Protease-Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota Is Associated With Visceral and Somatic Hypersensitivity

Abstract

Background & aims: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons.

Methods: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition.

Results: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2.

Conclusions: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.

Keywords: Abdominal Pain; Electrophysiology; Gut Microbiota; Gut-Brain Axis.

Graphical abstract

Figure 1

Vancomycin-induced changes in the murine stool microbiota. Gut dysbiosis promotes neuronal excitability (A) Beta-diversity PCoA plot demonstrating shifts in microbial populations in stool associated with 7-days of vancomycin exposure in drinking water. The observed shift in the gut microbiota persisted for at least an additional week (day 14) and then returned to pre-vancomycin population on days 21 and 28. There were no population-level shifts observed in the control group. (B) Prospective stacked aggregate plots demonstrating the changes in bacterial populations, at the family level, in each group of mice (controls vs. vancomycin-exposed). (C) Significant temporal changes in bacterial families following vancomycin exposure including a depletion in the Lachnospiraceae, (days 7 and 14) and a repletion  to pre-antibiotic levels once vancomycin is removed from the water (days 21 and 28). Vancomycin exposure was also temporally associated with a significant enrichment of Enterobacteriaceae on days 7 and 14; a family of Gram-negative bacteria that are intrinsically resistant to vancomycin.

Figure 2

(A, B) Example traces of rheobase recordings from control day 7 (left) and vancomycin day 7 (right). (C) Vancomycin-treatment led to a decrease in DRG neuronal rheobase at day 7 that reversed at day 28 (N = 10 in each group). Comparisons made using unpaired t tests. ∗∗∗P < .001.

Figure 3

Vancomycin treatment causes a reversible increase in visceral pain. (A) Representative EMG traces from control and vancomycin treated animals during 80 µL colorectal distention. (B) Prior to vancomycin treatment both cohorts of mice show no difference in visceral pain sensation in response to colorectal distention. (C) The vancomycin treated mice exhibit greater VMR response to colorectal distention compared to vehicle-controlled mice. (D) After a 21-day recovery period the vancomycin treated animals no longer exhibit elevated VMR response to colorectal distention. Control N = 10; Vancomycin N = 10. Comparisons made using two-way ANOVA with Bonferroni multiple comparison test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 4

Vancomycin treatment increased colonic afferent nerve activity at rest and during distension. (A) Representative traces showing the spontaneous activity and the distension-induced multiunit afferent response to colonic distension in preparations from control (left) and vancomycin-treated mice (right). (B) Basal firing frequency of colonic afferent nerves is significantly increased in mice treated with vancomycin for 7 days compared to vehicle control animals (Control N = 13, Vancomycin N = 10). (C) The total colonic afferent nerve activity during distension is significantly increased in the animals treated with vancomycin. (D, E) The discriminated units show the high threshold units are significantly more active during distension, while the wide dynamic range units are unaltered. Compared with unpaired t test and two-way ANOVA. ∗P < .05, ∗∗P < .01.

Figure 5

Effects of dysbiosis on voltage-gated ion currents. (A) Example sodium current density traces from control (Left) and vancomycin treated (right) DRG neurons. (B) Representative traces of potassium current density recordings from control DRG neurons (left) and DRG neurons from a vancomycin treated animal (right). Traces cropped to remove negative capacitance conductance. (C) Voltage-gated Na+ current density was increased in DRG neurons from vancomycin-treated animals (N = 7) compared to controls (N = 7). (D) Vancomycin-treatment had no effect on voltage-gated K+ current density (Control n = 8; Vancomycin n = 12). Comparisons made using two-way ANOVA. ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Figure 6

Widespread effects of vancomycin treatment on DRG neuron excitability. (A) Cervical DRG neurons from vancomycin-treated mice (N = 8) are more excitable than neurons from vehicle controls (N = 8). (B) Serum from vancomycin-treated mice applied to lumbar DRG neurons from control animals decreases rheobase (N = 6) compared to neurons incubated in serum from control mice (N = 6). (C) Overnight incubation of DRG neurons in vancomycin had no effect on rheobase (Control N = 4; Vancomycin N = 4). Comparisons made using unpaired t tests. ∗P < .05, ∗∗∗P < .001.

Figure 7

Effects of gut microbial dysbiosis on hindpaw somatosensation.(A) Mice treated with vancomycin exhibited increased thermal sensitivity at 7-days relative to control-treated animals using the Hargreaves test (p_time = .0064, p_treatment = .0537, p_interaction = .0691; P = .015 at day 7). (B) Vancomycin treatment did not affect mechanosensitivity, as assayed by von Frey threshold. (C) Vancomycin treatment had no effect on cold sensitivity, as assayed using the acetone test. Control N = 10; Vancomycin N = 10; comparisons made using Two-way repeated-measures ANOVA with Sidak's multiple comparison test. ∗∗P < .01.

Figure 8

The excitation of DRG neurons by serum from vancomycin-treated mice is blocked by a cysteine protease inhibitor and a PAR-2 antagonist. (A) DRG neurons incubated with serum from control animals shows no change in excitability (N = 9), while incubation with vancomycin treated animal serum leads to hyperexcitability (N = 7), which is blocked by E64 (10 µM) (N = 5). (B) The excitatory effect of serum from vancomycin-treated mice (Control N = 7; Vancomycin serum N = 6) is also blocked by incubation with GB83 (10 µM) (N = 6). (C) The serum from vancomycin treated animals excites Cre control mouse DRG neurons (N = 7), but not the DRG neurons from PAR2 KO mice (N = 7). (D) Quantification of fecal protease activity showed a significant decrease in proteolytic activity following vancomycin treatment relative to vehicle controls (Control N = 9; Vancomycin N = 9). Comparisons made using Kruskal Wallis test with Dunn's multiple comparisons test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 9

Supernatants from stool (SS) of vancomycin treated animals increases excitability of DRG neurons while colonic tissue supernatants (TS) from the same mice do not. (A) Supernatants from both control and vancomycin treated mouse colon tissue does not alter DRG neuron excitability (N = 6). (B) Stool supernatant from control mice does not change excitability, supernatants from vancomycin treated animal stool significantly increases DRG neuron excitability (N = 7), an effect that was blocked by the PAR-2 antagonist GB83 (10 μM) (N = 4). (C) Hyperexcitability induced by vancomycin treated animal SS was blocked by the cysteine protease inhibitor E64 ( (D) Treatment with vancomycin (N = 8) increased gut mucosal permeability, shown by increased FITC dextran in serum compared to control (N = 6). A, B One-way ANOVA with Tukey’s multiple comparison test. C, D Mann-Whitney test. ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.