Fig. 7

The beneficial effects of combined administration of CX3CL1 and M-CSF are specifically blocked in CX3CR1^−/− mice. (A) Schematic diagram illustrating in situ injection of 2 µl of CX3CL1 and M-CSF into the epicenter immediately after SCI in CX3CR1^−/− (CX3CR1^GFP/GFP) and CX3CR1^+/− (CX3CR1^+/GFP) mice. Histological analysis was performed at 28 dpi. (B) Representative immunofluorescence images of GFAP (red) and GFP (green) from sagittal sections of the injured spinal cord of CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. (C) Quantification of the number of GFP⁺ microglia in the epicenter of CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi (n = 3 mice per group). (D) Representative images of sections of the injured spinal cord of CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi, stained with antibodies against GFAP (green), PDGFRβ (red) and NeuN (purple). (E and F) Quantification of the percentage of GFAP⁻ area (E) and PDGFRβ⁺ area (F) within the spinal cord segment spanning the injured core in CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi (n = 3 mice per group). (G) Quantification of NeuN⁺ neurons in Z1–Z3 zones adjacent to central lesion core in CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi (n = 3 mice per group). (H) Immunofluorescence staining of 5-HT (red) and DAPI (blue) in sagittal sections of the injured spinal cord from CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi. White arrowheads indicate the lesion site. (I) Quantification of 5-HT density within the spinal cord segment spanning the injured core in CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi. (n = 3 mice per group). (J) Time course of functional recovery of CXCR1^−/− and CX3CR1^+/− mice assessed by the BMS score after the combined treatment of CX3CL1 and M-CSF post-SCI (n = 6 mice per group). (K) Footprint analysis performed at 28 dpi reveals improved functional recovery in the CX3CR1^+/− mice after the combined treatment of CX3CL1 and M-CSF. (L) Quantification of the footprint analysis parameters (stride length, stride width, and paw rotation) in CX3CR1^−/− and CX3CR1^+/− mice at 28 dpi (n = 6 mice per group). The data are presented as means ± SEM (error bars). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001, the CX3CR1^+/− mice versus the CX3CR1^−/− mice. Statistical analysis was performed using two-way ANOVA (G and J) followed by Bonferroni post hoc tests’, or Student’s two-tailed unpaired t-test (C, E, F, I and L). Scale bars = 200 μm (D and H), 100 μm (B), 20 μm (ROI)