Volume 128, Issue 7, Pages 1996-2008 (June 2005) Rectal Instillation of Butyrate Provides a Novel Clinically Relevant Model of Noninflammatory Colonic Hypersensitivity in Rats  Sophie Bourdu, Michel Dapoigny, Eric Chapuy, Fabrice Artigue, Marie-Paule Vasson, Pierre Dechelotte, Gilles Bommelaer, Alain Eschalier, Denis Ardid  Gastroenterology  Volume 128, Issue 7, Pages 1996-2008 (June 2005) DOI: 10.1053/j.gastro.2005.03.082 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Schematic drawing illustrating the technique used to determine the referred lumbar cutaneous hyperalgesia (the gray area represents the lumbar shaving zone). Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Effect of 6 enemas (twice daily) of 1 mL saline solution or of 8, 40, 200, or 1000 mmol/L butyrate solution (A) on the pressure thresholds inducing specific behavior following CRD and (C) on forces exerted by the lumbar application of von Frey hairs inducing a reaction (lumbar von Frey scores) in rats. These parameters were monitored every 3 days up to 27 days. n = 8 per group. Effect of 6 enemas (twice daily) of 1 mL saline or 200 mmol/L butyrate solution (B) on the pressure thresholds and (D) on lumbar von Frey scores in male and female rats. n = 10–12 per group. *P < .05 versus saline-instilled group, (A) 2-way ANOVA followed by a Student-Newman-Keuls post-hoc test, or (C) Kruskal-Wallis one-way ANOVA on ranks followed by a Dunn’s post-hoc test. +P < .05 versus saline-instilled group, (B) one-way ANOVA followed by a Bonferroni post-hoc test, or (D) Mann-Whitney rank sum test. #P < .05 versus female butyrate-instilled group, (B) one-way ANOVA followed by a Bonferroni post-hoc test, or (D) Mann-Whitney rank sum test. Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effect of 6 enemas (twice daily) of 1 mL saline solution or a 200 mmol/L butyrate solution on pain score using the method of Al Chaer et al measured at several CRD pressures (every 10 mm Hg from 0 to 60 mm Hg). n = 7–8 per group. *P < .05 versus saline-instilled group, Mann-Whitney rank sum test. Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Macroscopic and microscopic (histologic HES staining; original magnification 100×) images of the colon taken from rats receiving (A) 6 enemas (twice daily) of 1 mL saline solution, (B) 6 enemas (twice daily) of 200 mmol/L butyrate solution, or (C) one enema of TNBS solution. The bowel of TNBS-treated animals presents a complete destruction of the epithelial architecture, with an almost complete loss of crypts, and epithelial integrity. Edema and intense inflammation are present in all layers. The colons of butyrate-treated animals are normal. n = 7 per group. Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of (A) morphine (0.03, 0.1, 0.3, 1, 3, and 10 mg/kg subcutaneously), (B) U50448H (0.3, 1, 3, and 10 mg/kg intraperitoneally), and (C) trimebutine (3, 10, 30, and 100 mg/kg, intraperitoneally) on the colonic hypersensitivity and referred lumbar mechanical hyperalgesia induced by 6 enemas (twice daily) containing 1 mL of a 200 mmol/L butyrate solution. Colonic hypersensitivity and lumbar mechanical hyperalgesia were determined 7 days after the beginning of butyrate enemas using the CRD test (squares) and the von Frey test (circles). Scores of butyrate-treated animals receiving saline (0.9% NaCl) are presented as gray squares (for the CRD test) and gray circles (for the von Frey test) on their respective y-axes. n = 8 in each group. Black squares or black circles show a significant difference (P < .05) versus the control group (butyrate-instilled rats treated with saline in the same conditions); one-way ANOVA followed by a Bonferroni post-hoc test (CRD test; squares) or Kruskal-Wallis one-way ANOVA on ranks followed by a Dunn’s post-hoc test (von Frey scores; circles). Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Effect of 6 enemas (twice daily) of 1 mL saline or 200 mmol/L butyrate solution (A) on the pressure thresholds inducing specific behavior following CRD and (B) on lumbar von Frey scores in male rats. Colonic hypersensitivity and lumbar mechanical hyperalgesia were determined 7 days after the beginning of butyrate instillations using the CRD test and the von Frey test. Animals were pretreated at 2 days of age either with vehicle or with capsaicin. n = 10 in each group. *P < .05 versus vehicle-treated group instilled with saline. +P < .05 versus vehicle-treated group instilled with butyrate. One-way ANOVA followed by (A) Bonferroni post-hoc test or (B) Mann-Whitney rank sum test. Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effect of a CGRP receptor antagonist (CGRP8–37) and an NK1 receptor antagonist (L733060) on the (A) colonic hypersensitivity and (B) referred lumbar mechanical hyperalgesia induced by 6 enemas (twice daily) of 1 mL of 200 mmol/L butyrate solution. Colonic hypersensitivity and lumbar mechanical hyperalgesia were determined 7 days after the beginning of butyrate instillation using the CRD test and the von Frey test, respectively. n = 8 in each group. *P < .05 versus respective control group (treated with saline), one-way ANOVA followed by (A) Bonferroni post-hoc test or (B) Mann-Whitney rank sum test. Gastroenterology 2005 128, 1996-2008DOI: (10.1053/j.gastro.2005.03.082) Copyright © 2005 American Gastroenterological Association Terms and Conditions