High-resolution colonic motility recordings in vivo compared with ex vivo recordings after colectomy, in patients with slow transit constipation.

BACKGROUND: The pathogenesis of slow transit constipation (STC) remains poorly understood, with intrinsic and extrinsic abnormalities implicated. Here, we present high-resolution colonic manometry recordings from four STC patients recorded before total colectomy, and subsequently, ex vivo, after excision. METHODS: In four female, treatment-resistant STC patients (median age 35.5 years), a fiber-optic manometry catheter (72 sensors spaced at 1 cm intervals) was placed with the aid of a colonoscope, to the mid-transverse colon. Colonic manometry was recorded 2 h before and after a meal. After the colectomy, ex vivo colonic manometry was recorded in an organ bath. Ex vivo recordings were also made from colons from 4 patients (2 male; median age 67.5 years) undergoing anterior resection for nonobstructive carcinoma ('control' tissue). KEY RESULTS: A large increase in 'short single propagating contractions' was recorded in STC colon ex vivo compared to in vivo (ex vivo 61.3 ± 32.7 vs in vivo 2.5 ± 5/h). In STC patients, in vivo, the dominant frequency of contractile activity was 2-3 cycle per minute (cpm), whereas 1-cpm short-single propagating contractions dominated ex vivo. This same 1-cpm frequency was also dominant in control colons ex vivo. CONCLUSIONS & INFERENCES: In comparison to control adults, the colon of STC patients demonstrates significantly less propagating motor activity. However, once the STC colon is excised from the body it demonstrates a regular and similar frequency of propagating activity to control tissue. This paper provides interesting insights into the control of colonic motor patterns.

Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability.

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1ß and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1ß and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10(-5)M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10(-4)M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10µg/ml) increased permeability over 24-48h. These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

The neurochemical changes in the innervation of human colonic mesenteric and submucosal blood vessels in ulcerative colitis and Crohn's disease.

BACKGROUND: Neurogenic inflammation involves vasodilation, oedema and sensory nerve hypersensitivity. Extrinsic sensory nerves to the intestinal wall mediate these effects and functional subsets of these extrinsic nerves can be characterized by immunohistochemical profiles. In this study such profiles were examined in samples from patients with inflammatory bowel disease (IBD), in particular ulcerative colitis (UC) and Crohn's disease (CD). METHODS: Healthy margins from cancer patients were compared to specimens from IBD patients. All nerve fibres were labelled by PGP 9.5. Double and triple labelling with TH, NPY, SP, SOM, NOS, VIP, VAChT, CGRP, TRPv1 were performed. Perivascular nerve fibres in the mesentery, and submucosa, were examined. The percentage of all labelled nerve fibres was calculated with a transect method. KEY RESULTS: Total number of varicosities on mesenteric vessels increased in IBD but decreased around submucosal vessels. The percentage of nerve fibres around submucosal arteries labelled by SP increased from 11% in controls to 20% (UC) and 24% (CD) and mesenteric artery nerve fibres were unchanged. Nerve fibres labelled by SOM were markedly reduced surrounding submucosal arteries, from 22% to 1% (UC) and 2% (CD), but not perivascular mesenteric nerve fibres. 87 to 93% of SP immunoreactive nerve fibres were also reactive for TRvP1. TRPv1 labelling without SP was 12%in controls and increased to 40% in CD submucosal specimens. CONCLUSIONS & INFERENCES: There is an increase in SP and TRPv1, and a reduction in SOM immunoreactive nerve fibres in IBD. Changes in the perivascular functional nerve subclasses may underlie the hyperaemia, and ulceration, characteristic of IBD. Furthermore, pain may relate to underlying neural changes.

Interleukin 17A evoked mucosal damage is attenuated by cannabidiol and anandamide in a human colonic explant model.

Interleukin 17A (IL-17A) is a cytokine linked to inflammatory bowel disease. We investigated IL-17A expression in human colonic mucosa, whether IL-17A can elicit colonic mucosal damage in a human explant model and modulate gastrointestinal epithelial permeability in cell culture. We also tested if select cannabinoid ligands, shown to be protective in colitis models could attenuate damage caused by IL-17A. In addition, the ability of pro-inflammatory cytokines TNF-α and IL-1ß to modulate levels of IL-17A in the explant colitis model was also explored. IL-17A incubation caused significant mucosal epithelial and crypt damage which were attenuated following hydrocortisone treatment, and also reduced following anandamide or cannabidiol incubation. IL-17A-evoked mucosal damage was also associated with an increase in matrix metalloprotease activity. However, IL-17A did not induce any significant changes in epithelial permeability in confluent Caco-2 cell monolayers over a 48h incubation period. IL-17A was located predominantly in human mucosal epithelium together with IL-17C, but both IL-17A and IL-17C were also expressed in the lamina propria and submucosa. Incubation of human colonic mucosal tissue or Caco-2 cells with pro-inflammatory cytokines TNF-α and IL-1ß however did not alter IL-17A expression. These results indicate IL-17A has a widespread distribution in the human colon and the capacity to elicit mucosal damage which can be attenuated by cannabinoid ligands.

Peristalsis and propulsion of colonic content can occur after blockade of major neuroneuronal and neuromuscular transmitters in isolated guinea pig colon.

We recently identified hexamethonium-resistant peristalsis in the guinea pig colon. We showed that, following acute blockade of nicotinic receptors, peristalsis recovers, leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question: what mechanisms underlie hexamethonium-resistant peristalsis? We investigated whether blockade of the major receptors that underlie excitatory neuromuscular transmission is required for hexamethonium-resistant peristalsis. Video imaging of colonic wall movements was used to make spatiotemporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea pig distal colon was studied in hexamethonium, atropine, ω-conotoxin (GVIA), ibodutant (MEN-15596), and TTX. Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, ω-conotoxin abolished peristalsis in some preparations and reduced the velocity of propagation in all remaining specimens. Peristalsis could still occur in some animals in the presence of hexamethonium + atropine + ibodutant + ω-conotoxin. Peristalsis never occurred in the presence of TTX. The major finding of the current study is the unexpected observation that peristalsis can occur after blockade of the major excitatory neuroneuronal and neuromuscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/neurotransmitter(s) that generate(s) peristalsis and facilitate(s) natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuroneuronal and neuromuscular junction remains to be identified.

By what mechanism does ondansetron inhibit colonic migrating motor complexes: does it require endogenous serotonin in the gut wall?

BACKGROUND: 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5-HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT-depleted preparation. METHODS: Mice were injected with reserpine, 24 h prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT. KEY RESULTS: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was not different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally or significantly more effective at inhibiting CMMC frequency compared with control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs. CONCLUSIONS & INFERENCES: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus or 5-HT in myenteric neurons.

Is serotonin in enteric nerves required for distension-evoked peristalsis and propulsion of content in guinea-pig distal colon?

Recent studies have shown genetic deletion of the gene that synthesizes 5-HT in enteric neurons (tryptophan hydroxylase-2, Tph-2) leads to a reduction in intestinal transit. However, deletion of the Tph-2 gene also leads to major developmental changes in enteric ganglia, which could also explain changes in intestinal transit. We sought to investigate this further by acutely depleting serotonin from enteric neurons over a 24-h period, without the confounding influences induced by genetic manipulation. Guinea-pigs were injected with reserpine 24h prior to euthanasia. Video-imaging and spatio-temporal mapping was used to record peristalsis evoked by natural fecal pellets, or slow infusion of intraluminal fluid. Immunohistochemical staining for 5-HT was used to detect the presence of serotonin in the myenteric plexus. It was found that endogenous 5-HT was always detected in myenteric ganglia of control animals, but never in guinea-pigs treated with reserpine. Interestingly, peristalsis was still reliably evoked by either intraluminal fluid, or fecal pellets in reserpine-treated animals that also had their entire mucosa and submucosal plexus removed. In these 5-HT depleted animals, there was no change in the frequency of peristalsis or force generated during peristalsis. In control animals, or reserpine treated animals, high concentrations (up to 10 µM) of ondansetron and SDZ-205-557, or granisetron and SDZ-205-557 had no effect on peristalsis. In summary, acute depletion of serotonin from enteric nerves does not prevent distension-evoked peristalsis, nor propulsion of luminal content. Also, we found no evidence that 5-HT3 and 5-HT4 receptor activation is required for peristalsis, or propulsion of contents to occur. Taken together, we suggest that the intrinsic mechanisms that generate peristalsis and entrain propagation along the isolated guinea-pig distal colon are independent of 5-HT in enteric neurons or the mucosa, and do not require the activation of 5-HT3 or 5-HT4 receptors.