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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.