TRPV1 receptors on unmyelinated C-fibres mediate colitis-induced sensitization of pelvic afferent nerve fibres in rats

Abstract

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis-induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single-fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 0.25-5 mg kg(-1)) or its vehicle (hydroxypropyl-beta-cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A-fibre neurons, was performed in the dorsal root ganglia L6-S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C-fibres. BCTC did not significantly affect the C-fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C-fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Adelta-fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up-regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis-induced sensitization of pelvic afferent C-fibres to CRD, while Adelta-fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Figure 1. The effect of TNBS-induced colitis on the compliance of the colorectal wall

Results are expressed as intracolonic pressure (mmHg). *P < 0.05, significantly different from controls, unpaired Student's t test.

Figure 2. Effect of TNBS-induced colitis on the response to colorectal distension

Effect of TNBS-induced colitis on the response to CRD of all single pelvic afferent neurons identified (A), on pelvic afferent neurons with a low threshold response (B) or a high threshold response (C), and with low conduction velocity (C-fibres, D) or high conduction velocity (Aδ-fibres, E). Results are expressed as spike rate (Hz). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from controls, two-way ANOVA with post hoc Student–Newman–Keuls test.

Figure 3. Pelvic afferent nerve response to repeated colorectal distension

A, reproducibility of the pelvic afferent nerve response to repeated colorectal distension (CRD; 60 mmHg, 20 s, 4 min interval between consecutive distensions) in healthy controls and in rats with TNBS-induced colitis. Two-way ANOVA only showed significance for the effect of TNBS colitis (*P < 0.05, n= 6). B, typical tracings showing the effect of the specific TRPV1 receptor antagonist BCTC (0.25, 0.5, 1, 2 and 5 mg kg⁻¹i.v.) or its vehicle (25% hydroxypropyl-β-cyclodextrin) on the frequency response of a pelvic afferent neuron to repeated CRD (60 mmHg, 20 s, 4 min interval) in a control rat and in a rat with colitis. Both tracings are of unmyelinated C-fibres.

Figure 4. Effect of BCTC on the frequency response to repeated colorectal distension and spontaneous activity of pelvic afferent neurons

Effect of BCTC (0.25, 0.5, 1, 2 and 5 mg kg⁻¹i.v.) or its vehicle (25% hydroxypropyl-β-cyclodextrin) on the frequency response to repeated colorectal distension (60 mmHg, 20 s, 4 min interval) (A–C) and the spontaneous activity (D–F) of pelvic afferent neurons in controls and in rats with TNBS-induced colitis. Results are shown for all neurons (A and D), for C-fibres only (B and E) and for Aδ-fibres only (C and F). *P < 0.05, significantly different from controls; #P < 0.05, significantly different from vehicle treatment, repeated measures two-way ANOVA with post hoc Student–Newman–Keuls analysis.

Figure 5. Immunocytochemical detection of NF200 and TRPV1 receptors

A–C, immunocytochemical detection of NF200 (red, A) and TRPV1 receptors (green, B) on cryosections of the Fast Blue-traced (blue, C) dorsal root ganglion (DRG). D is the merged picture of A, B and C. This representative example of the triple stain was obtained in a control rat. Arrowheads indicate colon-derived C-fibres.