Rectal instillation of butyrate provides a novel clinically relevant model of noninflammatory colonic hypersensitivity in rats

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 kappa 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.     

Visceral hyperalgesia and intestinal dysmotility in a mouse model of postinfective gut dysfunction

BACKGROUND & AIMS: We established the concept that transient enteric infection may lead to persistent gut dysfunction, evident in vitro, in nematode-infected mice. The present study determined whether gut dysfunction in this model involves motor and sensory changes reminiscent of changes found in patients with postinfective irritable bowel syndrome (PI-IBS) and investigated underlying mechanisms. METHODS: Mice infected up to 70 days previously with Trichinella spiralis (Tsp) underwent videofluoroscopy with image analysis to assess upper gastrointestinal motility. Pseudoaffective responses to colorectal distention (CRD) were assessed using a barostat and validated by single fiber recordings from spinal nerves during CRD. Tissues were examined at different time points for histology, immunohistochemistry, and cytokine analysis. Some mice received dexamethasone intraperitoneally on days 23-25 PI or Tsp antigen orally on days 29, 43, and 57 PI. RESULTS: From day 28 PI, no discernible inflammation was present in the gut. Frequency and propagation velocity of intestinal contractions decreased, and retroperistalsis increased at days 28 to 42 PI. CRD induced an allodynic and hyperalgesic response in PI mice, which was accompanied by increased single unit discharge. Gavage of Tsp antigen induced T-cell responses and sustained gut dysfunction for 70 days PI. Administration of dexamethasone postinfection normalized dysmotility and visceral hyperalgesia. CONCLUSIONS: Long-lasting gut dysmotility and hyperalgesia develop in mice after transient intestinal inflammation. These changes are maintained by luminal exposure to antigen and reversed by corticosteroid treatment. The findings prompt consideration of this as a model of PI-IBS.     

Trypsin mediates nociception via the proteinase-activated receptor 2: a potentially novel role in pancreatic pain

BACKGROUND & AIMS: The pathogenesis of pain in pancreatitis remains poorly understood. We hypothesized that trypsin, a key inflammatory mediator in this condition, can also activate nociceptive neurons via the proteinase-activated receptor 2. METHODS: Double immunohistochemical staining of T8 to T12 dorsal root ganglia sections was performed with antibodies against proteinase-activated receptor 2 and vanilloid receptor 1, a marker for primary nociceptive neurons. In vivo nociceptive activity was measured by FOS immunoreactivity in thoracic spinal dorsal horn segments after intrapancreatic administration of proteinase-activated receptor 2 agonists. Pain behavior was assessed by visceromotor reflex activity in response to noxious stimulation of the pancreas with proteinase-activated receptor 2 agonists. RESULTS: Proteinase-activated receptor 2 was expressed by virtually all nociceptive neurons in thoracic dorsal root ganglia. Intraductal trypsin, in subinflammatory concentrations, activated spinal dorsal horn neurons in a dose-dependent manner, as measured by FOS expression. Both trypsin and a proteinase-activated receptor 2-specific peptide agonist induced a behavioral pain response when infused into the pancreatic duct of awake rats. Preinfusion of the pancreatic duct with proteinase-activated receptor 2-specific activating peptide desensitized the response to trypsin. CONCLUSIONS: Our findings suggest a novel proteinase-activated receptor 2-mediated role for trypsin in the pathogenesis of pancreatic pain and one that is independent of its inflammatory effect.     

Nerve growth factor mediates alterations of colonic sensitivity and mucosal barrier induced by neonatal stress in rats

BACKGROUND & AIMS: Maternal deprivation (MD) increases nerve growth factor (NGF) expression and colonic mast cell density and alters visceral sensitivity. This study aimed to establish whether NGF overexpression induced by neonatal stress is involved in altered visceral sensitivity and gut mucosal integrity in adult rats. METHODS: Male Wistar rat pups were either submitted to MD and treated with anti-NGF antibodies or left with their dam and treated daily with NGF. All rats were tested 10 weeks later for visceral sensitivity and 12 weeks later for gut permeability, myeloperoxidase activity, and mast cell numbers. Colonic NGF and NGF receptor expression were determined at 14 days and 12 weeks of age. To determine the involvement of colonic NGF overexpression and mast cell hyperplasia in visceral hyperalgesia induced by MD, neonatally deprived adult rats received anti-NGF antibodies or doxantrazole. RESULTS: MD increased visceral sensitivity to rectal distention, gut permeability, colonic myeloperoxidase activity, and mast cell density, and anti-NGF antibodies abolished these effects. Neonatal daily treatment with NGF mimicked the alterations induced by MD on both rectal sensitivity and mucosal barrier. In deprived compared with nondeprived rats, colonic NGF immunostaining and NGF messenger RNA were increased at 14 days and 12 weeks. Overexpression of NGF receptor messenger RNA, present at 14 days, was not observed later. Moreover, adult deprived rats treated with doxantrazole or anti-NGF antibodies exhibited normal gut permeability and visceral sensitivity to rectal distention. CONCLUSIONS: These data indicate that NGF triggers and maintains long-term alterations of visceral sensitivity and gut mucosal integrity induced by MD.     

慢性内脏高敏性大鼠结肠内P物质及其NK1受体表达的改变

目的　研究慢性应激刺激所致内脏高敏性大鼠和正常大鼠结肠内P物质 (substanup)及其NK1受体表达的改变 ,探讨内脏高敏感性产生的可能机制。方法　 40只新生SD大鼠 ,随机分为 2组 :慢性应激模型组、正常对照组 ,每组 2 0只。模型组大鼠在出生后第 8天到 2 1天 ,每天接受结直肠扩张刺激 ;分别在第 8周、12周观察大鼠的腹部回缩反射 (AWR)和玻璃小球排出情况 ,进行肠道敏感性评估 :采用RT PCR方法检测二组大鼠结肠组织内P物质及其NK1受体mRNA的表达 ,并运用免疫组化SABC法观察NK1受体的分布。结果　模型组较正常对照组大鼠腹部收缩阈值明显降低 (P <0 .0 1) ,玻璃小球排出时间缩短 (P <0 .0 1) ;P物质及其NKl受体mRNA的表达明显增加 (P <0 .0 1) ,NK1受体免疫反应阳性产物显著浓密 ,以平滑肌层和黏膜层为著。结论　SP及其NK1受体表达增加在大鼠肠道高敏感性、动力紊乱的产生机制中可能起重要作用     

The vanilloid receptor initiates and maintains colonic hypersensitivity induced by neonatal colon irritation in rats

BACKGROUND & AIMS: Robust chemical or mechanical irritation of the colon of neonatal rats leads to chronic visceral hypersensitivity. The clinical and physiologic relevance of such noxious stimulation in the context of human irritable bowel syndrome is questionable. The aims of this study were to determine whether mild chemical irritation of the colon of neonatal rats produced persistent changes in visceral sensitivity and to evaluate the role of transient receptor potential vanilloid 1 (TRPV1) in the initiation and maintenance of visceral hypersensitivity. METHODS: Ten-day-old rat pups received an intracolonic infusion of 0.5% acetic acid in saline. TRPV1 inhibitors were administered 30 minutes before acetic acid sensitization. Sensitivity of the colon to balloon distention (CRD) in adults was measured by grading their abdominal withdrawal reflex and electromyographic responses. In adult rats, TRPV1 antagonist was injected intraperitoneally 30 minutes before CRD. RESULTS: Neonatal acetic acid treatment resulted in higher sensitivity to CRD in adult rats compared with controls in the absence of histopathologic signs of inflammation. Treatment of colons of adult rats with acetic acid did not produce persistent sensitization. Antagonism of the TRPV1 before neonatal administration of acetic acid and after established visceral hypersensitivity attenuated sensitivity to CRD. TRPV1 expression was increased in dorsal root ganglia-containing colon afferent neurons. CONCLUSIONS: We have described a new model for persistent colonic sensory dysfunction following a transient noxious stimulus in the neonatal period and a potentially important role for TRPV1 in initiation and maintenance of persistent visceral hypersensitivity.     

The role of leukotriene B4 in Clostridium difficile toxin A-induced ileitis in rats

BACKGROUND & AIMS: Clostridium difficile toxin A is a potent intestinal inflammatory agent that has been shown to act at least partially by neurogenic mechanisms involving activation of the transient receptor potential vanilloid 1 (TRPV1) (capsaicin) receptor. We tested the hypothesis that leukotriene B4 (LTB4) mediates the effects of toxin A via activation of the TRPV1 receptor. METHODS: Isolated rat ileal segments were pretreated with pharmacologic agents before intraluminal injection of toxin A or LTB4. After 3 hours, the treated segments were removed and inflammation was assessed by luminal fluid accumulation, myeloperoxidase activity, and histology. RESULTS: LTB4 caused ileitis similar to that caused by toxin A and antagonism of TRPV1 receptors but not LTB4 receptors inhibited LTB4-induced inflammation. LTB4 also stimulated TRPV1-mediated substance P release and pretreatment with a specific substance P-receptor antagonist blocked LTB4-induced substance P action and ileitis. Inhibition of the LTB4 biosynthetic enzyme 5-lipoxygenase inhibited toxin A-induced increases in ileal LTB4 levels and toxin A- but not LTB4-induced ileitis. CONCLUSIONS: LTB4 mediates the inflammatory effects of toxin A via activation of TRPV1 receptors.     

Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles

BACKGROUND & AIMS: The aim of this study was to compare the response of symptoms and cytokine ratios in irritable bowel syndrome (IBS) with ingestion of probiotic preparations containing a lactobacillus or bifidobacterium strain. METHODS: Seventy-seven subjects with IBS were randomized to receive either Lactobacillus salivarius UCC4331 or Bifidobacterium infantis 35624, each in a dose of 1 x 10 10 live bacterial cells in a malted milk drink, or the malted milk drink alone as placebo for 8 weeks. The cardinal symptoms of IBS were recorded on a daily basis and assessed each week. Quality of life assessment, stool microbiologic studies, and blood sampling for estimation of peripheral blood mononuclear cell release of the cytokines interleukin (IL)-10 and IL-12 were performed at the beginning and at the end of the treatment phase. RESULTS: For all symptoms, with the exception of bowel movement frequency and consistency, those randomized to B infantis 35624 experienced a greater reduction in symptom scores; composite and individual scores for abdominal pain/discomfort, bloating/distention, and bowel movement difficulty were significantly lower than for placebo for those randomized to B infantis 35624 for most weeks of the treatment phase. At baseline, patients with IBS demonstrated an abnormal IL-10/IL-12 ratio, indicative of a proinflammatory, Th-1 state. This ratio was normalized by B infantis 35624 feeding alone. CONCLUSIONS: B infantis 35624 alleviates symptoms in IBS; this symptomatic response was associated with normalization of the ratio of an anti-inflammatory to a proinflammatory cytokine, suggesting an immune-modulating role for this organism, in this disorder.     

Oral baclofen reduces visceral pain-related pseudo-affective responses to colorectal distension in rats: Relation between plasma exposure and efficacy

Abstract

Objective. We previously showed that activation of GABA_B receptors by intravenous baclofen reduces pseudo-affective responses to colorectal distension in rats. Here we evaluate the potential clinical significance of these observations. Material and Methods. Clinically relevant colorectal distension protocols were used to assess the effects of oral baclofen on visceromotor and autonomic cardiovascular responses in conscious rats. Plasma levels of baclofen were monitored to provide clinical relevance to the doses used. Conscious female Sprague–Dawley rats were subjected to repeated noxious colorectal distension (12 × 80 mmHg), ascending-phasic colorectal distension (10–80 mmHg, 10 mmHg increments) or ramp colorectal distension (10 min ramp at 8 mmHg/min). Visceromotor and cardiovascular responses (mean arterial blood pressure and heart rate) were monitored. Pain-related response thresholds were assessed using ascending-phasic and ramp colorectal distension. Results. Baclofen (1–10 μmol/kg, p.o.) reduced the visceromotor response to colorectal distension, reaching a 40% maximal inhibition (p < 0.05). The highest dose (10 μmol/kg, p.o.) also inhibited pain-related cardiovascular responses in telemetrized rats (50–55% reduction in colorectal distension-evoked hypertensive and tachycardic responses; p < 0.05). Similar thresholds for pain-related visceromotor responses were determined during ramp or ascending-phasic colorectal distension (34.1 ± 1.9 and 31.7 ± 3.2 mmHg, respectively). Baclofen (10 μmol/kg, p.o.) increased thresholds to 71.1 ± 3.7 and 77.5 ± 1.8 mmHg during ramp and ascending-phasic colorectal distension, respectively (p < 0.001). Plasma levels of baclofen were 3.3 ± 0.2 μmol/l at 90 min post-dosing, corresponding to the end of the colorectal distension procedure. Conclusions. Oral baclofen, at plasma levels similar to those reported safe and within a therapeutic range in humans, produced significant visceral anti-nociceptive effects in rats.     

A model of neural cross-talk and irritation in the pelvis: implications for the overlap of chronic pelvic pain disorders

BACKGROUND & AIMS: Irritable bowel syndrome, interstitial cystitis, and other chronic pelvic pain (CPP) disorders often occur concomitantly. Neural cross-talk may play a role in the overlap of CPP disorders via the convergence of pelvic afferents. We investigated the hypothesis that afferent irritation of one pelvic organ may adversely influence and sensitize another via neural interactions. METHODS: We measured pelvic organ smooth muscle and striated muscle reflexes during micturition and colorectal distention (CRD) in urethane-anesthetized rats. The effects of acute cystitis on distal colonic sensory thresholds to CRD and the effects of acute colonic irritation on micturition parameters were assessed. RESULTS: External urethral sphincter (EUS) electromyography (EMG) was typical for the rat, with phasic firing during micturition. External anal sphincter EMG also showed phasic firing during micturition in synchrony with EUS activity but, in addition, showed both tonic bursts and phasic firing independent of EUS activity. Before bladder irritation, graded CRDs to 40 cm H2O produced no notable changes in abdominal wall EMG activity. Following acute bladder irritation, dramatic increases in abdominal wall EMG activity in response to CRD were observed at much lower distention pressures, indicating colonic afferent sensitization. Analogously, following acute colonic irritation, bladder contraction frequency increased 66%, suggesting sensitization of lower urinary tract afferents. CONCLUSIONS: We report compelling evidence of bidirectional cross-sensitization of the colon and lower urinary tract in a novel experimental model. This cross-sensitization may account for the substantial overlap of CPP disorders; however, further studies are needed to fully characterize these pathways.     

Alterations in the brain-gut axis underlying visceral chemosensitivity in Nippostrongylus brasiliensis-infected mice

BACKGROUND & AIMS: Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. METHODS: Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3-4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 micromol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. RESULTS: In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. CONCLUSIONS: Alterations in the intestinal brain-gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.     

High-sensitivity C-reactive protein and atherosclerotic disease: From improved risk prediction to risk-guided therapy

There is compelling experimental and clinical evidence suggesting a crucial role for inflammation in the initiation and also the progression of atherosclerosis. Numerous biomarkers involved at various levels of the inflammation cascade have been shown to be associated with adverse cardiovascular outcomes. Yet, to date, it is not clear whether inflammation simply accompanies the atherosclerotic process or represents a major driver. Among all blood biomarkers, C-reactive protein (CRP), the classical acute phase reactant that can be measured with high-sensitivity (hs) assays seems to be the most promising candidate. It has already found its way into the guidelines in primary prevention. Hs-CRP can also be used to identify a high-risk group for recurrent events in patients with manifest atherosclerosis. Several post hoc analyses of large-scale randomized clinical trials testing various statins have indicated that, besides low density lipoprotein (LDL) cholesterol, hs-CRP levels might also further aid in tailoring statin treatment. The large JUPITER trial has prospectively confirmed these findings in primary prevention in patients with elevated hs-CRP but normal LDL cholesterol levels. Still, statin therapy is not a specific anti-inflammatory regime acting on the inflammation cascade. Thus, to directly test the inflammation hypothesis, a novel, more proximally located cytokine-based approach is needed. Canakinumab, a fully human monoclonal antibody against interleukin-1β, might represent a promising compound in this regard and provide a proof of concept. If successful, this may become a novel strategy to treat high-risk patients with stable atherosclerotic disease to prevent recurrent events on top of standard medical care.