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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: Reduced microglia activation following metformin administration or microglia ablation is sufficient to prevent functional deficits in a mouse model of neonatal stroke

Fig. 3

H-I, in the presence or absence of metformin, increases astrocyte numbers in the cortex and striatum. 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 astrocytes (GFP+, green) and proliferating astrocytes (GFP + (green) EdU + (white) double-positive cells; yellow arrows) at P9 in the cortex in Sham (×20 magnification). C Representative image of astrocytes (GFP+, green) and proliferating astrocytes (GFP + (green) EdU + (white) double-positive cells; yellow arrows) at P9 in the cortex in Met-treated mice (×20 magnification). D Representative image of astrocytes (GFP +) and proliferating astrocytes (GFP + EdU+, yellow arrows) at P9 in the cortex after H-I (×20 magnification). E Representative image of astrocytes (GFP +) and proliferating astrocytes (GFP + EdU+, yellow arrows) at P9 in the cortex after H-I + Met (×20 magnification). F Quantification of the number of astrocytes (GFP +) in the cortex across time points and groups expressed as fold change. Astrocyte numbers were significantly increased after H-I at P9 relative to Sham (1.00 ± 0.17-fold change of GFP + cells in Sham mice vs. 2.27 ± 0.19 in H-I-injured mice, p = 0.0026). This increase was not prevented by HI + Met (1.45 ± 0.16-fold change of GFP + cells in Met-treated mice vs. 2.94 ± 0.17-fold change of GFP cells in H-I + Met-treated mice, p = 0.0044). Average number of GFP + cells/unit area: Sham = 27.94 ± 4.81; Met = 40.50 ± 4.57; H-I = 63.30 ± 5.17, H-I + Met = 82.00 ± 4.87. G Quantification of proliferating astrocytes (GFP + EdU + /GFP +) in the cortex at P9 across groups. Astrocyte numbers were significantly increased after H-I at P9 relative to Sham (2.64 ± 2.27% GFP + EdU + cells in Sham mice vs. 15.36 ± 2.37% in H-I-injured mice, p = 0.035). There was no significant difference in GFP + EdU + cells in Met-treated mice relative to and H-I + Met (7.63 ± 3.25% GFP + EdU + cells in Met-treated mice vs. 15.28 ± 2.77% GFP + EdU + cells in H-I + Met-treated mice, p = 0.27). H Representative image of astrocytes (GFP +) and proliferating astrocytes (GFP + EdU+, yellow arrows) at P12 in the striatum in Sham mice (×20 magnification). I Representative image of astrocytes (GFP+, green) and proliferating astrocytes (GFP + (green) EdU + (white) double-positive cells; yellow arrows) at P12 in the striatum in Met-treated mice (×20 magnification). J Representative image of astrocytes (GFP +) and proliferating astrocytes (GFP + EdU+, yellow arrows) at P12 in the striatum after H-I injury (×20 magnification). K Representative image of astrocytes (GFP +) and proliferating astrocytes (GFP + EdU+, yellow arrows) at P12 in the striatum after H-I + Met (×20 magnification). L Quantification of the number of astrocytes (GFP +) in the striatum across time points and groups expressed as fold change. Astrocyte numbers were significantly increased after H-I at P12 relative to Sham (1.00 ± 0.63-fold change of GFP + cells in Sham mice vs. 9.95 ± 2.46 in H-I-injured mice, p = 6.69 × 10–9). This increase was not prevented by HI + Met (2.49 ± 0.26-fold change of GFP + cells in Met-treated mice vs. 6.58 ± 0.74-fold change of GFP cells in H-I + Met-treated mice, p = 6.69 × 10–9). Average number of GFP + cells/unit area: Sham = 5.67 ± 3.56; Met = 14.09 ± 1.46; H-I = 56.38 ± 13.96, H-I + Met = 37.27 ± 4.18. M Quantification of proliferating astrocytes (GFP + EdU + /GFP +) in the striatum at P12 across groups. No significant differences were recorded across groups at P12 (p = 0.054). n = 3–7 mice per group. Data presented as mean ± SEM. Scale Bar: 50um. Unit area (cortex, striatum): 3 × 0.105 mm2. Statistics: (D, H) Two-way ANOVA; (E, I) One-way ANOVA. *p ≤ 0.050

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