Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus.

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Relationships between the results of anorectal investigations and symptom severity in patients with faecal incontinence.

PURPOSE: Anorectal dysfunction is the focus of diagnostic investigations for faecal incontinence. However, severity of incontinence and anorectal investigation results can be discordant. The aim of this study was to define the relationships between anorectal investigation results and incontinence severity to determine which measures, if any, were predictive of incontinence severity. METHODS: Patients presenting for investigation of faecal incontinence completed a symptom questionnaire, anorectal manometry, rectal sensation, pudendal nerve terminal motor latency, and endoanal ultrasound. Bivariate analyses were conducted between the Jorge-Wexner score and investigation results. Subgroup analyses were performed for gender and symptom subtypes (urge, passive, mixed). A multiple regression analysis was performed. RESULTS: Five hundred and thirty-eight patients were included. There were weak correlations between the Jorge-Wexner score and maximal squeeze pressure [r = - 0.24, 95%CI(- 0.31, - 0.16), p < 0.001], and resting pressure [r = - 0.18, (95%CI(- 0.26, - 0.10), p < 0.001]. In men only, there were significant associations between the Jorge-Wexner score and endoanal sonography [IAS defects: t(113) = - 2.26, p = 0.03, d = 0.58, 95%CI(- 4.38, - 0.29)] and rectal sensation (MTV: rs = - 0.24, 95%CI(- 0.41, - 0.06), p = 0.01). No substantial differences were observed in the urge/passive/mixed subgroup analyses. Multiple regression analysis included three variables: age (ß = 0.02, p = 0.17), maximal resting pressure (ß = - 0.01, p = 0.28), and maximal squeeze pressure (ß = - 0.01, p < 0.01). The variance in the Jorge-Wexner score accounted for by this model was < 10%, (R2 = 0.07, p = < 0.01, adjusted R2 = 0.06). CONCLUSION: Anorectal investigations cannot predict the severity of faecal incontinence. This may be due to limitations of diagnostic modalities, the heterogeneity of anorectal dysfunction in these patients, or contributing factors which are extrinsic to the anorectum.

Distribution, projections, and association with calbindin baskets of motor neurons, interneurons, and sensory neurons in guinea-pig distal colon.

Normal gut function relies on the activity of the enteric nervous system (ENS) found within the wall of the gastrointestinal tract. The structural and functional organization of the ENS has been extensively studied in the guinea pig small intestine, but less is known about colonic circuitry. Given that there are significant differences between these regions in function, observed motor patterns and pathology, it would be valuable to have a better understanding of the colonic ENS. Furthermore, disorders of colonic motor function, such as irritable bowel syndrome, are much more common. We have recently reported specialized basket-like structures, immunoreactive for calbindin, that likely underlie synaptic inputs to specific types of calretinin-immunoreactive neurons in the guinea-pig colon. Based on detailed immunohistochemical analysis, we postulated the recipient neurons may be excitatory motor neurons and ascending interneurons. In the present study, we combined retrograde tracing and immunohistochemistry to examine the projections of circular muscle motor neurons, myenteric interneurons, and putative sensory neurons. We focused on neurons with immunoreactivity for calbindin, calretinin and nitric oxide synthase and their relationship with calbindin baskets. Retrograde tracing using indocarbocyanine dye (DiI) revealed that many of the nerve cell bodies surrounded by calbindin baskets belong to motor neurons and ascending interneurons. Unique functional classes of myenteric neurons were identified based on morphology, neuronal markers and polarity of projection. We provide evidence for three groups of ascending motor neurons based on immunoreactivity and association with calbindin baskets, a finding that may have significant functional implications.

Morphological evidence for novel enteric neuronal circuitry in guinea pig distal colon.

The gastrointestinal (GI) tract is unique compared to all other internal organs; it is the only organ with its own nervous system and its own population of intrinsic sensory neurons, known as intrinsic primary afferent neurons (IPANs). How these IPANs form neuronal circuits with other functional classes of neurons in the enteric nervous system (ENS) is incompletely understood. We used a combination of light microscopy, immunohistochemistry and confocal microscopy to examine the topographical distribution of specific classes of neurons in the myenteric plexus of guinea-pig colon, including putative IPANs, with other classes of enteric neurons. These findings were based on immunoreactivity to the neuronal markers, calbindin, calretinin and nitric oxide synthase. We then correlated the varicose outputs formed by putative IPANs with subclasses of excitatory interneurons and motor neurons. We revealed that calbindin-immunoreactive varicosities form specialized structures resembling 'baskets' within the majority of myenteric ganglia, which were arranged in clusters around calretinin-immunoreactive neurons. These calbindin baskets directly arose from projections of putative IPANs and represent morphological evidence of preferential input from sensory neurons directly to a select group of calretinin neurons. Our findings uncovered that these neurons are likely to be ascending excitatory interneurons and excitatory motor neurons. Our study reveals for the first time in the colon, a novel enteric neural circuit, whereby calbindin-immunoreactive putative sensory neurons form specialized varicose structures that likely direct synaptic outputs to excitatory interneurons and motor neurons. This circuit likely forms the basis of polarized neuronal pathways underlying motility.

Discriminating movements of liquid and gas in the rabbit colon with impedance manometry.

BACKGROUND: High-resolution impedance manometry is a technique that is well established in esophageal motility studies for relating motor patterns to bolus flow. The use of this technique in the colon has not been established. METHODS: In isolated segments of rabbit proximal colon, we recorded motor patterns and the movement of liquid or gas boluses with a high-resolution impedance manometry catheter. These detected movements were compared to video recorded changes in gut diameter. Using the characteristic shapes of the admittance (inverse of impedance) and pressure signals associated with gas or liquid flow we developed a computational algorithm for the automated detection of these events. KEY RESULTS: Propagating contractions detected by video were also recorded by manometry and impedance. Neither pressure nor admittance signals alone could distinguish between liquid and gas transit, however the precise relationship between admittance and pressure signals during bolus flow could. Training our computational algorithm upon these characteristic shapes yielded a detection accuracy of 87.7% when compared to gas or liquid bolus events detected by manual analysis. CONCLUSIONS & INFERENCES: Characterizing the relationship between both admittance and pressure recorded with high-resolution impedance manometry can not only help in detecting luminal transit in real time, but also distinguishes between liquid and gaseous content. This technique holds promise for determining the propulsive nature of human colonic motor patterns.

Neurogenic and myogenic patterns of electrical activity in isolated intact mouse colon.

BACKGROUND: Relatively little is known about the electrical rhythmicity of the whole colon, where long neural pathways are preserved. METHODS: Smooth muscle electrical activity was recorded extracellularly from the serosa of isolated flat-sheet preparations consisting of the whole mouse colon (n=31). KEY RESULTS: Two distinct electrical patterns were observed. The first, long intense spike bursts, occurred every 349±256 seconds (0.2±0.2 cpm), firing action potentials for 31±11 seconds at 2.1±0.5 Hz. They were hexamethonium- and tetrodotoxin-sensitive, but persisted in nicardipine as 2 Hz electrical oscillations lacking action potentials. This pattern is called here neurogenic spike bursts. The second pattern, short spike bursts, occurred about every 30 seconds (2.0±0.6 cpm), with action potentials firing at about 1 Hz for 9 seconds (1.0±0.2 Hz, 9±4 seconds). Short spike bursts were hexamethonium- and tetrodotoxin-resistant but nicardipine-sensitive and thus called here myogenic spike bursts. Neurogenic spike bursts transiently delayed myogenic spike bursts, while blocking neurogenic activity enhanced myogenic spike burst durations. External stimuli significantly affected neurogenic but not myogenic spike bursts. Aboral electrical or mechanical stimuli evoked premature neurogenic spike bursts. Circumferential stretch significantly decreased intervals between neurogenic spike bursts. Lesioning the colon down to 10 mm segments significantly increased intervals or abolished neurogenic spike bursts, while myogenic spike bursts persisted. CONCLUSIONS & INFERENCES: Distinct neurogenic and myogenic electrical patterns were recorded from mouse colonic muscularis externa. Neurogenic spike bursts likely correlate with neurogenic colonic migrating motor complexes (CMMC) and are highly sensitive to mechanical stimuli. Myogenic spike bursts may correspond to slow myogenic contractions, whose duration can be modulated by enteric neural activity.

Colorectal cancer treatment and survival over three decades at four major public hospitals in South Australia: trends by age and in the elderly.

Data from registries at four major public hospitals in South Australia indicate increased 5-year disease-specific survivals for colorectal cancer from 48% to 63% between 1980-1986 and 2005-2010. For 80+ year olds, the increase was smaller, from 47% to 52%. Risk of case fatality halved overall, adjusting for age, gender, stage, differentiation and sub-site. Patients aged 80+ years had a lower risk reduction of about a third (hazards ratio: 0.69; 95% confidence limits, 0.52-0.92). Percentages having surgery and other specified treatments were lower for 80+ year olds than younger cases, although increases in treatment intensity occurred in this age range during 1980-2010, as seen in younger ages, in accordance with guidelines. The study illustrates the important feedback clinical registries can provide to clinicians on care patterns and outcomes in their hospital settings. Feedback can be the subject of local deliberations on how to achieve the best outcomes, including in the elderly by considering the best trade-offs between optimal cancer care and accommodations for co-morbidity and frailty. Clinical registry data can be used in comparative effectiveness research in local settings where there are sufficient case numbers.

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.

Colonic motor abnormalities in slow transit constipation defined by high resolution, fibre-optic manometry.

BACKGROUND: Slow transit constipation (STC) is associated with colonic motor abnormalities. The underlying cause(s) of the abnormalities remain poorly defined. In health, utilizing high resolution fiber-optic manometry, we have described a distal colonic propagating motor pattern with a slow wave frequency of 2-6 cycles per minute (cpm). A high calorie meal caused a rapid and significant increase in this activity, suggesting the intrinsic slow wave activity could be mediated by extrinsic neural input. Utilizing the same protocol our aim was to characterize colonic meal response STC patients. METHODS: A fiber-optic manometry catheter (72 sensors at 1 cm intervals) was colonoscopically placed with the tip clipped at the ascending or transverse colon, in 14 patients with scintigraphically confirmed STC. Manometric recordings were taken, for 2 h pre and post a 700 kCal meal. Data were compared to 12 healthy adults. KEY RESULTS: Prior to and/or after the meal the cyclic propagating motor pattern was identified in 13 of 14 patients. However, the meal, did not increase the cyclic motor pattern (preprandial 7.4 ± 7.6 vs postprandial 8.3 ± 4.5 per/2 h), this is in contrast to the dramatic increase observed in health (8.3 ± 13.3 vs 59.1 ± 89.0 per/2 h; p < 0.001). CONCLUSIONS & INFERENCES: In patients with STC a meal fails to induce the normal increase in the distal colonic cyclic propagating motor patterns. We propose that these data may indicate that the normal extrinsic parasympathetic inputs to the colon are attenuated in these patients.

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.

Prolonged pudendal nerve terminal motor latency is associated with decreased resting and squeeze pressures in the intact anal sphincter.

AIM: To determine the contribution of the pudendal nerve to the anal continence mechanism by determining the correlation between pudendal nerve terminal motor latency (PNTML) and resting and squeeze anal canal pressures. METHOD: In all, 1051 patients were investigated with anorectal physiology studies between January 1998 and July 2010. Of these, 213 patients had intact anal sphincters on endoanal ultrasound and had undergone PNTML testing and anal manometry with measurement of resting and squeeze pressures. The relationship between PNTML and mean resting and squeeze pressures was compared in these patients with an intact anal sphincter. Values were compared using a two-sample t test with equal variances. A P value of < 0.05 was considered significant. RESULTS: Of these patients 40.8% had normal PNTML bilaterally, 9.9% had slow PNTML bilaterally and 21.6% had a unilateral slow PNTML. Mean resting pressure was significantly reduced in patients with unilateral slow and bilateral slow PNTML compared with normal. The magnitude of the reduction was 28% and 19% respectively. Mean squeeze pressure was significantly reduced in patients with unilateral slow and bilateral slow PNTML compared with normal. The magnitude of the reduction was 18% and 23% respectively. CONCLUSION: In patients with an intact anal sphincter, either unilaterally or bilaterally prolonged PNTMLs are associated with significantly decreased resting and squeeze pressures. Our results suggest that both internal and external sphincter function is impaired with pudendal nerve injury. The inhibition of internal sphincter function may be due to damage of autonomic, principally sympathetic fibres carried in the pudendal nerve.

Ascending excitatory neural pathways modulate slow phasic myogenic contractions in the isolated human colon.

BACKGROUND: In animal models, enteric reflex pathways have potent effects on motor activity; their roles have been much less extensively studied in human gut. The aim of this study was to determine if ascending excitatory interneuronal pathways can modulate spontaneous phasic contractions in isolated preparations of human colonic circular muscle. METHODS: Human colonic preparations were cut into T shapes, with vertical bar of the 'T' pharmacologically isolated. Electrical stimulation and the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), were applied to the isolated region and circular muscle contractile activity was measured from the cross-bar of the T, more than 10 mm orally from the region of stimulation. KEY RESULTS: The predominant form of spontaneous muscle activity consisted of tetrodotoxin-resistant, large amplitude, slow phasic contractions (SPCs), occurring at average intervals of 124 ± 68 s. Addition of a high concentration of hexamethonium (1 mmol L(-1)) to the superfusing solution significantly increased the interval between SPCs to 278.1 ± 138.3 s (P < 0.005). Focal electrical stimulation more than 10 mm aboral to the muscle recording site advanced the onset of the next SPC, and this effect persisted in hexamethonium. However, the effect of electrical stimulation was blocked by tetrodotoxin (TTX, 1 µmol L(-1)). Application of the nicotinic agonist DMPP (1 mmol L(-1)) to the aboral chamber often stimulated a premature SPC (n = 4). CONCLUSIONS & INFERENCES: The major form of spontaneous contractility in preparations of human colonic circular muscle is SPCs, which are myogenic in origin. Activation of ascending excitatory neural pathways, which involve nicotinic receptors, can modulate the timing of SPCs and thus influence human colonic motility.

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.

The efficacy of treatment of patients with severe constipation or recurrent pseudo-obstruction with pyridostigmine.

OBJECTIVE: Disorders of colonic motility, such as severe constipation and pseudo-obstruction, remain difficult to treat. The pathophysiology of these conditions is not completely understood, but previous studies suggest a deficiency of cholinergic innervation and an imbalance in autonomic regulation of colonic motor function as contributing factors. Therefore, increasing the availability of acetylcholine in the bowel wall with a cholinesterase inhibitor, such as pyridostigmine, may improve symptoms. METHOD: We studied thirteen patients with severe constipation (slow transit type) or recurrent pseudo-obstruction. The six patients with slow transit constipation had mechanical obstruction and pelvic floor dysfunction excluded, and normal calibre colon and slow transit confirmed. These patients were offered pyridostigmine in an attempt to avoid surgery. The seven patients with pseudo-obstruction had dilated bowel on imaging, and mechanical obstruction was excluded. These patients received pyridostigmine when symptoms recurred, despite previous treatments. Pyridostigmine was initiated at 10 mg b.i.d. and increased if required. RESULTS: One of the six patients with slow transit constipation reported improvement of symptoms and had concurrently weaned anti-psychotic medications. Pyridostigmine was ceased in the remaining five patients due to lack of efficacy and/or side effects. Four patients proceeded to surgery for refractory symptoms. All seven patients with pseudo-obstruction had some improvement of symptoms with few side effects. Of these, two later had surgery for recurrent symptoms. CONCLUSION: In patients with slow transit constipation, treatment with pyridostigmine does not improve symptoms. However, it does improve symptoms in patients with recurrent pseudo-obstruction with few side effects, offering an extra treatment option for these patients.

Clinical trial: the impact of cyclooxygenase inhibitors on gastrointestinal recovery after major surgery - a randomized double blind controlled trial of celecoxib or diclofenac vs. placebo.

BACKGROUND: Ileus occurs after abdominal surgery and may be severe. Inhibition of prostaglandin release reduces post-operative ileus in a rat model. AIM: To determine whether prostaglandin inhibition by cyclooxygenase inhibitors, celecoxib or diclofenac, could enhance gastrointestinal recovery and reduce post-operative ileus in humans. METHODS: Two hundred and ten patients undergoing elective major abdominal surgery were randomized to receive twice daily placebo (n = 67), celecoxib (100 mg, n = 74) or diclofenac (50 mg, n = 69), preoperatively and continuing for up to 7 days. Primary outcomes were hallmarks of gut recovery. Secondary outcomes were paralytic ileus, pain and complications. RESULTS: There was no clinically significant difference between the groups for restoration of bowel function. There was a significant reduction in paralytic ileus in the celecoxib-treated group (n = 1, 1%) compared with diclofenac (n = 7, 10%) and placebo (n = 9, 13%); P = 0.025, RR 0.20, CI 0.01-0.77. Pain scores, analgesia, transfusion requirements and adverse event rates were similar between study groups. CONCLUSIONS: Perioperative low dose celecoxib, but not diclofenac, markedly reduced the development of paralytic ileus following major abdominal surgery, but did not accelerate early recovery of bowel function. This was independent of narcotic use and had no increase in post-operative complications.

Colovesical fistula following an open preperitoneal "Kugel" mesh repair of an inguinal hernia.

Erosion of the "Kugel" mesh into intraperitoneal organs has not been previously reported in the medical literature. We report such an occurrence in a 54-year-old male, 4 years following a "Kugel" preperitoneal repair of a left-sided inguinal hernia. The patient presented with septicaemia, pneumaturia and left iliac fossa pain. His computed tomography (CT) scan indicated the presence of gas in the bladder and a thickened loop of sigmoid colon attached to the region of the dome of the bladder. Colonoscopy showed some scattered diverticula in the sigmoid colon but no tumour. On surgical exploration, the "Kugel" mesh was found to erode the sigmoid colon and the bladder wall, leading to a colovesical fistula. An anterior resection of the rectum with removal of the mesh with closure of the bladder wall defect was performed.

Regional variation in the neurochemical coding of the myenteric plexus of the human colon and changes in patients with slow transit constipation.

There are differences in the structure and function between regions of the colon. In patients with slow transit constipation the activity of all regions is markedly slowed. Counts of colonic neurones in slow transit constipation have been semiquantitative and led to varying results. We have applied new methods of quantification of markers in whole mounts of the colonic myenteric plexus to compare density of innervation between regions and between normal patients and those undergoing resection for severe constipation. Whole mounts of colonic myenteric plexus were made from specimens removed for cancer treatment (controls) and cases of severe constipation. All neurones were labelled by anti-human neuronal protein antibodies. Neurones synthesizing acetyl choline were labelled for choline acetyltransferase (ChAT) and those for nitric oxide by antisera to nitric oxide synthase (NOS). Four populations of neurones were distinguished and quantified according to the two selective markers, ChAT and NOS. In the normal major populations were NOS alone (51% of ascending colon neurones and 44% of descending colon neurones) and ChAT alone (41% ascending colon, 48% descending colon). Nitric oxide synthase/ChAT and NOS-/ChAT-comprised only small populations. In all regions in severe constipation, the percentage of NOS-only colonic myenteric neurones was raised (54% ascending colon, 49% descending colon) and ChAT only was reduced (36% ascending colon, 42% descending colon). The other populations were not changed. Accurate quantification of neuronal populations in whole mounts of human colon reveals inter-regional differences in innervation and marked changes in innervation in cases of very severe constipation.