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. 2023 Oct 4;16:1144614. doi: 10.3389/fnmol.2023.1144614

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Copyright © 2023 Li, Liu, Hu, Yang, Ma, Tian, Cao, Pan, Jiang, Liu, Wu, Luo and Xie.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CFA and CCL2 induces upregulation of CCR2, HCN2, and CGRP expression in DRG. (A–C) Representative 20× and 40× (magnified) IHC images of CCR2 (red) and HCN2 (green) in DRGs of control, DRG injection of CCL2 and intraplantar CFA injection mice. Scale bar, 50 μm. (D–F) Size distribution and CCR2⁺/HCN2⁺ co-expression characterization of CCR2-positive neurons in DRGs of control, intervertebral foramen injection CCL2 and intraplantar CFA injection mice. (H–J) Representative 20× and 40× (magnified) IHC images of CGRP (red) and CCR2 (green) in DRGs of control, intervertebral foramen injection CCL2 and intraplantar CFA injection mice. Scale bar, 50 μm. (K–M) Size distribution and CCR2⁺/CGRP⁺ co-expression characterization of CCR2-positive neurons in DRGs of control, intervertebral foramen injection CCL2 and intraplantar CFA injection mice. Note that for CFA-inflamed group, ipsilateral L4/L5 DRGs were obtained and analyzed at 24 h after intraplantar CFA injection; for CCL2-inflamed group, ipsilateral L4/L5 DRGs were obtained and analyzed at 5 h after intervertebral foramen injection CCL2. (G) Percentage of CCR2⁺/HCN2⁻, CCR2⁺/HCN2⁺, CCR2-cells in the total number of cells. Percentage of CCR2⁺/HCN2⁺ in DRG after intraplantar injection of CFA or DRG injection of CCL2 were significantly increased vs. control mice (by one-way ANOVA, Newan–Keuls test, n = 3 per group). (N) Percentage of CCR2⁺/CGRP⁻, CCR2⁺/CGRP⁺, CCR2-cells in the total number of cells (n = 3 per group). (O–Q) The mRNA expression of HCN2 was identified by RT-qPCR in 12 groups (n = 6 per group). (R) The expression of HCN2 protein in 4 groups of CFA 1 d mice (6 cases in each group) was detected by Western blot. (S) The expression of HCN2 protein in 4 groups of DRG injection of CCL2 5 h mice (6 cases in each group) were detected by Western blot. (T) The expression of CCR2 protein in 4 groups of CFA 1 d mice (6 cases in each group) was detected by Western blot. (U) The expression of CCR2 protein in 4 groups of DRG injection of CCL2 5 h mice (6 cases in each group) were detected by Western blot (by nonparametric Mann–Whitney test). CCL2 represents for DRG injection CCL2 5 h in B,E,I,L,G,N–P,S–U. All data are presented as mean ± SEM. *p < 0.05 and **p < 0.01.