Macrophage accumulation in dorsal root ganglion is associated with neuropathic pain in experimental autoimmune neuritis

Abstract

Background: Neuropathic pain is a common symptom of Guillain-Barré syndrome (GBS). The infiltration of macrophages in the dorsal root ganglion (DRG) contributed to neuropathic pain in nerve injury. The underlying mechanisms of neuropathic pain in patients with GBS remain unknown. Experimental autoimmune neuritis (EAN) is a useful mice model of GBS. Our study aimed to explore whether the infiltration of macrophages in DRG is associated with neuropathic pain of EAN.

Methods: Male C57BL/6 mice were randomly divided into two groups, the EAN group (n = 12) and the control group (n = 12). Six mice in each group were sacrificed after anesthetization in the attack and remission phase, respectively. The 50% paw withdrawal threshold and clinical score were measured, and macrophages with its subtypes were detected in the spleen and DRG tissue.

Results: More macrophages infiltrated the DRG of the EAN group in the attack phase and mostly surrounded neurons in the DRG. The proportion of macrophages and pro-inflammatory macrophages in the spleen of mice with EAN was significantly higher than the control group in the attack phase.

Conclusion: The infiltration of macrophages in DRG might be associated with neuropathic pain of EAN and pro-inflammatory macrophages may involve in neuropathic pain of EAN.

Keywords: dorsal root ganglion; experimental autoimmune neuritis; macrophages; neuropathic pain.

Figure 1

Clinical information in different time courses of the experimental autoimmune neuritis (EAN) and control groups. (a) The weight data of the EAN and control groups after immunization. (b) The clinical score of the two groups after immunization. (c) Comparison of 50% withdrawal threshold detection of the two groups. ^# p ＜ 0.05; *p ＜ 0.01.

Figure 2

Immunohistochemical analysis of the four groups of DRG. Macrophages were stained by F4/80 antibodies and were shown as cells in the figures with brown membrane surface staining. The pictures with the black border are enlarged in the upper right corner, and macrophages are indicated with red arrows. (a) DRG section of the attack phase of mice with EAN. Abundant macrophages were observed around the neurons in the DRG of mice with EAN in the attack phase. (b) DRG section of the control group (Ctr1) for the attack phase. (c) DRG section of the remission phase of mice with EAN. (d) DRG section of the control group (Ctr2) for the remission phase. (e) Statistical plot for the percentage of the area of each group of sections positively expressed by macrophages. DRG, dorsal root ganglion; EAN, experiment autoimmune neuritis. **p ＜ 0.01; ns, p ＞ 0.05.

Figure 3

Immunofluorescence staining of the EAN and the control group in the attack phase was performed to verify the pathological changes in the attack phase. The pictures with the white border are enlarged in the upper right corner, and macrophages are indicated with white arrows Distribution of individual nuclei in DAPI blue fluorescent chromosomes now in DRG tissue; FITC green dye staining for intracellular NF200 shows the morphology of neurons and nerve fibers; Cy3 red dye stains the F4/80 membrane surface, reflecting macrophage morphology. Abundant macrophage infiltration was observed around neurons of the DRG of mice with EAN in the attack phase.

Figure 4

The proportions of macrophages in mice spleen MNCs were analyzed by flow cytometry. (a) The proportions of macrophages (CD11b + F4/80 + cells), pro-inflammatory macrophage (iNOS + CD40 + cells), anti-inflammatory macrophage (Arg + CD206 + cells) in mice spleen MNCs at attack phages; (b) the proportions of macrophages (CD11b + F4/80 + cells), pro-inflammatory macrophage (iNOS + CD40 + cells), and anti-inflammatory macrophage (Arg + CD206 + cells) in mice spleen MNCs at remission phages. M, macrophage; M1, pro-inflammatory macrophage; M2, anti-inflammatory macrophage. *p ＜ 0.05; **p ＜ 0.001; ***p ＜ 0.001;.