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Rectal Instillation of Butyrate Provides a Novel Clinically Relevant Model of Noninflammatory Colonic Hypersensitivity in Rats

2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2005.03.082

1528-0012

Sophie Bourdu, Michel Dapoigny, Eric Chapuy, Fabrice Artigue, Marie‐Paule Vasson, Pierre Déchelotte, Gilles Bommelaer, Alain Eschalier, Denis Ardid,

Neuropeptides and Animal Physiology

Background & Aims: The treatment of irritable bowel syndrome (IBS), characterized by abdominal pain and bloating, is empirical and often poorly efficient. Research lacks suitable models for studying the pathophysiologic mechanisms of the colonic hypersensitivity and new pharmacologic targets. The present study aimed to develop a novel model of colonic hypersensitivity possessing several of the characteristics encountered in patients with IBS. Methods: Rats received enemas of a butyrate solution (8–1000 mmol/L) twice daily for 3 days. A time course was determined for colonic hypersensitivity (colorectal distention test) and referred cutaneous lumbar hyperalgesia (von Frey hairs). Macroscopic and histologic analyses were performed on colonic mucosa. The efficacy of morphine, U50488H (a κ opioid agonist), and trimebutine on the 2 pain parameters was determined. Finally, the involvement of peptidergic C-fibers was evaluated using capsaicin-pretreated animals and treatments with calcitonin gene-related peptide (CGRP) and neurokinin 1 receptor antagonists. Results: Butyrate enemas induced a sustained, concentration-dependent colonic hypersensitivity and, to a lesser extent, a referred cutaneous mechanical hyperalgesia, particularly in female rats, but no macroscopic and histologic modifications of the colonic mucosa, as observed in patients with IBS. Both pain parameters were sensitive to morphine, U50488H, trimebutine, neonatal capsaicin treatment, and the CGRP receptor antagonist but not to the neurokinin 1 receptor antagonist. Conclusions: These results present our noninflammatory model of chronic colonic hypersensitivity as a useful novel tool for studying IBS. The CGRP receptor antagonist-induced reduction of colonic hypersensitivity suggests that CGRP receptors may provide a promising target for treatment of IBS. Background & Aims: The treatment of irritable bowel syndrome (IBS), characterized by abdominal pain and bloating, is empirical and often poorly efficient. Research lacks suitable models for studying the pathophysiologic mechanisms of the colonic hypersensitivity and new pharmacologic targets. The present study aimed to develop a novel model of colonic hypersensitivity possessing several of the characteristics encountered in patients with IBS. Methods: Rats received enemas of a butyrate solution (8–1000 mmol/L) twice daily for 3 days. A time course was determined for colonic hypersensitivity (colorectal distention test) and referred cutaneous lumbar hyperalgesia (von Frey hairs). Macroscopic and histologic analyses were performed on colonic mucosa. The efficacy of morphine, U50488H (a κ opioid agonist), and trimebutine on the 2 pain parameters was determined. Finally, the involvement of peptidergic C-fibers was evaluated using capsaicin-pretreated animals and treatments with calcitonin gene-related peptide (CGRP) and neurokinin 1 receptor antagonists. Results: Butyrate enemas induced a sustained, concentration-dependent colonic hypersensitivity and, to a lesser extent, a referred cutaneous mechanical hyperalgesia, particularly in female rats, but no macroscopic and histologic modifications of the colonic mucosa, as observed in patients with IBS. Both pain parameters were sensitive to morphine, U50488H, trimebutine, neonatal capsaicin treatment, and the CGRP receptor antagonist but not to the neurokinin 1 receptor antagonist. Conclusions: These results present our noninflammatory model of chronic colonic hypersensitivity as a useful novel tool for studying IBS. The CGRP receptor antagonist-induced reduction of colonic hypersensitivity suggests that CGRP receptors may provide a promising target for treatment of IBS. Irritable bowel syndrome (IBS) is nowadays a health care burden, because it represents one of the most common disorders encountered in gastrointestinal practice and highly affects quality of life.1Drossman D.A. Camilleri M. Mayer E.A. Whitehead W.E. AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1218) Google Scholar The major features for diagnosing IBS have been defined as (1) abdominal pain or discomfort, (2) altered bowel habits, and (3) the absence of any detectable structural or biochemical abnormality.2Read N.W. Al Janabi M.N. Bates T.E. Holgate A.M. Cann P.A. Kinsman R.I. McFarlane A. Brown C. Interpretation of the breath hydrogen profile obtained after ingesting a solid meal containing unabsorbable carbohydrate.Gut. 1985; 26: 834-842Crossref PubMed Scopus (119) Google Scholar Chronic or recurrent abdominal pain represents a symptom common to all patients with IBS, and rectal or colonic hypersensitivity is frequently encountered in IBS.1Drossman D.A. Camilleri M. Mayer E.A. Whitehead W.E. AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1218) Google Scholar, 3Delvaux M. Role of visceral sensitivity in the pathophysiology of irritable bowel syndrome.Gut. 2002; 51: i67-i71Crossref PubMed Scopus (128) Google Scholar The underlying causes of the pathophysiologic changes responsible for these changes in colonic sensitivity remain unclear. Animal models must therefore be developed to investigate the pathophysiologic mechanisms underlying the colonic hypersensitivity observed in IBS,4Mayer E.A. Collins S.M. Evolving pathophysiologic models of functional gastrointestinal disorders.Gastroenterology. 2002; 122: 2032-2048Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar which could lead to more adapted treatments because current treatments have thus far proved unsatisfactory. Some models have been developed to obtain colonic hypersensitivity in adult rats after colonic irritation either induced by neonatal repeated colonic distention or inflammation5Al Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar or following repeated neonatal stressful separation of rat pups from their mother.6Coutinho S.V. Plotsky P.M. Sablad M. Miller J.C. Zhou H. Bayati A.I. McRoberts J.A. Mayer E.A. Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G307-G316Crossref PubMed Scopus (419) Google Scholar Diop et al7Diop L. Raymond F. Fargeau H. Petoux F. Chovet M. Doherty A.M. Pregabalin (CI-1008) inhibits the trinitrobenzene sulfonic acid-induced chronic colonic allodynia in the rat.J Pharmacol Exp Ther. 2002; 302: 1013-1022Crossref PubMed Scopus (93) Google Scholar developed a model in adult rats in which distant colonic hypersensitivity (distal part of the colon) was induced using an injection of trinitrobenzene sulfonic acid (TNBS) in the proximal part. Recently, we showed a visceral hypersensitivity in rats elicited by enemas with butyrate, a short-chain fatty acid produced by the colonic degradation of alimentary fibers.8Tarrerias A.L. Millecamps M. Alloui A. Beaughard C. Kemeny J.L. Bourdu S. Bommelaer G. Eschalier A. Dapoigny M. Ardid D. Short-chain fatty acid enemas fail to decrease colonic hypersensitivity and inflammation in TNBS-induced colonic inflammation in rats.Pain. 2002; 100: 91-97Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar This is in line with the observation that colonic butyrate levels were increased in patients with IBS.9Treem W.R. Ahsan N. Kastoff G. Hyams J.S. Fecal short-chain fatty acids in patients with diarrhea-predominant irritable bowel syndrome in vitro studies of carbohydrate fermentation.J Pediatr Gastroenterol Nutr. 1996; 23: 280-286Crossref PubMed Scopus (127) Google Scholar These observations argue for the use of butyrate as an etiologic factor, in link with nutrition, to create a novel, particularly relevant animal model of colonic hypersensitivity as observed in patients with IBS. The aim of this study was first to characterize whether butyrate enemas could provoke a distention-induced colonic hypersensitivity in rats and/or an alteration of bowel habits and if these modifications were independent of any detectable abnormality of the mucosa as observed in patients with IBS.10Schuster M.M. Irritable bowel syndrome.in: Sleisenger M.H. Fordtran J.S. Gastrointestinal disease. WB Saunders, New York, NY1989: 1402-1418Google Scholar Hence, macroscopic, microscopic, or biochemical modifications of the colon mucosa were evaluated in our experimental conditions. After having determined the characteristics and clinical relevance of our model, it was important to test its pharmacologic sensitivity to both reference analgesic drugs such as opioids and compounds such as trimebutine maleate, which is commonly used in patients with IBS. One factor that could explain the difficulty in proposing new treatments in patients with IBS is the poor understanding of the pathophysiologic mechanisms involved in the development of this pathology. Hence, using this model, we looked to research the pathophysiologic mechanisms that could be involved in noninflammatory colonic hypersensitivity. We chose to determine whether peptidergic C-fibers were involved because these fibers are known to be involved in several chronic pain contexts, including neuropathic11Meller S.T. Gebhart G.F. Maves T.J. Neonatal capsaicin treatment prevents the development of the thermal hyperalgesia produced in a model of neuropathic pain in the rat.Pain. 1992; 51: 317-321Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 12Rashid M.H. Inoue M. Bakoshi S. Ueda H. Increased expression of vanilloid receptor 1 on myelinated primary afferent neurons contributes to the antihyperalgesic effect of capsaicin cream in diabetic neuropathic pain in mice.J Pharmacol Exp Ther. 2003; 306: 709-717Crossref PubMed Scopus (112) Google Scholar and inflammatory13Chiang C.Y. Kwan C.L. Hu J.W. Sessle B.J. Effects of GABA receptor antagonist on trigeminal caudalis nociceptive neurons in normal and neonatally capsaicin-treated rats.J Neurophysiol. 1999; 82: 2154-2162PubMed Google Scholar, 14Kwan C.L. Hu J.W. Sessle B.J. Neuroplastic effects of neonatal capsaicin on neurons in adult rat trigeminal nucleus principalis and subnucleus oralis.J Neurophysiol. 1996; 75: 298-310PubMed Google Scholar pain, and also in bronchial hypersensitivity.15Kummer W. Fischer A. Kurkowski R. Heym C. The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry.Neuroscience. 1992; 49: 715-737Crossref PubMed Scopus (264) Google Scholar This involvement was assessed using neonatal capsaicin-treated animals; in fact, both peptidergic C-fibers, which synthesize and release substance P and calcitonin gene-related peptide (CGRP), and nonpeptidergic C-fibers, which do not express these peptides, are sensitive to capsaicin.16Le Bars D. Adam F. [Nociceptors and mediators in acute inflammatory pain].Ann Fr Anesth Reanim. 2002; 21: 315-335Crossref PubMed Scopus (27) Google Scholar Thus, to show the involvement of the peptidergic C-fiber population, we studied the involvement of the major peptides peripherally released from these fibers, substance P, and CGRP. The role of substance P has been demonstrated in several inflammatory bowel models17Nguyen C. Coelho A.M. Grady E. Compton S.J. Wallace J.L. Hollenberg M.D. Cenac N. Garcia-Villar R. Bueno L. Steinhoff M. Bunnett N.W. Vergnolle N. Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism.Can J Physiol Pharmacol. 2003; 81: 920-927Crossref PubMed Scopus (80) Google Scholar, 18Traub R.J. Hutchcroft K. Gebhart G.F. The peptide content of colonic afferents decreases following colonic inflammation.Peptides. 1999; 20: 267-273Crossref PubMed Scopus (73) Google Scholar or in acute visceral hyperalgesia.19Gschossmann J.M. Coutinho S.V. Miller J.C. Huebel K. Naliboff B. Wong H.C. Walsh J.H. Mayer E.A. Involvement of spinal calcitonin gene-related peptide in the development of acute visceral hyperalgesia in the rat.Neurogastroenterol Motil. 2001; 13: 229-236Crossref PubMed Scopus (45) Google Scholar Moreover, substance P receptors (neurokinin 1 [NK1] receptors) have been proposed as targets for IBS treatment.20Lecci A. Maggi C.A. Peripheral tachykinin receptors as potential therapeutic targets in visceral diseases.Expert Opin Ther Targets. 2003; 7: 343-362Crossref PubMed Scopus (55) Google Scholar, 21Tough I.R. Lewis C.A. Fozard J. Cox H.M. Dual and selective antagonism of neurokinin NK(1) and NK(2) receptor-mediated responses in human colon mucosa.Naunyn Schmiedebergs Arch Pharmacol. 2003; 367: 104-108Crossref PubMed Scopus (25) Google Scholar We also chose to investigate the involvement of CGRP, which is colocalized with substance P in peptidergic C-fibers and has been poorly investigated in the context of colonic hypersensitivity. Male and female Sprague-Dawley rats (Charles River, L’Arbresle, France) weighing 200–220 g were used in this study. Rats were maintained in laboratory conditions for 1 week before each experiment. The animals were housed 5 per cage with food and water available ad libitum. All studies were performed in accordance with the proposal of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.22Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals.Pain. 1983; 16: 109-110Abstract Full Text PDF PubMed Scopus (6958) Google Scholar Great care was taken, particularly with regard to housing conditions, to avoid or minimize discomfort to the animals. For each enema, a catheter (2-mm Fogarty catheter) was placed into the colon at 7 cm from the anus, and the animals received 1 mL of sodium butyrate solution at neutral pH (pH 6.9) twice daily for 3 days. A TNBS model was used as a positive control for inflammation. Colitis was induced under anesthesia (acepromazine 4 mg/kg intraperitoneally, ketamine 30 mg/kg intraperitoneally) in 24-hour fasted rats by a single intracolonic enema with 0.5 mL of 50 mg/kg TNBS dissolved in 30% ethanol solution. TNBS was introduced through a 7-cm-long Fogarty probe inserted into the descending colon. Nociception in the animals was assessed by measuring the intracolonic pressure required to induce a behavioral response during colorectal distention (CRD) due to the inflation of a balloon introduced in the colon. This response was characterized by an elevation of the hind part of the animal body and a clearly visible abdominal contraction corresponding to the severe contractions described by Al Chaer et al5Al Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar and used as a pain marker by Tarrerias et al.8Tarrerias A.L. Millecamps M. Alloui A. Beaughard C. Kemeny J.L. Bourdu S. Bommelaer G. Eschalier A. Dapoigny M. Ardid D. Short-chain fatty acid enemas fail to decrease colonic hypersensitivity and inflammation in TNBS-induced colonic inflammation in rats.Pain. 2002; 100: 91-97Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar This method was chosen to avoid the surgery needed to implant recording electrodes and wires in the abdominal muscles, which may cause additional sensitization and disrupt the evaluation of lumbar cutaneous referred hyperalgesia. Distention balloons were prepared using a 2-mm Fogarty catheter cut at 9 cm. A 2-cm flexible latex balloon was ligated to the tip of the catheter. Rats were anesthetized with volatile anesthesia (2% isoflurane), the balloon was inserted intrarectally in a minimally invasive manner to 7 cm from the anus, and the catheter was taped to the base of the tail. After 5 minutes, rats were placed in the middle of a 40 × 40-cm Plexiglas box and the catheter was connected to an electronic barostat apparatus (Synectics Visceral Stimulator; Medtronic, Boulogne-Billancourt, France). Increasing pressure was continuously applied until pain behavior was displayed or a cutoff pressure of 80 mm Hg was reached. The influence of volatile anesthesia on motor activity (Actimeter Apelex panlab 0602 and 0603; Apelex, Massy, France) was controlled to make sure that there was no residual effect on the motor reaction of animals to CRD. There was no difference in the spontaneous motor activity of nonanesthetized and anesthetized rats (453 ± 22.53 vs 406 ± 20.17 arbitrary units, respectively) between 5 and 10 minutes following isoflurane exposure. To confirm the butyrate-induced colonic hypersensitivity, another pain evaluation method was used. Using the same protocol for distention, we performed semiquantitative scoring using the behavioral scale described by Al Chaer et al.5Al Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar This consisted of visual observation of the animal’s response to the graded CRD by a blind observer and assignment of a score: 0, no behavioral response to CRD; 1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of the abdomen; 4, body arching and lifting of the pelvic structures. Behavioral measurements were scored at several pressures (10, 20, 30, 40, 50, and 60 mm Hg) during distention. Mechanical lumbar hyperalgesia, a marker of referred cutaneous pain, was measured by applying von Frey hairs to the lower back of rats (Figure 1). Rats were shaved on the lower back the day before the test. On the day of the test, they were acclimatized to a grid platform (30 × 45 × 25 cm) for 15 minutes and then calibrated von Frey hairs of increasing diameter were applied 5 times for 1 second, ranging from 2.1 to 72.3 mN (cutoff). The reaction thresholds (named “lumbar von Frey scores”) corresponded to the force in mN of the von Frey hair that induced skin wrinkling in the lumbar area, followed or not by an avoidance behavior characterized by the rat escaping. Image analysis software (Image Tool, developed in the Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, TX) was used to obtain a macroscopic scoring of lesions. After removal, the bowel was laid on a white surface and a digital picture was taken. The picture was read using the software and converted into a gray scale. Lesion areas were determined over a length of 15 cm from the rectum by measuring surfaces that showed an increased gray level. A second evaluation used a slightly modified Morris scoring method23Morris G.P. Beck P.L. Herridge M.S. Depew W.T. Szewczuk M.R. Wallace J.L. Hapten-induced model of chronic inflammation and ulceration in the rat colon.Gastroenterology. 1989; 96: 795-803Abstract Full Text PDF PubMed Scopus (1597) Google Scholar: 0, no damage; 1, localized hyperemia without ulcers; 2, one site of ulceration with inflammation; 3, 2 or more major sites of ulceration and/or inflammation. After removal, the fixed tissues (colonic tissue) were processed into paraffin, cut into 4-μm sections, and stained with HES or Giemsa colorations. Eosinophil and mast cell count within the lamina propria was systematically performed at a 400× magnification in 20 different areas. For each animal, results were expressed as the mean number of cells per area. Myeloperoxidase (MPO), a marker of polymorphonuclear neutrophil primary granules, was determined according to the method of Mazelin et al24Mazelin L. Theodorou V. More J. Fioramonti J. Bueno L. Protective role of vagal afferents in experimentally-induced colitis in rats.J Auton Nerv Syst. 1998; 73: 38-45Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar but slightly modified. Briefly, a small section of colon (about 0.5–1 cm, taken at 7 cm from the anus) was rinsed with 0.1 mol/L cold phosphate buffer and suspended in hexadecyltrimethylammonium bromide buffer (0.5% wt/vol in 50 mmol/L phosphate buffer, pH 6.0; 50 mg of tissue/mL), a detergent that releases MPO from the primary granules. Intestinal tissue was homogenized using a Turrax (Ika-Werke, Staufen, Germany) and sonicated on ice for 30 seconds. The homogenate was then centrifuged at 1500g for 10 minutes at 4°C, freeze thawed 3 times, and centrifuged again. Supernatants were assayed by spectrophotometry for MPO activity and protein measurements. Each supernatant was mixed with 0.22% (vol/vol) aqueous guaiacol and 10 mmol/L phosphate buffer (pH 6.0). Three percent peroxide (H2O2) was added to start the reaction. Absorbance at 470 nm was determined with a spectrophotometer at 10-second intervals over 2 minutes. As a standard, MPO of human neutrophils (0.1 U/10 μL) was combined with the reaction buffer. The absorbance changes at 470 nm for 1 μmol peroxide/min were calculated from the standard curve, which equals 1 unit of MPO activity. Protein concentrations were determined by the bicinchoninic acid method,25Lleu P.L. Rebel G. Interference of Good’s buffers and other biological buffers with protein determination.Anal Biochem. 1991; 192: 215-218Crossref PubMed Scopus (23) Google Scholar and MPO activity was expressed as unit MPO per gram of protein. Time-dated pregnant Sprague-Dawley rats were housed with free access to food and water until delivery. Two-day-old male pups received capsaicin (50 mg/kg in 10/10/80 Tween 80/EtOH/saline [NaCl 0.9%]) or its vehicle subcutaneously. This technique is a well-established method of denervation, allowing destruction of more than 90% of C-fibers.26Kihara N. de la Fuente S.G. Fujino K. Takahashi T. Pappas T.N. Mantyh C.R. Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats.Gut. 2003; 52: 713-719Crossref PubMed Scopus (185) Google Scholar The effectiveness of capsaicin pretreatment to destroy C-fibers was assessed in adult rats (7–8 weeks) before any experiment using 3 different noxious stimuli: thermal (tail immersion test in a water bath at 46°C), mechanical (paw pressure test), and chemical (ocular application of 10 μL capsaicin in 0.1% EtOH) tests. Animals that responded positively to 2 of the 3 tests (delay >15 seconds for the tail immersion test, vocalization threshold >600 g for the paw pressure test, and no sign of eye scratching after ocular application of capsaicin) were selected for the experiment. All experiments were performed in a blind manner by the same experimenter using the block method, which consists of distributing one animal per treatment or control (saline-treated rats) in the same block. The number of blocks corresponds to the number of rats receiving each treatment. The order of treatments was randomized in each block. Different animals were used for each experimental series. All animals in a same block were treated in the same period of time. This procedure avoids any uncontrollable environmental influences. Five groups of 8 male rats were used. A group was instilled twice daily for 3 days with 1 mL saline and others with different concentrations of butyrate solution (8, 40, 200, and 1000 mmol/L). Lumbar von Frey scores and pressure-induced behavioral response to CRD were successively assessed on days 3, 6, 9, 12, 15, 18, 21, 24, and 27 after the first enema for the same animals. Assessment of sex-dependent hypersensitivity was performed by instilling both male and female rats with saline or butyrate (200 mmol/L; n = 10–12 in each of the 4 groups). The same parameters were assessed on day 7 after the first enema. Two other groups of 7–8 rats were used to confirm the colonic hypersensitivity using the method described by Al Chaer et al.5Al Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar One group was instilled twice daily for 3 days with 1 mL saline and the other with a 200 mmol/L butyrate solution. Behavioral scoring was performed on day 7 after the first enema. Male rats were placed in individual metabolism cages with free access to food and water. They were allowed to acclimatize in these cages for 5 days. Two groups of 8 rats were used. The rats were instilled twice daily for 3 days with 1 mL saline or butyrate solution (200 mmol/L). The animals’ body weight, the mass of wet and dry feces, and the volume of urine of each rat produced during the previous 24 hours were determined on days 0, 3, 6, 9, 12, and 15 after the first enema. The amount of food and water consumed was also determined. The dry mass of the feces was determined after drying at 60°C for 48 hours. The water content (%) was then calculated using the following formula: 100 × (1 − [Dry Mass/Wet Mass]). Three groups of 7 male rats were used. The first 2 groups were instilled twice daily for 3 days with 1 mL saline or butyrate solution (200 mmol/L). At day 3, the third group was instilled with a single intracolonic TNBS enema. This group was used as a positive control group with colonic inflammation. At day 5, when inflammatory response was potentially maximal in the 2 models, all of the animals were killed by cervical dislocation. A midline laparotomy was performed, and the total colon was removed. The Morris score was determined, and a digital picture was taken for postmacroscopic analysis using Image Tool. The colon was then divided in half by a longitudinal cut. One part of the colon was frozen in liquid nitrogen and stored until MPO activity assay, and the other part was immersion fixed in 10% formalin for histologic study. All the groups of 8 rats were instilled with 1 mL butyrate solution (200 mmol/L) twice daily for 3 days. Seven days after the beginning of butyrate instillations, 3 experimental series corresponding to the different treatments were performed. Animals were treated with morphine (0.03, 0.1, 0.3, 1, 3, and 10 mg/kg subcutaneously), U50488H (0.3, 1, 3, and 10 mg/kg intraperitoneally), or trimebutine (3, 10, 30, and 100 mg/kg intraperitoneally). The von Frey and CRD tests were performed respectively in the same animals 25 and 35 minutes after the injections. Each control group was injected with saline. Four experimental groups of 10 male rats were used. Rats were pretreated with capsaicin (and selected after noxious tests) and instilled twice daily for 3 days with 1 mL saline or butyrate solution (200 mmol/L). Mechanical lumbar hyperalgesia and pressure-induced behavioral response to CRD were determined on day 7 after the first enema for the same animals. Four experimental groups of 8 rats were used. All of the groups were instilled with 1 mL butyrate solution (200 mmol/L) twice daily for 3 days. Seven days after the beginning of butyrate instillations, they received an intravenous injection of saline, CGRP8–37 (20 μg/kg), or L733060 (1 and 3 mg/kg) as described by Gschossmann et al19Gschossmann J.M. Coutinho S.V. Miller J.C. Huebel K. Naliboff B. Wong H.C. Walsh J.H. Mayer E.A. Involvement of spinal calcitonin gene-related peptide in the development of acute visceral hyperalgesia in the rat.Neurogastroenterol Motil. 2001; 13: 229-236Crossref PubMed Scopus (45) Google Scholar and Rupniak et al,27Rupniak N.M. Carlson E. Boyce S. Webb J.K. Hill R.G. Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils.Pain. 1996; 67: 189-195Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar respectively, 10 minutes before the CRD test. The same injection protocol was used 10 minutes before von Frey testing on different animals. Results are expressed as mean ± SEM of raw data. Results of the CRD testing were analyzed using a one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test to compare several treatments or by a 2-way ANOVA followed by a Student-Newman-Keuls post-hoc test when several treatments were compared over the same time course. von Frey scores were analyzed using nonparametric Kruskal-Wallis ANOVA on ranks followed by a Dunn’s test when several treatments were compared over the same time course or by a Mann-Whitney rank sum test to compare 2 groups. Differences were considered significant at P < .05. The following agents were used in this study: acepromazine (Vetoquinol, Lure, France); ketamine (Panpharma, Luitre Fougères, France); n-sodium salt butyrate, TNBS, Evan’s blue, acetone, sodium sulfate, hexadecyltrimethylammonium bromide buffer, phosphate buffer, ibuprofen, naloxone, U50488H, MPO of human neutrophils, guaiacol, capsaicin, L733060, and CGRP8–37 (Sigma Aldrich, Lyon, France); morphine (Cooperation Pharmaceutique Française); bicinchoninic acid (Interchim, Paris, France); and trimebutine (Debridat; Pfizer, Paris, France). Butyrate induced a significant concentration-dependent decrease of thresholds to CRD (Figure 2A) and of von Frey scores (Figure 2C) in 100% of butyrate-instilled animals, while scores for saline-treated male rats remained stable throughout the experiment (50.6 ± 2.1 mm Hg and 45.7 ± 1.3 mN for CRD and von Frey scores, respectively). A significant decrease in CRD thresholds was observed for the lowest butyrate concentration (8 mmol/L) at days 3 and 6 after the start of butyrate enemas. The intensity and duration of these changes increased with the butyrate concentrations. For the 200-mmol/L butyrate concentration, selected for further investigations, the maximal decrease was obtained at day 6 (−26.1% ± 3.