Fig. 3

Ablation of resident microglia reduces formalin induced phase-II persistent inflammatory pain. a Schematic diagram showing the timeline of TM and 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. Ablation or repopulation efficiency were checked at four time points before DT injection and days 2,3, 4 after the last DT injection. b–d Iba1-positive resident macrophages in DRGs can also be ablated acutely along with resident microglia in the spinal cord. However, DRG macrophages repopulated faster than that of spinal microglia. The number of DRG macrophages reached control levels 3 days after last DT injection, while spinal microglia were still largely absent. F4/80-positive macrophages in hind paw skin remain preserved after TM and DT treatment. b Representative images of Iba1-positive cells in spinal cord and DRG before DT injection and 2,3, 4 days after last DT injection. 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 = 6–12 mice/group) and DRG (***p < 0.001, unpaired 2-tailed Student’s t test, n = 6–8 mice/group) from control and microglia ablation (MG Abl) mice. Data are presented as mean ± SEM. d Representative images of F4/80-positive macrophages in hind paw skin in control and microglia ablation POD3 mice. Scale bar, 200 μm. Quantitative data showing the density of F4/80-positive macrophages in skin from control and microglia ablation mice. (n.s., unpaired 2-tailed Student’s t test, n = 5 mice/group). e Analysis of rotarod test in control and microglia ablation mice indicating that CNS microglia ablation did not affect basal motor function (n = 6 mice/group). f Basal pain threshold for heat sensitivity (Tail immersion tests, n = 6 mice/group; Thermal Withdrawal Latency with Hargreaves Method, n = 9 mice/group) and mechanical sensitivity (Mechanical Withdrawal Threshold with von Frey tests, n = 8 mice/group) in control and microglia ablation POD3 mice. Data are presented as mean ± SEM; n.s., unpaired 2-tailed Student’s t test. g Time course (0–60 min) of formalin-induced spontaneous pain behavior (licking/flinching) in control and microglia ablation mice, as measured every 5 min. Histogram representing formalin induced Phase-I (1–10 min) and Phase-II inflammatory pain responses (10–60 min) in control and microglia ablation mice. Formalin-induced Phase-II persistent inflammatory pain is reduced in microglia ablation mice at POD3, while formalin-induced Phase-I acute inflammatory pain was not affected. 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 = 7–8 mice/group