Fig 2: Microglia influence astrocytic transformation via the GAS6/AXL axis. (A) Immunofluorescence staining of GAS6 (red) with IBA1, GFAP, or NeuN (green) after TBI; nuclei were stained by DAPI (blue). (B) GAS6 level was suppressed by minocycline administration at 3 dpi. *p < 0.05 **p < 0.01 versus sham, ##p < 0.01 versus vehicle by one-way ANOVA. (C) ABCA1 level was suppressed after minocycline treatment at 3 dpi. *p < 0.05, **p < 0.01 versus sham, ##p < 0.01 versus vehicle by one-way ANOVA. (D) The effect of GAS6 specific targeted siRNA or scramble siRNA after transfected into BV2 cells. *p < 0.05 by two-tailed Student’s t-test. (E) Brief workflow of in-vitro cell crosstalk analysis. (F) BV2 supernatant enhanced primary astrocyte phagocytosis after 24 h co-culture, as detected by flow cytometry. **p < 0.01 by two-tailed Student’s t-test. (G) Flow cytometry analysis of BV2 supernatant-induced astrocytic phagocytosis showed it was abolished after GAS6 siRNA transfection. **p < 0.01 versus scramble by one-way ANOVA

Fig 3: Time-dependent response of glial cells to traumatic brain injury. (A) Western blot of the time course of expression changes in GFAP and CD68. (B, C) The bar graphs show relative intensities for GFAP and CD68. The results are shown as the mean ± SD. **p < 0.01 by one-way ANOVA. (D) Reactive astrocytes surround damaged regions while microglia populate the center of the damaged region at 3 dpi. (E) Quantitative analysis for distances of IBA1 + activated microglia, and GFAP + reactive astrocytes from the center of the lesion in (D) from 4 mice. *p < 0.05 by two-tailed Student’s t-test. (F) Western blot samples reflect the changes in expression of AXL and GAS6 after TBI. (G, H) Quantitative analyses of AXL and GAS6 bands. *p < 0.05, **p < 0.01 by one-way ANOVA. (I) Immunofluorescence staining of GFAP, IBA1, NeuN (green), and AXL (red) in the ipsilateral cortex at 3 dpi; the nuclei were stained by DAPI (blue). (J) Immunofluorescence staining of GFAP (green), and AXL (red) at different time points post injury; the nuclei were stained by DAPI (blue). (K) Quantification analysis of the reactive astrocytes which expressed AXL kinase (GFAP+ & AXL+) after TBI in (J). n = 3 per group. **p < 0.05 by two-tail Student’s t-test