Fig 2: Effects of single administrations of CXCL3 neutralizing antibody (A,B) on nociceptive transmission in CCI-exposed mice. The effects of single intrathecal administrations of CXCL3 neutralizing antibody (1, 4, or 8 µg/5 µl) on mechanical hypersensitivity (von Frey test, A) and thermal hypersensitivity (cold plate test, B) were measured at 1.5, 5, 24, and 48 h after administration at 7 days after CCI. Data are presented as the means ± SEM (6–8 mice per group). The results were evaluated using one-way ANOVA followed by Bonferroni's test for comparisons of selected pairs. *p < 0.05, **p < 0.01, ***p < 0.001 for the comparison of CCI-exposed vehicle-treated animals with all groups at the indicated time points. Additionally, the results were evaluated using two-way ANOVA to determine the time × drug interaction (please see results in chapter 3.6). V, vehicle.

Fig 3: The spinal localization of CXCR2 and its ligand CXCL3 in naive and CCI-exposed rats. Immunofluorescent staining was performed on paraffin-embedded 7 µm (A,B) co-staining of CXCR2 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). (C,D) co-staining of CXCL3 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). White arrows indicate representative CXCL3-positive cells that co-localize with IBA1-positive cells. Scale bar for all pictures: 25 µm.

Fig 4: Effects of single NVP CXCR2 20 administration on a single CXCL3 injection and nociceptive transmission in naive mice (B,C). Single intrathecal administrations of vehicle (V) or NVP CXCR2 20 (60 µg/5 µl) were performed 120 min before a single intrathecal administration of V or CXCL3 (2 ng/5 µl). The effects of administrations on mechanical (von Frey test; B) and thermal (cold plate test; C) hypersensitivity were measured 3.5, 7 and 26 h after the NVP CXCR2 20 injection (1.5, 5, and 24 h after the CXCL3) (A). Data are presented as the means ± SEM (6–8 mice per group). The results were evaluated using one-way ANOVA followed by Bonferroni's test for comparisons of selected pairs. **p < 0.01, ***p < 0.001 indicate differences in comparison with V+V-treated animals at the indicated time points. ##p < 0.01, ###p < 0.001 indicate differences in comparison with V+CXCL3-treated animals at the indicated time points. V, vehicle; NVP, NVP CXCR2 20.

Fig 5: The time course of changes in CXCR2, CXCL1, CXCL2, CXCL3 mRNAs (A–D) and proteins (E–H) in the spinal cord tissues on the 2nd, 7th, 14th, and 28th days after chronic constriction injury (CCI) in rats. The RT-qPCR and Western blot data are presented as the means ± SEM of 6–10 and 4–6 samples per group in each method, respectively. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. *p < 0.05, ***p < 0.001 indicate differences vs. naive rats. CCI, chronic constriction injury; N, naïve.