Fig. 4

Treatment cessation following microglia ablation, which had no effects on astrocytes or sham motor behaviour, causes rapid microglia repopulation. A Experimental timeline. Mice received PLX administration from P8 to P15 (SAC on P15 or P22) or P8 to P22 (SAC on P22). Immunohistochemistry (IHC) was performed on P15 and P22. Hindlimb suspension was performed on P12 and Grip Strength on P15. B Representative image of microglia (Iba1+, red) at P15 in the cortex of vehicle-treated mice (×20 magnification). C Representative image of microglia (Iba1+, red) at P15 in the cortex of mice treated with PLX from P8 to P15 (×20 magnification). D Representative image of microglia (Iba1+, red) at P22 in the cortex of vehicle-treated mice (×20 magnification). E Representative image of microglia (Iba1+, red) at P22 in the cortex of mice treated with PLX from P8 to P15. F Representative image of microglia (Iba1+, red) at P22 in the cortex of vehicle-treated mice (×20 magnification). G Representative image of microglia (Iba1+, red) at P22 in the cortex of mice treated with PLX from P8 to P22. H Quantification of the number of microglia (Iba1 +) in the cortex across groups expressed as fold change. There was a significant decrease in microglia after PLX treatment relative to vehicle at P15 (1.00 ± 0.11-fold change of Iba1 + cells in vehicle-treated mice vs. 0.19 ± 0.05 in H-I-injured mice, p = 0.0008). There was no difference in microglia numbers between vehicle vs. PLX at P22 following treatment from P8 to P15 (1.00 ± 0.11-fold change of Iba1 + cells in vehicle-treated mice vs. 0.80 ± 0.10 in H-I-injured mice, p = 0.646). There was a significant decrease in microglia after treatment relative to vehicle at P22 following two weeks of treatment (PLX from P8 to P22) (1.00 ± 0.14-fold change of Iba1 + cells in vehicle-treated mice vs. 0.30 ± 0.04 in H-I-injured mice, p = 0.003). Average number of Iba1 + cells/unit area: P15 Vehicle = 67.17 ± 7.21; P15 PLX = 12.42 ± 3.2; P8-P15 Sac 22 Vehicle = 19.83 ± 3.06; P8-P15 Sac 22 PLX = 21.42 ± 2.69; P8-P22 Sac 22 Vehicle = 40.68 ± 5.60, P8-P22 Sac 22 PLX = 12.22 ± 1.49. I Representative image of microglia (Iba1 +) at P15 in the striatum of vehicle-treated mice (×20 magnification). J Representative image of microglia (Iba1 +) at P15 in the striatum of PLX-treated mice (×20 magnification). K Representative image of microglia (Iba1+, red) at P22 in the striatum of vehicle-treated mice (×20 magnification). L Representative image of microglia (Iba1+, red) at P22 in the striatum of mice treated with PLX from P8 to P15. M Representative image of microglia (Iba1+, red) at P22 in the striatum of vehicle-treated mice (×20 magnification). N Representative image of microglia (Iba1+, red) at P22 in the striatum of mice treated with PLX from P8 to P22. O Quantification of the number of microglia (Iba1 +) in the striatum across groups expressed as fold change. There was a significant decrease in microglia after PLX treatment relative to vehicle at P15 (1.00 ± 0.08-fold change of Iba1 + cells in vehicle-treated mice vs. 0.31 ± 0.04 in H-I-injured mice, p = 0.005). There was no difference in microglia numbers between vehicle vs. PLX at P22 following treatment from P8 to P15 (1.00 ± 0.17-fold change of Iba1 + cells in vehicle-treated mice vs. 0.91 ± 0.05 in H-I-injured mice, p = 0.982). There was a significant decrease in microglia after treatment relative to vehicle at P22 following two weeks of treatment (PLX from P8 to P22) (1.00 ± 0.08-fold change of Iba1 + cells in vehicle-treated mice vs. 0.32 ± 0.06 in H-I-injured mice, p = 0.005). Average number of Iba1 + cells/unit area: P15 Vehicle = 32.19 ± 2.65; P15 PLX = 9.83 ± 1.36; P8-P15 Sac 22 Vehicle = 17.83 ± 3.03; P8-P15 Sac 22 PLX = 16.25 ± 0.87; P8-P22 Sac 22 Vehicle = 20.33 ± 1.53, P8-P22 Sac 22 PLX = 6.44 ± 1.25. P Representative image of microglia (Iba1 +) at P15 in the SEZ of vehicle-treated mice (×20 magnification). Q Representative image of microglia (Iba1 +) at P15 in the SEZ of PLX-treated mice (×20 magnification). R Representative image of microglia (Iba1+, red) at P22 in the SEZ of vehicle-treated mice (×20 magnification). S Representative image of microglia (Iba1+, red) at P22 in the SEZ of mice treated with PLX from P8 to P15. T Representative image of microglia (Iba1+, red) at P22 in the SEZ of vehicle-treated mice (×20 magnification). U Representative image of microglia (Iba1+, red) at P22 in the SEZ of mice treated with PLX from P8 to P22. V Quantification of the number of microglia (Iba1 +) in the SEZ across groups expressed as fold change. There was a significant decrease in microglia after PLX treatment relative to vehicle at P15 (1.00 ± 0.04-fold change of Iba1 + cells in vehicle-treated mice vs. 0.31 ± 0.01 in H-I-injured mice, p = 0.001). There was no difference in microglia numbers between vehicle vs. PLX at P22 following treatment from P8 to P15 (1.00 ± 0.13-fold change of Iba1 + cells in vehicle-treated mice vs. 1.20 ± 0.09 in H-I-injured mice, p = 0.463). There was a significant decrease in microglia after treatment relative to vehicle at P22 following two weeks of treatment (PLX from P8 to P22) (1.00 ± 0.04-fold change of Iba1 + cells in vehicle-treated mice vs. 0.50 ± 0.05 in H-I-injured mice, p = 0.016). Average number of Iba1 + cells/unit area: P15 Vehicle = 20.03 ± 0.87; P15 PLX = 6.19 ± 0.10; P8-P15 Sac 22 Vehicle = 17.83 ± 3.03; P8-P15 Sac 22 PLX = 16.25 ± 0.87; P8-P22 Sac 22 Vehicle = 13.22 ± 0.48, P8-P22 Sac 22 PLX = 6.67 ± 0.67. W No significant differences between the latency to fall (s) on the hindlimb suspension task at P12 were recorded between vehicle- vs. PLX-treated mice (p = 0.42). X No significant differences between the angle at fall (degree) on the grip strength task at P15 were recorded between vehicle- vs. PLX-treated mice (p = 0.91). n = 3–4 mice per group for immunohistochemistry. n = 20–21 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, G, J) Two-way ANOVA; (K, L) Unpaired t-test. *p ≤ 0.050