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Aims Exclusion of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) from the diet is effective in alleviating symptoms of irritable bowel syndrome (IBS) in adults. Rapid-transit constipation (RTC) is a recently discovered subset of chronic constipation and has been linked to food intolerance. The aim of this study was to audit the effect of specific FODMAP elimination diets in children with RTC. Methods This was an audit of children presenting to a tertiary children's hospital surgeon with refractory chronic constipation who had rapid transit in the proximal colon on nuclear imaging; had hydrogen/methane breath tests for fructose, lactose, and/or sorbitol intolerance; and were advised to exclude positive sugar under clinical supervision. Patients filled in a questionnaire rating severity of constipation, abdominal pain, and pain on defecation with a visual analogue scale (VAS, 0 = none, 10 = high) and stool consistency for 6 months before and after dietary exclusion. Results In responses from 29 children (5-15 years, 21 males), 70% eliminated fructose, and 40% eliminated lactose. There was a significant reduction in the severity of constipation (VAS mean ± SEM, pre 5.8 ± 0.5 vs post 3.3 ± 0.6, P

Background and Aim: Polyethylene glycol (PEG) is the gold standard for fecal disimpaction in constipation. A regimen of PEG combined with the stimulant laxative sodium picosulphate (SPS) produced fecal disimpaction in chronically constipated children in the community, but it is unknown if it is effective for more severe constipation. To determine the stool output and effect of a combined PEG and SPS regimen on fecaloma in children with severe constipation and impaction. Methods: Children with symptoms for a duration of >= 2 years, a palpable fecaloma, and enlarged rectum on X-ray (rectal: pelvic ratio > 0.6) were recruited from a tertiary hospital. Daily diaries recorded laxative dose, stool frequency, volume, and consistency (Bristol stool scale, BSS). Abdominal X-rays were taken on day 1 and day 8, and stool loading was assessed using the Leech score. Laxative doses were based on the child's age. The dose of PEG with electrolytes taken was 2-8 sachets (14.7 g/sachet) on days 1-2, reducing to 2-6 sachets on day 3. The SPS dose was 15-20 drops on days 2-3. Results: Eighty-nine children (4-18 years) produced a large volume of soft stool (median/inter-quartile-range: 2.2/1.6-3.1 L) over 7 days. Stool volume on X-rays decreased significantly in the colon (P < 0.001). Fecalomas resolved in 40 of 89 children, while 49 needed a second high dose. Rectal: pelvic ratios did not change. Conclusions: A combined high dose of PEG and SPS on days 1 and 2 was effective in removing the fecaloma in half of the children. Administering high doses for a longer period should be tested to provide outpatient disimpaction for severe fecalomas. Rectums remained flaccid after emptying.

Aim Constipation is a common cause of admission to hospital for disimpaction, as oral laxatives are often inadequate. High-dose oral laxative protocols are used for complete bowel clearance prior to colonoscopy, but have not been reported for treating faecal impaction. The aim of this study was to assess the effectiveness of a high-dose oral protocol using polyethylene glycol with electrolytes (PEG + E) (Movicol Rx) combined with sodium picosulphate (SP) (Dulcolax SP Rx) in faecal impaction in children presenting to a suburban clinic. Methods Forty-four children presented with acute/chronic faecal impaction were given six to eight sachets of PEG + E were given on day 1, with decreasing doses on subsequent 3 days, while 15–20 SP drops were given on days 2 and 3. Compliance with medication was achieved using a simple method of motivation, with the child drinking the laxatives in a race. On day 4, PEG + E was reduced to one sachet and SP to 10 drops as an ongoing maintenance dose. Defecation, soiling, diet and water intake was monitored daily for 7 days in a diary. Results Forty-four children (aged 2–17 years) seen over 8 months were reviewed retrospectively. Children began defecating within 10–12 h reaching a maximum volume of stool/day (four cups) on day 2. All patients were disimpacted successfully and in the week following disimpaction there was no reported faecal soiling or complications. Conclusions A high-dose oral protocol combining PEG + E sachets and SP drops successfully and safely disimpacted a cohort of children with acute/chronic constipation presenting to a suburban continence clinic. This protocol appears to be useful to control faecal disimpaction in an outpatient setting, thereby avoiding hospital admission.

Aim Constipation is common, with severe symptoms requiring hospitalisation. Constipation can be a primary (present at admission and requires treatment or investigation) or principal (first listed) diagnosis for hospitalisation. In the USA, constipation is the second most common ambulatory care digestive diagnosis with total costs >US$1.7 billion/year. Incidence of hospitalisation for constipation in children peaks at toilet-training age. This study determined the burden of paediatric constipation to hospital care in Victoria, Australia. Method The Victorian Admitted Episodes Dataset was analysed retrospectively, examining hospital admissions with a primary diagnosis of constipation in the 7-year period 2002/2003 to 2008/2009. Results For children, constipation was recorded as a primary diagnosis in 8688 admissions (3.6/1000 of population). In-hospital prevalence was ∼1.0%. Mean length of stay was 4.4 days (median 1.0, range 0–993, standard deviation 16.7). There were 1121 readmissions in 668 children. Average treatment cost was A$4235/admission (median A$1461, range A$0–$278 816), with annual costs of ∼A$5 505 500. Children in the highest socio-economic area had ∼50% fewer admissions (P < 0.0001). Predictors of readmission included age 10–18, male gender, rural residence, severe socio-economic disadvantage, public hospital, planned admission, longer length of stay and association with other medical conditions. Conclusions This study identified that constipation in children is a significant cost burden in Victoria (costing public hospitals ∼A$5.5 million/year). Hospitalisation in Victoria is 10-fold higher than in the USA with 10% readmissions within a month. We conclude that strategies aimed at reducing hospitalisation for constipation could result in significant savings for the paediatric public health system in Victoria, Australia.

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5–10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

Congenital and acquired undescended testes are two distinct entities. Current management is surgery in the first 6-12 months of life for congenital undescended testes. Current management of acquired undescended testes is surgery at the time of diagnosis. Accurate diagnoses and expedient management are imperative in this condition to minimize the long-term sequelae of infertility and testicular cancer.

Comparisons between developmental studies rely on embryonic staging systems. It is important for comparison of molecular, immunohistochemical, and physiological studies of the developing chick intestine that the developmental stage of embryos is reliably determined. Good staging systems exist for the external features of the chick embryo but not for development of internal organs. To facilitate precise comparisons of chick embryo intestine development, we prepared an intestinal staging system. Embryos were fixed, other tissues dissected away, and the intestine and associated organs were then drawn to scale using a camera lucida. This produced black-and-white drawings with features of the gut clearly visible. The detailed drawings of intestine from chick embryos aged 2.5 to 10 days were correlated with age of embryos and developmental stages described in three common staging systems, Hamilton and Hamburger, Thompson and Fitzharris, and Allan and Newgreen. Descriptions of key changes in gut morphology and position are given for each stage. This staging of chick gut development will allow future studies to quote and compare development of the gut rather than external features or incubation time. This will allow much more precise reporting and comparisons in developmental studies of cell migration and gene expression.

Background: Chronic constipation in children may have organic or behavioral causes. The purpose of the present study was to investigate the effect of treatment with transcutaneous electrical stimulation (using interferential current) in children with chronic treatment-resistant constipation with proven organic disorders. Methods: Eight children (7–16 years) with at least 4 years of chronic treatment-resistant constipation and soiling, who had failed diet, laxative treatment and behavioral therapy were given 1 month of transcutaneous electrical stimulation. The three most severe cases had appendicostomies with antegrade washouts every 2–3 days to prevent impaction and reduce their soiling. Children and carers kept a daily diary of bowel habits, recording number of spontaneous defecations, episodes of soiling, use of bowel washouts and medications. Transcutaneous stimulation using interferential current was applied three times per week for 3–4 weeks using four surface electrodes, two to the paraspinal area of T9–10 to L2 and one to either side of the anterior abdominal wall beneath the costal margin. Diaries were recorded for 1 month before, during, and after stimulation and for 2 weeks 3 months later. Results: Transcutaneous electrical stimulation using interferential current stopped soiling in 7/8 children and increased the frequency of spontaneous defecations in 5/8. Defecations remained high and soiling low for 3 months in 3/6 children (with data). Conclusions: These results suggest that transcutaneous electrical stimulation using interferential current has a beneficial effect for children with chronic treatment-resistant constipation. Further trials using larger series of patients are needed to confirm this benefit, to determine the ideal stimulation parameters and to investigate why electrical stimulation might be effective. © 2005 Blackwell Publishing Asia Pty Ltd

Diagnostic tools for paediatric chronic constipation have been limited, leading to over 90% of patients with treatment-resistant constipation being diagnosed with chronic idiopathic constipation, with no discernible organic cause. Work in our institution suggests that a number of children with intractable symptoms actually have slow colonic transit leading to slow transit constipation. This paper reviews recent data suggesting that a significant number of the children with chronic treatment-resistant constipation may have organic causes (slow colonic transit and outlet obstruction) and suggests new approaches to the management of children with chronic treatment-resistant constipation.

Chronic constipation is a common condition in both adults and children. Children with chronic constipation frequently have symptoms that continue into adulthood. In the second part of the review we describe advances in the identification of abnormalities in the control of motility. The role of neurotransmitters in both paediatric and adult constipation is examined and the radical rethink of colonic dysmotility caused by the re-emergence of interstitial cells of Cajal is discussed. The recognition of chronic constipation as an heterogenous condition has led to the introduction of new therapies. Antegrade washouts through appendix stomas and an exciting new treatment with electrical interferential therapy may, in the future, result in a less invasive approach to the management of chronic constipation. An improved understanding of the assessment and management of chronic constipation in childhood is also likely to reduce the frequency and morbidity of chronic constipation in adults.

Constipation is very common in all age groups and can be resistant to standard therapies, producing chronic morbidity. Childhood onset constipation frequently produces symptoms that persist into adulthood. Recent advances have been made in the diagnosis and treatment of childhood constipation that could have application in adult practice. In the first part of the present review, the methods of differentiation of patients with a disorder of colonic motility from those with an anorectal hold-up are discussed. Slow transit constipation, with distinct features on colonic manometry and scintigraphy, has only recently been recognized in children. This diagnosis, together with a novel method of placement of a manometric catheter, is described. While the cause of slow transit constipation remains unclear, clinical features that differ between children and adults may provide an insight into the aetiology. The diagnosis of intestinal neuronal dysplasia is explained and the controversy surrounding the diagnosis outlined. We propose that the traditional histological criteria exclude many other clinically significant forms of dysplasia of the enteric nervous system and should be extended.

Tachykinins (TKs) colocalize with acetylcholine in excitatory motor neurones supplying human colonic circular muscle (CCM). Some children with slow-transit constipation (STC) have reduced TK-immunoreactivity in nerve terminals in CCM suggesting a deficit in neuromuscular transmission. This study aimed to test this possibility. Seromuscular biopsies of transverse colon were obtained laparoscopically from STC children (37, 17 with low density of TK-immunoreactivity). Specimens of transverse (17) and sigmoid colon (20) were obtained from adults undergoing colonic resection for cancer. CCM contractions were measured isotonically and responses to carbachol, neurokinin A (NKA) and electrical field stimulation (EFS) recorded. Carbachol and NKA-evoked contractions in adult and STC colon. Hyoscine (2 micromol L-1) significantly depressed responses to EFS in all preparations. Blockade of NK2 receptors (SR 48968, 2 micromol L-1) significantly depressed EFS-evoked contractions of adult transverse CCM, but had no effect on STC preparations. Thus, neuromuscular transmission in both adults and STC children is predominantly cholinergic and this component is unimpaired in the latter, indicating that reduced TK-immunoreactivity is not a marker for depressed cholinergic responses. Although pharmacologically responsive TK receptors are present in STC colon, we did not detect neuromuscular transmission mediated by release of TKs in these preparations.

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are located between nerve fibres and muscle cells and have a role in neuromuscular transmission and muscle contractility. Protein kinase C (PKC) is involved in modulation of muscle contractility by neurotransmitters, but it is not known if PKC has a role in ICC. There are 11 different PKC isoforms. The presence of PKC isoforms in ICC in guinea-pig gastrointestinal tract was examined using fluorescence immunohistochemistry and confocal microscopy. Segments of guinea-pig stomach, duodenum, ileum, proximal and distal colon were fixed in zambonis fixative. Frozen sections and wholemounts were incubated with anti-PKC antibodies (alpha, beta, delta, epsilon, gamma, iota, lambda, mu, theta) followed by fluorescent secondary antibody. Only PKC theta (theta) immunoreactivity was found in ICC. None of the other PKC isoforms (alpha, beta, delta, epsilon, gamma, iota, lambda, mu) localized to the ICC. PKC theta immunoreactivity was prominent in ICC located between the circular and longitudinal muscle layers (ICC-MY) in all regions except stomach and within the circular muscle (ICC-IM) in the large intestine. PKC theta was not present in ICC in the deep muscular plexus in either duodenum or ileum. PKC theta immunoreactivity was present in the cell body and proximal processes of the ICC. The cells containing PKC theta also contained cKit confirming the cells were ICC. ICC-MY in the ileum also contained the neurokinin (NK) 1 receptor. In conclusion, PKC theta is present in pacemaker ICC, but its function is not yet known. Functional studies will be needed to determine the role of this kinase in ICC. Knowing the second messenger cascades and being able to manipulate subpopulations of ICC will add to our understanding of the molecular and cell biology of ICC networks within the gastrointestinal tract and may ultimately help in understanding the aetiology of some gastrointestinal motor pathologies.

In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. British Journal of Pharmacology (1999) 126, 131–136; doi:10.1038/sj.bjp.0702285

The distribution of immunoreactivity to the neurokinin3 receptor (NK3R) was examined in segments C7, T11-12, L1-2, and L4-6 of the rat spinal cord. NK3R immunoreactivity was visualized by using two antisera generated against sequences of amino acids contained in the C-terminal region of the NK3R. NK3R-immunoreactive cells were numerous in the substantia gelatinosa of all spinal segments examined as well as the dorsal commissural nucleus of spinal segments L1-2. Isolated, immunoreactive cells were scattered throughout other regions of the spinal cord. The relationship of NK3R-immunoreactivity with neurons was demonstrated by colocalization with microtubule associated protein 2-immunoreactivity in individual cells. Within neurons, NK3R-immunoreactivity was associated predominately with the plasma membrane of cell bodies and dendrites. Within the substantia gelatinosa, 86% of nitric oxide synthase (NOS)-immunoreactive neurons were also NK3R-immunoreactive. Although NOS-immunoreactive neurons were found throughout all other regions of the spinal cord in the segments examined, these were not NK3R-immunoreactive. When preganglionic sympathetic neurons in spinal segments T11-12 and L1-2 were visualized by intraperitoneal injection of Fluorogold, less than 1% of the Fluorogold-labeled neurons were also immunoreactive for NK3R. The large number of NK3R-immunoreactive neurons in the substantia gelatinosa suggests that some effects of tachykinins on somatosensation may be mediated by NK3R.

Tachykinins, including substance P, neurokinin A, and neuropeptides K and gama, are expressed widely in the peripheral nervous system where they affect smooth muscle contraction, exocrine gland secretion, vascular permeability, and neurotransmission. Substance P, the preferred ligand for the NK1 receptor, is found in high concentrations in the enteric nervous system. In the present study, the localisation and distribution of the NK1 receptor was studied throughout the gastrointestinal tract of the guinea-pig by using a polyclonal antiserum raised against the C-terminal 15 amino acids of the NK1 receptor. Co-localisation with other neuronal markers was examined in the ileum. Nerve cell bodies reactive for the NK1 receptor were found in the myenteric plexus of all regions and the submucous plexus of the small and large intestines. In the small intestine, the interstitial cells of Cajal were also immunoreactive. Immunoreactivity was largely confined to cell surfaces. Almost all immunoreactive myenteric nerve cells had Dogiel type I morphology, and most of these were immunoreactive for nitric oxide synthase, a transmitter of inhibitory neurons to the muscle and of descending interneurons. Neuropeptide Y-containing secretomotor neurons in the submucous and myenteric plexuses also exhibited NK1 receptor immunoreactivity. NK1 receptors were present on a minority of tachykinin immunoreactive neurons of submucous ganglia. The results suggest that receptors on the longitudinal muscle might not be conventional NK1 receptors, that excitation of the circular muscle of the ileum is indirect, perhaps via the interstitial cells of Cajal, and that enteric inhibitory neurons may be excited via NK1 receptors.

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine-binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359–R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta. The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5′ end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N-termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine-binding site in the central channel of transthyretin, since, at least in humans, the N-termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416–24241.

The study by Heredia et al. (2010) in a recent issue of The Journal of Physiology shows that elongation of colon longitudinal muscle results in slow colonic transit. This study was performed in mice, but the results are probably applicable to humans. The study by Heredia et al. presents a new mechanism that may explain the association of elongated colon and poor motility and might be manipulated to overcome bowel lengthening and stasis. Their study shows that elongation of the longitudinal muscle triggers inhibition of the colonic migrating motor complex (CMMC), resulting in slow colonic transit. In humans, slow colonic transit occurs in patients with chronic constipation and is known as slow transit constipation (STC) (Hutson, 2009). Interestingly, we have observed that many patients with slow transit constipation have an elongated transverse colon (Fig. 1). The results from Heredia et al. suggest this elongation would inhibit colonic motility. Figure 1 Differences in colon length in children with constipation Elongated (called redundant) colon often occurs in patients with constipation. These patients are notable during colonic endoscopy because it is very hard to get along the length of the colon (Rex et al. 2007). Both colonic transit time and constipation symptoms increase with the number of redundancies and these are the patients that are least likely to respond to treatments (Raahave et al. 2009). Redundant bowel is often removed surgically. Propagating contractions in the proximal colon are highly propulsive and are a major determinant of proximal colonic flow in normal human colon (Dinning et al. 2008). High amplitude propagating contractions (HAPCs) underlie mass movements along the colon and may be similar to CMMC. Children with STC have reduced numbers of propagating contractions in the proximal colon (King, 2008) and lack of HAPC in constipated patients is associated with poor outcomes (van den Berg et al. 2006). Heredia et al. show that lengthening the colon inhibits CMMC in mice. Possibly the same is occurring in humans with colonic lengthening inhibiting HAPCs and so reducing colonic motility and slowing transit. STC can be associated with abnormal recto/sigmoid function (outlet obstruction, anorectal retention or functional fecal retention) or occur with normal recto/sigmoid function (Southwell et al. 2009). There is a lot of discussion about whether the defect is primarily at the outlet (ano-rectum) producing slowing of motility upstream as a secondary effect or if a defect can occur in the proximal colon without an outlet defect. Heredia et al.'s study shows the effects of elongation are different in the proximal and distal colon, and provides evidence that the proximal colon can inhibit motility in the distal colon. Thus it is possible that elongation can inhibit motility in the proximal colon primarily and this can then affect the distal colon. Heredia et al. also investigated the mechanism and showed that nitric oxide was released and activated inhibitory neurons reducing pellet propulsion and amplitude of colonic migrating motor complex (CMMC). Ileus (lack of movement of the bowel) develops after surgery and has been shown to be due to release of nitric oxide that then activates inhibitory neurons. Removal of the tonic nitrergic inhibition is a potent stimulus for human proximal colonic propagating sequences (Dinning et al. 2006). STC often develops after childbirth and there may be a connection between surgery (anaesthesia and caesarean) inducing nitric oxide release within the bowel and then leading to stasis in the ileum and in the colon. This study reveals a previously unrecognised mechanism for control of colonic motility: lengthening of the longitudinal muscle. If this mechanism can be manipulated in people, we may have a treatment for a currently poorly treated condition. Further detailed physiological studies of the effects of longitudinal muscle lengthening on migrating motor complexes in proximal and distal colon in other species should be helpful.

Background Slow-transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples. Methods Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images. Key Results In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty-three percent of STC children had low SP nerve fiber density. Conclusions & Inferences There are age-related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.

Background Studies in animals suggest that enteric neurons decrease in density or number with increasing age. Neurons containing nitric oxide (NO), vasoactive intestinal peptide (VIP) and Substance P (SP) have been implicated. In human large intestine, NO-utilizing neurons decrease during childhood or early adulthood but it is not known if the innervation of the muscle changes. This study examined the density of nerve fibres containing these transmitters in sigmoid colon circular muscle from children and adults. Methods Fluorescence immunohistochemistry using antibodies to neuronal NO synthase (nNOS), VIP and SP was performed on sigmoid colon from 18 adults with colorectal cancer, two children with familial adenomatous polyposis, and normal colon from nine children with Hirschsprung’s disease. The percentage area of immunoreactive (IR) nerve fibres containing each transmitter in circular muscle was quantified in confocal images. Key Results In the adult sigmoid colon circular muscle, the percentage area of nerve fibres containing nNOS>VIP>SP (6 : 2 : 1). Paediatric groups had significantly higher percentage area of nerve fibres containing nNOS, VIP or SP-IR than adults, with the decrease in nerve fibre density occurring from birth to 30 years. Circular muscle thickness increased between 12 and 30 years. Total nerve fibre area remained constant, while the muscle increased in thickness. Conclusions & Inferences In human sigmoid colon circular muscle, there are reductions in nNOS-, VIP- and SP-IR nerve fibre density with growth from newborn to late adolescence but little further change with aging. The reduction in nerve density is due to an increase in circular muscle thickness rather than a loss of nerve fibres.

Background Muscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. The cellular localization of subtypes of MRs within enteric circuits mediating muscle and mucosal reflexes remains to be demonstrated. This study aimed to localize the three functionally significant subtypes of MRs in human colon. Methods Reverse transcriptase-PCR was used to determine expression levels of muscarinic receptor subtype (MRs) M1Rs, M2Rs and M3Rs in human colon. Indirect immunofluorescence and confocal microscopy was used to localize MRs in cryostat-cut sections of human colon. Sections were double labeled for multiple cellular and neurochemical markers. Western blotting was used to confirm specificity of the muscarinic antisera used. Key Results All three MR subtypes were expressed in human colon. Immunoreactivity (IR) for M2Rs and M3Rs was most abundant in circular and longitudinal muscle. M1R-IR was most abundant on myenteric and submucosal nerve cells, both cholinergic and nitrergic. M3R-IR was also present on populations on myenteric nerve cell bodies. Immunoreactivity for all three receptors was present on nerve fibers in the circular muscle. Conclusions & Inferences In the human colon, subtypes of MRs were present on multiple cell types within the enteric circuits underlying motility, secretory and vasoactive reflexes. The cellular distribution for MRs found in this study agrees with data from functional studies, providing insight into the role MRs have in mediating enteric cholinergic neurotransmission.

Aim There have recently been reports of reduced tachykinin (TK) levels in the colonic circular muscle (CCM) in both children and adults with slow-transit constipation (STC), suggesting a relationship between TK deficiency and impaired colonic motility. In this study, we have examined the role of TKs and acetylcholine in mediating contraction in the adult colon, in order to determine the relative importance of these excitatory neurotransmitters. Methods Isotonic contractile responses to carbachol (cholinomimetic, 10 μm), NKA (neurokinin A receptor agonist, 100 nmol) and electrical field stimulation (EFS) were measured, in transverse (n = 5) and sigmoid (n = 7) colon. EFS was repeated in hyoscine (2 μm); NK1, NK2 and NK3 receptor antagonists (SR 140333, SR 48968, SR 142801, 200 μm); and NOLA (NOS inhibitor, 100 μm). Responses were re-tested with tetrodotoxin (TTX, 1 mm). Results Carbachol and NKA dose-dependently induced contractions in all specimens. The maximal NKA induced contraction was similar to the maximal carbachol contraction in sigmoid colon, but only 1/4 of the maximal carbachol contraction in transverse colon. EFS alone elicited similar responses in the sigmoid and the transverse CCM (40 and 32% of the maximal carbachol response, respectively). Hyoscine reduced the contractile response to EFS by 2/3 in sigmoid CCM, and by 1/2 in transverse CCM. NOLA caused a small increase in EFS-induced contraction in both sigmoid and transverse CCM. Individually, the TK antagonists did not produce significant changes in contractile responses elicited by EFS. However, when incubated together, the three TK antagonists produced a large reduction in EFS-induced contraction, by 1/4–3/4 in sigmoid, and by 1/4 in transverse CCM. EFS-induced responses were abolished in TTX, suggesting that responses were neuro-neuronal. Conclusion There is both a muscarinic cholinergic and tachykininergic input to contraction in the human colon. Muscarinic input produces at least 1/2 of EFS-induced contraction. Tachykinins produce at least 1/4 of the contraction in response to EFS in transverse colon. Tachykinins have an even greater input to EFS-induced contraction in the sigmoid colon.