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

2.1 Animals and reagents

Male C57BL/6 mice (6–8 weeks, weight 16–18 g) were obtained from Slack Experimental Animal Co., Ltd. (Shanghai, China) and acclimated to the animal feeding room of Zhejiang University of Technology for 1 week before the experiments. The room temperature was maintained between 21 and 25℃. Regular feeding and free water were provided. The experiment was approved by the ethics committee of Zhejiang Provincial People’s Hospital (SYXK2018-0012). The 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 (day 14 post-immunization [p.i.]) and remission phase (day 32 p.i.), respectively.

2.2 Induction of EAN

The EAN group was immunized on day 2 and 7 p.i. by subcutaneous injection of P0 peptide 180-199 (GenScript, USA) mixed with Mycobacterium tuberculosis (BD, USA), emulsified saline, and Freund’s incomplete adjuvant (FIA; Sigma, USA). FIA plus M. tuberculosis is referred to as Freund’s complete adjuvant (FCA). Each mouse was given pertussis toxin (PTX; Millipore, USA) by tail intravenous injection on days 1, 3, and 5 p.i., respectively. The control group was managed in the same way as mentioned above but with phosphate-buffered saline (PBS) rather than P0 peptide.

2.3 The assessment of clinical severity and 50% Paw withdrawal threshold

The clinical severity of mice was assessed every day by two different examiners beginning on day 0 p.i. The severity for EAN was defined into 8 levels as follow: 0, normal; 1, flaccid tail; 2, ataxia or mild paraparesis; 3, moderate paraparesis; 4, severe paraparesis; 5, tetraparesis; 6, moribund; 7, death. Clinical severity for the intermediate level was set as 0.5. The “up–down” von Frey method was used to estimate the pain threshold, and monofilaments (Semmes-Weinstein monofilaments) with increasing force (g) from 0.07 to 4 g were used. Before the “up–down” method, the “ascending stimulus” method was taken to value the initial force that was near the 50% proportion responding and chose 1.4 g as our initial force [12].

2.4 Analysis of the proportions of macrophages in spleen mononuclear cells (MNCs) by flow cytometry

The mice were sacrificed at the attack and remission phase respectively. Single suspended macrophages were obtained from mice spleen, and centrifuged for 10 min. After red blood cell lysis, the splenocytes were harvested and adjusted the cell concentration to 1 × 10⁶/ml. The spleen MNCs were incubated with antibodies FITC-conjugated anti-mouse CD11b (BD Pharmingen™), APC-R700-conjugated anti-mouse F4/80 (BD Pharmingen™), PE-conjugated anti-mouse CD40 (BD Pharmingen™) for 30 min at 4°C in a dark environment. Then, cells were permeabilized with the fixation/permeabilization solution Kit (BD Pharmingen™) for 20 min. The intracellular molecules were stained with antibodies AF647-conjugated anti-mouse CD206 (BD Pharmingen™), eFlour450-conjugated anti-mouse arginase-1 (Arg-1) (Thermo), and PE-Cy7-conjugated anti-mouse inducible nitric oxide synthase (iNOS) (Thermo) for 30 min at 4°C in a dark environment. Flow cytometric analysis was performed by using FLOWJO 7.6.1 (Treestar). Both F4/80- and CD11b-positive were calculated as macrophages. In macrophages, both iNOS and CD40 positive were identified as pro-inflammatory macrophages, and either CD206 or Arg-1 positive was identified as anti-inflammatory macrophages.

2.5 Macrophages in DRG using immunohistochemical and immunofluorescence assays

The mice were anesthetized with 5% chloral hydrate and perfused with cold saline and 4% buffered formaldehyde. The lumbar DRG tissues were extracted and were cut lengthwise at a thickness of 8 µm and stored at 4°C in PBS containing 0.1% sodium azide. Macrophages were visualized using anti-F4/80 antibody (Servicebio, China) immunohistochemistry and immunofluorescence staining, respectively. The neurons and nerve fibers in DRG tissue are stained with anti-neurofilament heavy polypeptide antibodies (Servicebio, China). After blocking in PBS containing 30% BSA for 30 min at room temperature. Next, HRP-conjugated secondary antibody was added and incubated for 2 h at room temperature. Finally, the samples were fixed with PBS containing 10% DAPI for 2 min. The samples were observed using a confocal microscope (Zeiss LSM 800, Carl Zeiss Microimaging Inc., NY, USA) under a 20× objective. The percentage of the area of each group of sections positively expressed by macrophages was measured through ImageJ.

2.6 Statistical analysis

Statistical analysis was performed with Statistical Package for Social Sciences version 23.0 software (IBM, West Grove, PA, USA). The data was represented by mean ± standard and the means were compared using the rank sum test, and the Bonferroni–Dunn test for post hoc comparisons. For all statistical tests, p < 0.05 was considered to be significant.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. This study was approved by Ethics Committee of the Center for rehabilitation Medicine, Department of Neurology, Zhejiang Provincial People’s hospital.

3.1 Clinical information of EAN

The clinical course was compared between 12 mice with EAN induced by injecting P0 peptide 180-199 with FCA and the same number of control mice administered by PBS and FCA. After day 14 p.i., the clinical scores were compared between EAN and control group (n = 6, for each group). There was no significant difference in weight on day 0 p.i. In the early days (days 4–6 p.i.) and later days (days >20 p.i.), a tendency of higher weight was observed in the EAN group than in the control group (Figure 1a). Our data revealed that the symptoms of EAN mice started on day 10 p.i. and peaked on day 20 p.i., after which the clinical score showed a decreasing trend. (Figure 1b).

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.

3.2 Lower pain threshold observed in the EAN mice

50% withdrawal threshold measuring, reflecting the pain threshold, starts on day 0 p.i. and was performed every two days. As exhibited in Figure 1c, both the EAN group and the control group showed a falling trend in pain thresholds and became stable after day 10 p.i. The pain thresholds were lower in the EAN group than in the control group and were statistically significant after day 6 p.i.

3.3 Macrophage infiltration in the DRG of EAN mice

Immunohistochemistry staining with F4/80 antibody of lengthwise sections of DRG revealed that amounts of macrophages infiltrate the DRG tissue of EAN mice in the attack phase are significantly increased than remission phase as well as control group both in the attack and remission phases (p < 0.05, Figure 2a–e). In the remission phase, the amounts of macrophages infiltrate the DRG of the EAN group had no significant difference compared with the control group both in the attack and remission phases (p > 0.05). Immunofluorescence staining of the EAN and control groups in the attack phase was performed to verify the pathological changes in the attack phase, and as shown in Figure 3, abundant macrophage infiltration was observed in the DRG around the neurons of mice with EAN, consisting with the pathological result mentioned above.

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.

3.4 The proportion of macrophages in EAN

The proportion of macrophage, pro-inflammatory macrophage, and anti-inflammatory macrophage in mice spleen MNCs was analyzed by flow cytometric analysis in attack and remission phages. We found the proportions of macrophages (both CD11b and F4/80 positive) in EAN mice spleen MNCs were significantly higher than the control group (p < 0.01, Figure 4a) in the attack phase. The proportions of pro-inflammatory macrophages (both CD40 and iNOS positive) in macrophages of EAN mice spleen MNCs were increased significantly (p < 0.05, Figure 4a); however, the proportions of anti-inflammatory macrophages (both CD206 and Arg positive) had no significant difference compared with the control group (p > 0.05, Figure 4a) in the attack phase. In the remission phase, compared with the control group, we found the proportions of macrophages (both CD11b and F4/80 positive) in EAN mice spleen MNCs were higher (p < 0.05, Figure 4b). The proportions of pro-inflammatory macrophage (both CD40 and iNOS positive) in macrophages of EAN mice spleen MNCs were increased significantly (p < 0.001, Figure 4b), the proportions of anti-inflammatory macrophage (both CD206 and Arg positive) in macrophages of EAN mice spleen MNCs were decreased, while there was no statistical significance compared with the control group (p > 0.05, Figure 4b).

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;.