The effects of bradykinin and ATP on human visceral afferent fibre activity, a pilot study

Cian McGuire, Madusha Peiris, George Boundouki, James Hockley, Ashley Blackshaw, Charles Knowles, David Bulmer. Queen Mary University of London, London, UK.

The recent failure in clinical trials of several drugs designed to treat irritable bowel syndrome (IBS) despite these drugs having previously demonstrated antinociceptive effects in pre-clinical studies raises concerns over the translation of observations in animal models of visceral pain to clinical studies in humans. To help address these concerns we have developed in vitro electrophysiological recordings of visceral afferent fibre activity from isolated human appendix, colon and ileum to enable like for like comparisons to be made between responses to algogenic and inflammatory stimuli in human and animal tissue. The aim of the current study was to further validate our human visceral afferent fibre recordings by examining the effect of the prototypic inflammatory mediators bradykinin (BK) and ATP on human visceral afferent activity.

All experiments were performed in accordance with human ethics regulations [NREC 09/H0704/2 and 10/H0703/71]. Surgically resected human appendix, colon and ileum were obtained from consenting patients undergoing right hemicolectomy or pan colectomies and placed in carbogenated ice cold krebs buffer. Following gross dissection tissue was placed in a tissue bath superfused with carbogenated Krebs buffer (7ml/min, 32-34oC), pinned flat and mesenteric nerve bundles dissected free allowing activity to be recorded using suction electrodes. Recordings were allowed to stabilise over a 30min period before application of test agents. BK was applied either directly into the tissue bath (10µM, n = 1 or 100µM, n = 2 in 500µl buffer) or superfused (10µM in 10ml buffer, n = 2). ATP was applied directly into the tissue bath (3mM, n = 1 or 10mM, n = 1 in 500µl buffer). Afferent fibre activity was displayed as a rate histogram (20s bin width). Peak firing was determined before and after application of test compounds and expressed in spikes/20s. Only one dose of either BK or ATP was tested per experiment and responses were combined into a single data set irrespective of dose to allow changes in afferent fibre activity to be statistically analysed using Wilcoxon’s matched pairs test significance determined at p<0.05.

Recordings were made from 6 tissues (4 appendix, 1 colon, 1 ileum) obtained from 6 separate patients. Spontaneous activity (50±24.1 spikes/20s n = 6) was observed in all nerves recorded. Application of BK produced an increase in nerve activity in 5/5 preparations tested with an increase of 42 and 24±6.4 spikes/20s following bath application of 10µM and 100µM BK respectively, and 89.5±23.0 spikes/20s following superfusion with 10µM BK. Comparison of peak firing rates before and after application of BK for all the doses tested demonstrated a statistically significant increase (p = 0.03) to BK. Application of ATP produced an increase in nerve activity in 2/2 preparations tested (including 1 preparation that responded to both ATP and BK) with an increase of 64 and 69 spikes/20s following bath application of 3mM and 10mM ATP respectively.

The data generated confirms the feasibility of electrophysiological recordings of human visceral afferent fibre activity in vitro, and demonstrates that these preparations respond to application of the inflammatory mediators, bradykinin and ATP, with robust increases in nerve activity.