Short-chain fatty acids contribute to neuropathic pain via regulating microglia activation and polarization

Abstract

Microglia activation and subsequent pro-inflammatory responses play a key role in the development of neuropathic pain. The process of microglia polarization towards pro-inflammatory phenotype often occurs during neuroinflammation. Recent studies have demonstrated an active role for the gut microbiota in promoting microglial full maturation and inflammatory capabilities via the production of Short-Chain Fatty Acids (SCFAs). However, it remains unclear whether SCFAs is involved in pro-inflammatory/anti-inflammatory phenotypes microglia polarization in the neuropathic pain. In the present study, chronic constriction injury (CCI) was used to induce neuropathic pain in mice, the mechanical withdrawal threshold, thermal hyperalgesia were accomplished. The levels of microglia markers including ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation 11b (CD11b), pro-inflammatory phenotype markers including CD68, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and anti-inflammatory phenotype markers including CD206, IL-4 in the hippocampus and spinal cord were determined on day 21 after CCI. The results showed that CCI produced mechanical allodynia and thermal hyperalgesia, and also increased the expressions of microglia markers (Iba1, CD11b) and pro-inflammatory phenotype markers (CD68, IL-1β, and TNF-α), but not anti-inflammatory phenotype marker (CD206, IL-4) in the hippocampus and spinal cord, accompanied by increased SCFAs in the gut. Notably, antibiotic administration reversed these abnormalities, and its effects was also bloked by SCFAs administration. In conclusion, data from our study suggest that CCI can lead to mechanical and thermal hyperalgesia, while SCFAs play a key role in the pathogenesis of neuropathic pain by regulating microglial activation and subsequent pro-inflammatory phenotype polarization. Antibiotic administration may be a new treatment for neuropathic pain by reducing the production of SCFAs and further inhibiting the process of microglia polarization.

Keywords: SCFAs; gut microbiota; microglial polarization; neuropathic pain.

Figure 1.

Nociceptive manifestations before and after surgery and the changes of SCFAs in fecal samples of mouse treated with surgery.Time courses show mechanical thresholds to von Frey filaments (a) and withdrawal latency to heat stimulation in the plantar test (b). Measures are shown before CCI/sham surgery (day -1), after surgery (day 3,7,14,21). Plantar test showed a persistent decrease in the mechanical thresholds and withdrawal latency to heat stimulation in paws of all CCI-operated mice(***p < 0.001 vs. sham-operated mice).(c) total SCFAs heat map, (d) acetate, (e) propionate, (f) butyrate, (g) isobutyrate, (h) valeric acid, (i) isovaleric acid, (j) hexanoic acid, and (k) total SCFAs. acetate, propionate, butyrate valeric acid and total SCFAs in CCI mouse are significantly higher than those of sham mouse. ***p < 0.001, **p < 0.01 (n = 8, mean ± SEM).

Figure 2.

The changes of SCFAs in Intestinal contents of mouse treated with antibiotics and surgery.Immunofluorescence staining to Iba1 in the hippocampus and spinal cord on day 21 post-CCI. (a) There was no statistical difference in the body weight monitoring of the five groups of mice during the experiment. (b) The use of antibiotics significantly reduced the levels of total SCFAs. ***P < 0.001 compared to the Sham group. (c) The use of antibiotics improves in the mechanical thresholdsl, and exogenous SCFAs can reverse this effect. *P < 0.05 compared to the Sham group. (d) The use of antibiotics improves in the withdrawal latency, and exogenous SCFAs can reverse this effect.*P < 0.05 compared to the Sham group. (e) Representative images of Iba1 (green) in the hippocampus. (f) Representative images of Iba1 (green) in the spinal cord. (g) Antibiotics significantly attenuated CCI-induced fluorescence intensity increase of Iba1 in the hippocampus,but exogenous SCFAs can reverse this effect (Scale bar = 50 µm). (h) Antibiotics significantly attenuated CCI-induced fluorescence intensity increase of Iba1 in the spinal cord,but exogenous SCFAs can reverse this effect (Scale bar = 50 µm). The data are expressed as the mean ± SEM of 4 mouse in each group. *P < 0.05 compared to the Sham group, DAPI (4′, 6-diamidino-2-phenylindole) staining is shown in blue.

Figure 3.

Western blot results of the hippocampus and spinal cord. (a) A representative blot of CD11b, CD68, CD206 in the hippocampus. (b) A representative blot of CD11b, CD68, CD206 in the spinal cord. (c to e) A quantitative analysis of CD11b, CD68, CD206 in the hippocampus. (f to h) A quantitative analysis of CD11b, CD68, CD206 in the spinal cord. β-tubulin was included as control. The data are expressed as the mean ± SEM of 4 mice in each group. *P < 0.05 compared to the sham group. **P < 0.01 compared to the sham group. ***P < 0.001 compared to the sham group.

Figure 4.

mRNA expressions of pro-inflammatory/anti-inflammatory phenotypes microglia markers in the hippocampus and spinal cord. Fold increase of pro-inflammatory phenotype markers (CD11b, CD68) in the hippocampus (a and b); Fold increase of anti-inflammatory phenotype microglia markers (CD206) in the hippocampus (c). Fold increase of the ratio of CD68/CD206 in the hippocampus (d). Fold increase of pro-inflammatory phenotype markers (CD11b, CD68) in the spinal cord (e and f); fold increase of anti-inflammatory phrnotype microglia markers (CD206) in the spinal cord (g). Fold increase of the ratio of CD68/CD206 in the spinal cord (h). The data are expressed as mean ± SEM; n = 4 for each group; *P < 0.05 compared to the Sham group.

Figure 5.

Western blot results of the hippocampus and spinal cord. (a) A representative blot of IL-1β, TNF-α, IL-4 in the hippocampus. (b) A representative blot of IL-1β, TNF-α, IL-4 in the spinal cord. (c to e) A quantitative analysis of IL-1β, TNF-α, IL-4 in the hippocampus. (f to h) A quantitative analysis of IL-1β, TNF-α, IL-4 in the spinal cord. β-tubulin was included as control. The data are expressed as the mean ± SEM of 4 mice in each group. ***P < 0.001 compared to the sham group.

Figure 6.

mRNA expressions of different inflammatory cytokines in the hippocampus and spinal cord. Fold increase of pro-inflammatory cytokines (TNF-α, IL-1β) in the hippocampus (a and b); fold increase of M2 anti-inflammatory cytokine in the hippocampus (IL-4) (c). Fold increase of the ratio of IL-1β/IL-4 in the hippocampus (d). Fold increase of pro-inflammatory cytokines (TNF-α, IL-1β) in the spinal cord (e and f); fold increase of M2 anti-inflammatory cytokine in the spinal cord(IL-4) (g). Fold increase of the ratio of IL-1β/IL-4 in the spinal cord (h). The data are expressed as mean ± SEM; n = 4 for each group; **P < 0.01 compared to the Sham group, ***P < 0.001 compared to the Sham group.