Fig 2: Hypothalamic overexpression of soluble CX3CL1 reduces weight gain, food intake and improves leptin sensitivity in DIO mice. (A) Schematic diagram of a mouse coronal brain section indicating bilateral administration of AAVs into the mediobasal hypothalamus. (B,C) Representative image of a hypothalamic section from a mouse treated with AAV–CX3CL1-s. CX3CL1-s overexpression in the MBH is indicated by HA staining (red). Higher magnification view in C shows broad expression in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH). (D,E) Body weight gain (D) and 24 h food intake (E) in HFD-fed mice with hypothalamic overexpression of GFP (AAV–GFP) or the soluble isoform of CX3CL1 (AAV–CX3CL1-s) measured daily over the first week and then bi-weekly until day 25 of HFD. (F) Cumulative food intake over days 1–2 (left panel), 3–7 (middle panel) and 8–24 (right panel). (G) 24 h and 48 h food intake after i.p. saline or leptin injections (2 µg/g/day) in AAV–GFP and AAV–CX3CL1-s mice on HFD. Data are presented as mean ± SEM of at least 6 animals per group. (H) Hypothalamic mRNA expression of Pomc in chow-fed AAV–GFP and AAV–CX3CL1-s mice. Data are expressed as a percentage of AAV–GFP control; n = 6 per group. * p < 0.05; *** p < 0.001.

Fig 3: Central CX3CL1 does not reduce weight gain and food intake in mice treated with MC3R/MC4R antagonist. (A) Time scale of the experimental approach used for acute i.c.v. administration of SHU9119 and CX3CL1. (B,C) Average of cumulative 24 h food intake (A) and body weight gain (B) in mice treated with saline (Sal) or SHU9119 i.c.v. and 30 min later with Sal or CX3CL1 i.c.v. Data are presented as mean ± SEM of at least 5 animals per group. * p < 0.05. (D) Time scale for chronic i.c.v. administration of SHU9119 in mice previously subjected to hypothalamic viral microinjection of AAV–GFP and AAV–CX3CL1. (E,F) Body weight gain (E) and total food intake (F) measured in mice with hypothalamic overexpression of CX3CL1-s or GFP control that received SHU9119 i.c.v. infusion over 2 weeks (compare with Figure 1D). Data are presented as mean ± SEM of 10 animals per group. * p < 0.05.

Fig 4: Co-administration of CX3CL1 and M-CSF may exert beneficial effects by enhancing SYK levels in CX3CR1+ microglia and reducing foamy cell recruitment after SCI. (A) Immunofluorescence staining of SYK (green) and DAPI (blue) in sagittal sections from the CX3CL1 + M-CSF, CX3CL1, M-CSF, Control and PBS groups at 28 dpi. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. (B) Quantification of the percentage of SYK+ area within the spinal cord segment spanning the injured core at 28 dpi (n = 4 mice per group). (C) Lipid accumulation was determined by ORO staining at 28 dpi. (D) The quantification of the ratio of the mean density of ORO signal to the lesion area within the spinal cord segment spanning the injured core at 28 dpi (n = 3 mice per group). (E) Representative immunofluorescence images of SYK (red) and GFP (green) in sagittal sections of the injured spinal cord from CX3CR1−/− and CX3CR1+/− mice at 28 dpi. These mice were treated with an in situ injection of 2 µl of CX3CL1 and M-CSF into the epicenter immediately after SCI. (F) Quantification of the percentage of SYK+ area within the spinal cord segment spanning the injured core in CX3CR1−/− and CX3CR1+/− mice at 28 dpi (n = 3). The data are presented as means ± SEM (error bars). *P < 0.05 and **P < 0.01, ***P < 0.001 and ****P < 0.0001. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests (B and D), or Student’s two-tailed unpaired t-test (F). Scale bars = 200 μm (A, C and E), 20 μm (ROI)

Fig 5: The early centripetal migration of microglia alone does not significantly contribute to scar formation and neuron preservation after SCI. (A) Immunofluorescence staining of GFAP (green) and PDGFRβ (red) in sagittal sections from the CX3CL1, Control and PBS groups at 28 dpi. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. (B and C) Quantification of the percentage of GFAP− area (B) and PDGFRβ+ area (C) within the spinal cord segment spanning the injured core at 28 dpi (n = 4 mice per group). (D) Representative immunofluorescence images of NeuN+ neurons in Z1–Z3 zones adjacent to central lesion core in the CX3CL1, Control and PBS groups at 28 dpi. (E) Quantification of NeuN+ neurons in Z1–Z3 zones adjacent to central lesion core in the CX3CL1, Control and PBS groups at 28 dpi (n = 3 mice per group). The data are presented as means ± SEM (error bars). Statistical analysis was performed using either one-way (B and C) or two-way (E) ANOVA followed by Bonferroni post hoc tests. Scale bars = 200 μm (A and D), 20 μm (ROI)