Ex vivo study of human visceral nociceptors

Abstract

Objective: The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation.

Design: Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV₄) modulation on mechanical responses.

Results: Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (-20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (-34.9±10.0%, n=7, p<0.05), a TRPV₄ antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity.

Conclusions: Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Keywords: ABDOMINAL PAIN; ELECTROPHYSIOLOGY; NEUROGASTROENTEROLOGY; VISCERAL NOCICEPTION; VISCERAL SENSITIVITY.

Figure 1

Characterisation of isolated visceral afferent fibres from resected human bowel tissues into serosal nociceptor and muscular afferent subtypes based on responses to differing mechanical and noxious chemical stimuli. (A) Example image of resected bowel tissue pinned flat in the recording chamber. The bowel serosa can be seen below the dissected mesentery. (B) Proportions of muscular and serosal nociceptor subtypes characterised from identified mechanosensitive afferent recordings. (C) Stimulus-response curve to von Frey hair (VFH) probing (0.02–4 g) for serosal afferents in resected human bowel tissues. (D) Associated activation thresholds of VFH probing (0.02–4 g). Dashed line at 0.6 g VFH weight highlights differential activation thresholds of serosal nociceptor (100%) versus muscular subtypes (0%) to VFH probing, potentially allowing subpopulations to be discriminated by VFH probe threshold alone. (E) Example responses to 0.4 g VFH probe, circular and longitudinal tissue stretch and stroking of the gut mucosa in both serosal nociceptor and muscular afferent subtypes. Specifically, serosal nociceptors elicit action potential firing to a range of VFH probe weights tested (0.02–4 g), but are non-responsive to tissue stretch and mucosal stroking. Muscular afferents are responsive to tissue stretch and only respond to VFH probing at weights of >0.6 g. (F) Examples of action potential firing to prototypic algogenic mediators bradykinin and ATP in serosal nociceptor and muscular afferents, and the proportion of responders in each afferent subtype.

Figure 2

Spontaneous activity in serosal and muscular afferents innervating the human intestine. (A) Pie charts illustrating the proportion of spontaneously active serosal and muscular units. Muscular afferents were significantly more likely to exhibit spontaneous activity (p<0.01, Fisher's exact test). (B) Bar graph demonstrating the firing rate of serosal and muscular units that were spontaneously active. Activity was significantly greater in muscular compared with serosal afferents (**p<0.01, unpaired t-test). Mean±SEM.

Figure 3

‘Silent’ afferents were evoked after the application of the algogenic mediator bradykinin (n=2). Rate histograms and neurogram showing (A) the lack of response to mechanical probing before bradykinin application, (B) the increase in ongoing activity following application of bradykinin and (C) the acquired mechanosensitivity to von Frey hair probing postbradykinin.

Figure 4

Modulation of mechanosensitive human visceral nociceptors by tegaserod, and the transient receptor potential channel, vanilloid 4 (TRPV₄) antagonist HC067047. Example rate histogram and neurogram responses of individual von Frey hair probes at baseline (BL) and from the set of probes given within the respective minutes illustrated (eg, 5, 10, 15 min) following (Ai) vehicle (0.1% DMSO/Krebs), (Bi) the TRPV₄ agonist GSK1016790A, (Ci) the TRPV₄ antagonist HC067047 or (Di) the partial 5-HT₄ antagonist tegaserod. Bar graphs illustrating the normalised firing rate per 2 s probe before and after the application of (Aii) vehicle (0.1% DMSO/Krebs) (n=5), (Bii) GSK1016790A (n=6), (Cii) HC067047 (n=7) or (Dii) tegaserod (n=6). Mean±SEM. NS, not significant (p>0.05), *p<0.05, paired t-test.

Figure 5

Application of algogenic and disease mediators activates visceral afferents innervating the human intestine. Example of rate histograms illustrating the response profile, and pie charts illustrating the proportion of preparations responding to (A) bradykinin, (B) ATP, (C) capsaicin, (D) histamine, (E) prostaglandin E2 (PGE₂) and (F) 5-hydroxytryptamine (5-HT).

Figure 6

Repeated applications of bradykinin or ATP result in reproducible human afferent responses after initial desensitisation. Bar graphs illustrating the reproducibility of responses to (A) bradykinin (n=6), (B) ATP (n=4), after initial desensitisation to the first application of the respective mediator. A proportion of preparations responded to a second application of (C) 5-hydroxytryptamine (5-HT) (2/2) and (D) histamine (1/2), and no response was seen to a third application of either respective mediator. Mean±SEM. NS, not significant, p>0.05, paired t-test.

Figure 7

Investigation of receptors involved in the activation of afferents innervating the human intestine by bradykinin and ATP. Example of a rate histogram (A) and bar graph (B) demonstrating the inhibition of human afferent firing in response to bradykinin by pretreatment with the bradykinin receptor 2 antagonist HOE140 (300 nM, n=6, p<0.05; 1 µM, n=4, p<0.01). In contrast, the bradykinin receptor 1 antagonist R715 (n=6) failed to inhibit the human afferent response to bradykinin (B). Example of a rate histogram (C) and bar graph (D) showing the lack of human afferent inhibition in response to ATP when pretreated with the P1 adenosine receptor antagonist CGS15943 (n=6). Similarly, the P2X2/3, 3 receptor antagonist RO4 (n=3) failed to reduce the human afferent response to ATP (D). Mean±SEM. NS, not significant (p>0.05). *p<0.05, **p<0.01, paired t-test.

Figure 8

The effect of age on visceral afferent mechanosensitivity and chemosensitivity. Scatter plots illustrating afferent responses to (A) von Frey hair probing at 0.4 and 2 g, (B) bradykinin or (C) ATP compared with the patient's age. Responses were plotted for tissues from cancer resections only, and presented for all tissue regions studied and sigmoid colon only. Pearson's or Spearman's correlations were performed based on data normality.