Fig. 1

CX₃CR1⁺ cell depletion reduces both formalin induced phase-I acute inflammatory pain and phase-II persistent inflammatory pain. a Schematic diagram showing the timeline of tamoxifen (TM) and diphtheria toxin (DT) administration, intraplantar injection of formalin (5% in PBS, 10 μl) or bradykinin (3 μg in PBS, 10 μl), behavioral tests (pain thresholds and rotarod test) and immunostaining in mice. b Iba1-positive cells were largely depleted 2 days after last DT injection in spinal cord dorsal horn (DH) and dorsal root ganglia (DRG). F4/80-positive macrophages in hind paw skin were also largely depleted at postoperative day (POD) 2 after the last DT injection, compared to the control group (mice without DT treatment). Left column, low magnification view of Iba1-positive cells in spinal cord DH of CX₃CR1^{CreER/ +}: R26^iDTR/+ mice in control and CX₃CR1⁺ cell ablation (Abl) mice. Inset: representative higher magnification images showing detailed microglia morphology. Scale bars represent 200 μm and 20 μm. Middle column, representative images of Iba1-positive cells in DRG in control and CX₃CR1⁺ cell ablation mice. Scale bar, 100 μm. Right column, representative images of F4/80-positive macrophages in hind paw skin in control and CX₃CR1⁺ cell ablation mice. Scale bar, 200 μm. c Quantitative data showing the density of Iba1-positive cells in the DH (***p < 0.001, unpaired 2-tailed Student’s t test, n = 8–12 mice/group), DRG (***p < 0.001, unpaired 2-tailed Student’s t test, n = 7–12 mice/group) and F4/80-positive cells in hind paw skin (*p < 0.05, unpaired 2-tailed Student’s t test, n = 5–7 mice/group) from control and CX₃CR1⁺ cell ablation mice. Data are presented as mean ± SEM. d–f CX₃CR1⁺ cell ablation does not affect acute pain responses and basal motor function. d Analysis of rotarod test in control and CX₃CR1⁺ cell ablation mice showing that CX₃CR1⁺ cell ablation did not affect basal motor function. (n = 6–7 mice/group). e, f Basal pain threshold for heat sensitivity (Tail immersion tests, Thermal Withdrawal Latency with Hargreaves Method) and mechanical sensitivity (Mechanical Withdrawal Threshold with von Frey tests) in control and CX₃CR1⁺ cell ablation mice. Data are presented as mean ± SEM. n.s., no significance, unpaired 2-tailed Student’s t test, n = 8–9 mice/group. g, h Time course (0–60 min) of formalin-induced spontaneous pain behavior (licking/flinching) in control and CX₃CR1⁺ cell ablation mice, as measured in every 5 min (g). Histogram representing formalin-induced Phase-I (1–10 min) and Phase-II inflammatory pain responses (10–60 min) in control and CX₃CR1⁺ cell ablation mice. Both formalin-induced Phase-I and Phase-II inflammatory pain are reduced in CX₃CR1⁺ cell depleted mice (h). Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, compared to control mice, unpaired 2-tailed Student’s t test, n = 9–11 mice/group