Fig 2: CCR2 is widely expressed in DRG neurons and displays upregulation following peripheral inflammation. a, b Typical examples and quantitative summary from dual immunofluorescence experiments showing CCR2 immunoreactivity IB4-binding non-peptidergic DRG neurons in the naïve and CFA-inflamed mice. c, d Immunofluorescence experiments and quantitative summary showing the coexistence of CCR2 immunoreactivity in CGRP-expressing peptidergic DRG neurons in naïve mice and its upregulation in CFA-inflamed mice. e, f Immunofluorescence staining and quantitative summary showing the colocalization of CCR2 immunoreactivity with NF200-positive large-diameter DRG neurons in naïve mice and no significant change in expression level after CFA inflammation. Note that in CFA-inflamed group, ipsilateral L3/L4 DRGs were obtained and analyzed at 24 h after intraplantar CFA injection. Arrows indicate the co-expressing cells. **P < 0.01. Two-Way ANOVA followed by post-hoc Bonferroni test (n = 4 mice/group). Data are expressed as mean ± SEM

Fig 3: Evaluation of the quality of regenerated nerves.(A) Longitudinal neurofilament 200 immunofluorescence staining (NF200: green, DAPI: blue) of the grafting segment in the CD + scCO2 NG group 3 weeks after the operation is shown. (B) The general view of the nerve grafts in each group at 12 weeks after the operation, as well as the immunofluorescence (NF200: red, MBP: green, DAPI: blue), toluidine blue staining and transmission electron microscopy (TEM) images of the farthest sections of the grafts. (C, D, E) The mean density and diameter of myelinated nerve fibers and the mean thickness of the myelin sheath in each group. Data are presented as the mean ± SD (n = 10). Statistical analysis: n.s. no significances, **p < 0.01, *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: Nerve injury-induced TSLP expression in both small-sized and large-sized DRG neurons, esp. nociceptive DRG neurons. Distribution of TSLP was detected when DRG neurons were double-labeled (yellow) with TSLP (green) and neuronal markers (red). (A) Detection of peripherin presented as a small-sized neuronal marker and (B) NF200 as a large-sized neuronal marker. TSLP-positive DRG neurons expressed not only peripherin but also NF200 in the ipsilateral (ipsi) injured side as compared to that in the contralateral (contra) side and sham group. (C) Detection of substance P presented as a peptidergic non-myelinated neuronal marker and (D) TRPV1 as a nociceptive marker. TSLP-positive DRG neurons expressed TRPV1 mostly in the ipsilateral (ipsi) injured side as compared to that in the contralateral (contra) side and sham group. Pairs of merged images are shown on the right panel. White arrows indicate doubled-labeled cells. Scale bars represent 50 μm (n = 6 rats).

Fig 5: Systemic AP treatment depletes macrophages in the DRG of MAFIA mice and delays mechanical allodynia.a FACS analysis of CX3CR1+ macrophage expansion in the L4/L5 DRG of wild-type mice after SNI (n = 6–7 per group). BL, baseline. POD, post-operative day. b FACS analysis of Ki67 expression in CX3CR1+ macrophages in the L4/L5 DRG of wild-type mice after nerve injury (n = 5 per group). c Representative images illustrating AP-induced depletion of GFP+ (green)/PU.1+(red) macrophages in the L4/L5 DRG of MAFIA mice injected with systemic AP (1.0 mg kg−1) for 3 days. NF200 (blue) marks myelinated neurons; scale bar: 50 μm. d FACS analysis of GFP+ DRG macrophages 1 day after the 3rd AP injection (nVEH = 9; nAP = 6). e, f SNI 1 day after AP or VEH treatment followed by FACS analysis of DRG macrophages on POD4 (e) and POD9 (f) (n = 5–6 per group). g qPCR analysis of Cx3cr1 gene expression in the L4/5 DRG of mice pretreated with AP or VEH on POD1 (n = 3 per group). h FACS analysis of GFP+ spinal cord microglia after a 3-day AP or VEH (n = 6 per group). i GFP+ (green) and Iba1+ (red) immunoreactive spinal cord microglia after a 3-day AP or VEH. Scale bar: 50 µm. j qPCR analysis of Cx3cr1 gene expression on POD1 in the lumbar spinal cord in mice pretreated with AP or VEH (n = 3 per group). k FACS analysis of GFP+ microglia in the lumbar spinal cord of mice pretreated with AP or VEH (nVEH = 5; nAP = 7) on POD4. l Effect on mechanical thresholds of systemic AP or VEH followed by SNI (n = 5 per group). Gray shading indicates injection days. Data presented as mean ± SEM. One-way ANOVA with Tukey’s correction in a, Student’s t-test in b, d, h, and k, and two-way ANOVA with Sidak’s correction in e–g, j, and l. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS nonsignificant compared with control. Source data are available as a Source Data file.