at P12 in the SEZ in Sham mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. O Representative image of microglia (Iba1+, red) and proliferating microglia (Iba1 + (red) EdU + (white) double-positive cells) at P12 in the striatum in Met-treated mice (×20 magnification). Proliferating microglia are depicted by yellow arrows. P Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the SEZ after H-I (×20 magnification). Proliferating microglia are depicted by yellow arrows. Q Representative image of microglia (Iba1 +) and proliferating microglia (Iba1 + EdU +) at P12 in the SEZ after H-I + Met treatment (×20 magnification). Proliferating microglia are depicted by yellow arrows. R Quantification of the number of microglia (Iba1 +) in the SEZ across time points and groups expressed as fold change. No significant differences were found between groups (p = 0.15, n = 3–7) mice per group. Data presented as mean ± SEM. Scale Bar: 50um. Unit area (cortex, striatum): 3 × 0.105 mm². Unit area (SEZ): 3 × 0.045mm2. Statistics: (D, H, L) Two-way ANOVA; (E, I) One-way ANOVA. *p ≤ 0.050