Fig. 5
Microglia ablation affords protection against functional deficits after H-I. A Experimental timeline. H-I was performed on P8 and PLX administration from P8 to P15. IHC was performed on P15. Behaviour tasks were performed on P8 (righting reflex), P12 (hindlimb suspension), P15 (grip strength) and P22 (cylinder task). B Representative image of microglia (Iba1+, red) at P15 in the cortex of Sham mice (×20 magnification). C Representative image of microglia (Iba1+, red) at P15 in the cortex of H-I mice (×20 magnification). D Representative image of microglia (Iba1 +) at P15 in the cortex of H-I + PLX mice (×20 magnification). E Quantification of the number of microglia (Iba1 +) in the cortex across groups expressed as fold change. There was no difference in Iba1 + microglia after H-I at P15 (1.00 ± 0.10-fold change of Iba1 + cells/unit area in Sham vs. 1.05 ± 0.05-fold change of Iba1 + cells/unit area after HI) (p = 0.93). There was a significant decrease in Iba1 + microglia after H-I + PLX relative to H-I (1.05 ± 0.05-fold change of Iba1 + cells/unit area after H-I vs. 0.45 ± 0.11-fold change of Iba1 + cells/unit area after H-I + PLX) (p = 0.0076). Average number of Iba1 + cells/unit area: Sham = 67.17 ± 7.21; H-I = 48.11 ± 2.38; H-I + PLX = 20.56 ± 4.83. F Quantification of the number of microglia (Iba1 +) in the striatum across groups, expressed as fold change. There was no difference in Iba1 + microglia after H-I at P15 (1.00 ± 0.09-fold change of Iba1 + cells/unit area in Sham vs. 0.99 ± 0.04-fold change of Iba1 + cells/unit area after H-I) (p = 0.99). There was a significant decrease in Iba1 + microglia after H-I + PLX relative to H-I (0.99 ± 0.04-fold change of Iba1 + cells/unit area after H-I vs. 0.30 ± 0.04-fold change of Iba1 + cells/unit area after H-I + PLX) (p = 0.00060). Average number of Iba1 + cells/unit area: Sham = 32.19 ± 2.65; H-I = 31.11 ± 1.46; H-I + PLX = 9.33 ± 1.35. G Quantification of the number of microglia (Iba1 +) in the SEZ across groups, expressed as fold change. There was no difference in Iba1 + microglia after H-I at P15 (1.00 ± 0.09-fold change of Iba1 + cells/unit area in Sham vs. 1.12 ± 0.11-fold change of Iba1 + cells/unit area after H-I) (p = 0.62). There was a significant decrease in Iba1 + microglia after H-I + PLX relative to H-I (1.12 ± 0.11-fold change of Iba1 + cells/unit area after H-I vs. 0.25 ± 0.06-fold change of Iba1 + cells/unit area after H-I + PLX) (p = 0.0022). Average number of Iba1 + cells/unit area: Sham = 20.03 ± 0.87; H-I = 17.89 ± 1.68; H-I + PLX = 6.00 ± 1.00. H Latency to prone (s) on the righting reflex across groups. Significant increases were found after injury between Sham vs. H-I (p = 3.86 × 10–10), Vehicle vs. H-I (p = 1.59 × 10–10), and PLX vs. H-I + PLX (p = 1.58 × 10–10). There was no difference in the righting reflex between H-I vs. H-I + PLX (p = 0.26). I Latency to fall (s) on the hindlimb suspension across groups. Significant decreases were found after injury between Sham vs. H-I (p = 6.69 × 10–7), Vehicle vs. H-I (p = 7.95 × 10–7) and H-I vs. H-I + PLX (p = 5.19 × 10–7). J Angle at fall (degree) on grip strength across groups. Significant decreases were found after injury between Sham vs. H-I (p = 2.46 × 10–6), Vehicle vs. H-I (p = 1.37 × 10–7) and H-I vs. H-I + PLX (p = 7.03 × 10–6). K Forepaw preference for the uninjured paw on the cylinder test. H-I-injured mice are significantly impaired compared to Shams (-0.29 ± 3.44% preference for the un-impaired paw in Sham mice vs. 19.56 ± 3.12% in H-I-injured mice, p = 0.0009). PLX-treated (p = 0.99) and vehicle-treated mice (p = 0.99) did not perform differently than Shams. H-I + PLX are significantly improved compared to H-I only (-1.96 ± 2.48% preference for the un-impaired paw in H-I + Met mice vs. 19.56 ± 3.12% in H-I-injured mice, p = 0.0009) and were not significantly different from Shams (p = 0.99). n = 3 mice per group for immunohistochemistry. n = 6–21 mice per group for behavioural testing. Data presented as mean ± SEM. Scale Bar: 50um. Unit area (cortex, striatum): 3 × 0.105 mm2. Unit area (SEZ): 3 × 0.045mm2. Statistics: (D-J) One-way ANOVA. *p ≤ 0.050