Volume 141, Issue 6, Pages 2098-2108.e5 (December 2011) Brain–Gut Interactions Increase Peripheral Nociceptive Signaling in Mice With Postinfectious Irritable Bowel Syndrome  Charles Ibeakanma, Fernando Ochoa–Cortes, Marcela Miranda–Morales, Todd McDonald, Ian Spreadbury, Nicolas Cenac, Fiore Cattaruzza, David Hurlbut, Stephanie Vanner, Nigel Bunnett, Nathalie Vergnolle, Stephen Vanner  Gastroenterology  Volume 141, Issue 6, Pages 2098-2108.e5 (December 2011) DOI: 10.1053/j.gastro.2011.08.006 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Chronic stress (WAS) increases HPA-SA activation. (A) Timeline of WAS. (B) Bar graph of serum epinephrine (left) and corticosterone (right) levels from nonstressed (control) or stressed (WAS) animals. (C) Schematic illustration of the apparatus (plus maze) used for assessment of anxiety-related behavior in WAS animals (left), and a summary graph showing the percentage of time spent in the open arm by control or WAS animals (right). Numbers in parentheses represent number of animals from each group. Data are reported as mean ± SEM, *P < .05, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Chronic stress (WAS) increases excitability of colonic DRG neurons. (A) Representative trace of action potentials evoked by a 500-millisecond depolarizing pulse at rheobase (left panel) and twice the rheobase (right panel) from neurons isolated from stressed (upper traces) and nonstressed (lower traces) animals. (B) Bar graphs summarizing changes in the electrical properties of colonic sensory neurons brought about by the 9-day paradigm of (WAS) stress. Neurons from stressed animals fired significantly more action potentials than controls. Numbers in parentheses represent number of neurons from each group. Data are reported as mean ± SEM, *P < .05, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Chronic stress (WAS) combined with prior C rodentium infection augmented HPA-SA axis activation and increased excitability of colonic DRG neurons. (A) Schematic drawing showing the timeline of the WAS and C rodentium infection and enzyme-linked immunosorbent assay measurements of epinephrine and corticosterone and electrophysiologic recordings (day 30). (B) Bar graph summarizing serum levels of corticosterone (left) and epinephrine (right) in C rodentium alone and C rodentium plus stress animals. (C) Summary data illustrating the combined effects of post C rodentium infection and stress on excitability of colonic DRG neurons. Stress plus post C rodentium infection not only significantly increased action potential discharges at twice the rheobase but also decreased the rheobase. Numbers in parentheses represent number of animals from each group. Data are reported as mean ± SEM, *P < .05, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Combined post C rodentium infection plus chronic stress (WAS) augmented colonic afferent mechanosensitivity and induced visceral hyperalgesia and allodynia in mice. (A) Representative trace of multi-unit afferent nerve discharges recorded from colons of day 30 postinfected C rodentium or C rodentium plus WAS animals in response to ramp distention. Top traces indicate ramp distention, middle traces indicate nerve activity histogram, and bottom traces indicate raw recording of whole nerve activity. (B) Summary of distention-response profiles of multi-unit afferents recorded from colons of C rodentium (solid circles) or C rodentium plus WAS (open circles) animals; ***P < .0001; 2-way analysis of variance. (C) Summary data (n = 7–8 animals per group) illustrating VMR responses to graded CRD (15, 30, 45, and 60 mm Hg pressure) in mice subjected to saline-only (open squares), C rodentium (solid circles), WAS (solid squares), or C rodentium plus WAS (open circles) treatment. Visceral hyperalgesia was induced in WAS plus C rodentium and WAS alone groups. Only the C rodentium plus WAS group exhibited evidence of allodynia (dashed box). Data are reported as mean ± SEM; ***P < .001 (one-way analysis of variance with Tukey posttests). Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Protease signaling is important in C rodentium–induced excitation of colon sensory neurons. (A) Summary graphs illustrating trypsin-like (left), serine protease (center), and cysteine protease (right) activities in mouse distal colonic tissues at day 30 after infection. Trypsin-like and serine protease activities, but not cysteine proteases, were elevated following C rodentium infection compared with water-gavaged controls. Ten colons were analyzed in each group. (B) Bar graphs illustrating decreased excitability (increased rheobase and decreased action potential firing at twice the rheobase) of neurons incubated in colonic supernatants from C rodentium infection plus a nonselective protease inhibitor compared with those incubated in supernatants without the inhibitor. (C) Summary data showing lack of effect of the nonselective protease inhibitor with colonic supernatants from WAS animals. Numbers in parentheses represent number of impaled neurons from each group. Data are reported as mean ± SEM; *P < .05, **P < .01, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Stress mediators can signal directly to colonic DRG neurons. (A) Reverse-transcription polymerase chain reaction products showing expression of glucocorticoid- (middle gel; 355–base pair product) and β2-adrenergic (right gel; 386–base pair product) messenger RNA transcript in colonic DRG neurons. Fast Blue–labeled colonic neurons were obtained using laser-capture microdissection. (B) Bar graphs summarizing evidence of direct activation of colonic sensory neurons by stress mediators corticosterone and epinephrine. Overnight incubation of colonic DRG neurons with 1 μmol/L corticosterone and 5 nmol/L epinephrine significantly decreased the rheobase and increased action potential discharges at twice the rheobase compared with control neurons. (C) Colonic DRG neurons were incubated overnight in colonic supernatants from control or WAS animals. There was no difference in the level of neuronal excitability between the 2 groups, suggesting WAS does not significantly increase the net signaling of pro-nociceptive tissue cytokines. Numbers in parentheses represent number of recorded neurons from each group. Data are reported as mean ± SEM; *P < .05, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Stress mediators and protease activation had a synergistic effect on colonic DRG neuronal excitability. Data summarizing rheobase and action potential numbers at twice rheobase of neurons from control or WAS neurons exposed to subthreshold levels of protease-activating peptide 2 (PAR2-AP; 30 μmol/L). PAR2-AP alone had no effect on rheobase or action potential discharge number but, in a separate experiment, when combined with neurons from WAS animals, PAR2-AP induced neuronal excitability indicated by the significantly decreased rheobase (left graph). Data are reported as mean ± SEM; **P < .002, Student t test. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Markers of colonic inflammation do not differ between C rodentium (black bars) and C rodentium plus WAS (white bars) animals. Data summarizing histopathology inflammation scores (total damage scores, inflammation, crypt epithelial proliferation/hyperplasia, recent and chronic damages) for colonic tissues from each group. Data are reported as mean ± SEM. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 The expression of the occludin tight junction protein was not decreased in the colons of C rodentium plus WAS animals compared with controls. (A) Western blot of the tight junction protein occludin in 4 groups of animals: controls (saline gavage), WAS and saline gavage, C rodentium, and C rodentium and WAS. The 3 bands for each group represent protein obtained from colons in a separate group of animals. (B) Graph summarizing the mean optical densities of the bands in the Western blot gel for each group. Data are expressed as the mean ± SEM of the 3 bands for each group (as shown in parentheses). Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Serine protease activity is increased in postinfected colonic tissues. Representative gel micrographs illustrating serine (left; ∼ 29-kilodalton mass) and cysteine protease (right) activities in control and C rodentium–infected mouse colonic tissues. Activities were detected using small molecule inhibitor-base (activity-based) probes that covalently interact with the activated enzymes. β-actin was used as a reference protein to ensure equal amounts of proteins are loaded in the gels. Gastroenterology 2011 141, 2098-2108.e5DOI: (10.1053/j.gastro.2011.08.006) Copyright © 2011 AGA Institute Terms and Conditions