Effect of Microglial Activity on Gut Microbiota in Rats with Neuropathic Pain

Abstract

This study aimed to investigate the relationship between microglial activity and gut microbiota composition in a rat model of neuropathic pain (NP), and to evaluate how pregabalin treatment may influence these interrelated parameters. NP was simulated in rats via ligation and transection of the sciatic nerve. After confirming NP, the rats were randomly divided into treatment and control groups. Pregabalin (10 mg/kg) and the same dose of normal saline were administered to the treatment and control groups, respectively, on scheduled days. Microglial activity, cytokine levels, and the composition of the gut microbiota (assessed by the Firmicutes/Bacteroidetes (F/B) ratio) were evaluated. Pregabalin treatment significantly reduced microglial activity (which was notably lower in the treatment group than in the control group) and modulated pro-inflammatory and anti-inflammatory cytokine levels. While the F/B ratio in the control group significantly increased after NP surgery, the treatment group showed an initial increase followed by a notable decrease, approaching pre-surgery levels by day 28. This finding suggests that pregabalin treatment in rats with NP ameliorates microglial activity and is associated with a beneficial shift in the gut microbiota composition.

Keywords: allodynia; brain–gut axis; microbiota; microglia; neuropathic pain; pregabalin.

Figure 1

Assessment of mechanical allodynia for neuropathic pain before and after the treatment of normal saline or pregabalin. (A) Contralateral side for the surgery, (B) ipsilateral side for the surgery. () Control group, () treatment group. Data are presented as means ± standard deviation (n = 6 per group). Statistical significance was determined by two-way repeated analysis of variance (ANOVA) followed by Tukey’s post hoc test for intra-group comparisons and unpaired t-test for inter-group comparisons. Abbreviations: Control, control group; treatment, treatment group; D0, before the surgery; D, day after the surgery; before, before the treatment of normal saline or pregabalin; after, 60 min after the treatment of normal saline or pregabalin. * p < 0.05 compared with D0 within the same group. † p < 0.05 compared with the control group at the same time point.

Figure 2

Assessment of cold allodynia for neuropathic pain before and after the treatment with normal saline or pregabalin. (A) Contralateral side for the surgery, (B) ipsilateral side for the surgery. () Control group, () treatment group. Abbreviations: Control, control group; treatment, treatment group; D0, before the surgery; D, day after the surgery; before, before the treatment of normal saline or pregabalin; after, 60 min after the treatment of normal saline or pregabalin. Data are presented as means ± standard deviation (n = 6 per group). Statistical significance was determined by two-way repeated ANOVA followed by Tukey’s post hoc test for intra-group comparisons and unpaired t-test for inter-group comparisons.* p < 0.05 compared with D0 within the same group. † p < 0.05 compared with the control group at the same time point.

Figure 3

Microglial activity in the L5 segment of the spinal cord. Merged images represent overlay of Iba1 (green), TMEM119 (red), and DAPI (blue). Consistent with the quantitative data presented, microglial activation (indicated by Iba1 and TMEM119 expression) was markedly higher on the ipsilateral side in the control group compared to the attenuated activation observed in the treatment group. Images were taken under identical magnification and exposure settings using an upright microscope. Data are presented as means ± standard deviation (n = 6 per group). Statistical significance was determined by unpaired t-test with Bonferroni correction for inter-group comparisons (control vs. treatment for each side) and paired t-test for within-group comparisons (ipsilateral vs. contralateral within each group). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; Iba1, ionized calcium-binding adapter molecule 1; TMEM119, transmembrane protein 119; Merged, merged image of DAPI, Iba1, and TMEM119; control, control group; treatment, treatment group; contralateral, contralateral side for surgery; ipsilateral, ipsilateral side for the surgery. * p < 0.05 compared with the control group. † p < 0.05 compared with the contralateral side in each group.

Figure 4

The change in gut microbiota composition. (A) Firmicutes, (B) Bacteroidetes, and (C) Firmicutes/Bacteroidetes ratio. () Control group, () treatment group. Data are presented as means ± standard deviation (n = 6 per group). Statistical significance was determined by two-way repeated ANOVA (followed by Tukey’s post hoc test for within-group comparisons over time and for between-group comparisons at specific time points). Abbreviations: D0, before the surgery; D, day after the surgery. * p < 0.05 compared with D0 within the same group. † p < 0.05 compared with D15 within the same group. ‡ p < 0.05 compared with the control group at the same time point.