vasoactive-intestinal-peptide and Hirschsprung-Disease

Vasoactive intestinal peptide (VIP), first isolated from the gut, was originally considered a candidate gastrointestinal hormone. Since about 1975, however, it has become increasingly clear that it is primarily a neurotransmitter or neuromodulator and that it exerts its functions mainly by local release from nerve endings. VIP plays a hormonal role only when it is released in large amounts from a tumor, with a consequent overflow into the circulation and grossly elevated plasma concentrations of the peptide. Moderately increased VIP plasma and tissue concentrations that cause mainly local effects are found in intestinal ischemia. Crohn's disease and some other chronic inflammatory diseases of the bowel. VIP is also measured in increased amounts in the normal fetus and neonate, where it may play an important physiological role. Such an increase of VIP levels in the circulation could enhance perfusion and metabolic activity of tissues during their rapid-growth period. On the other hand, disorders with a disturbed VIP function such as achalasia and Hirschsprung's disease and possibly also asthma and cystic fibrosis seem to be characterized mainly by a derangement of smooth muscle activity and/or exocrine secretion. Considering this list of disorders where VIP has either a proven or suspected role, it is easy to imagine the significance of this peptide in pediatric pathophysiology.

Topics: Asthma; Bronchodilator Agents; Celiac Disease; Child; Child, Preschool; Crohn Disease; Cystic Fibrosis; Digestive System; Esophageal Achalasia; Hirschsprung Disease; Humans; Hypoxia; Infant; Infant, Newborn; Placenta; Vasoactive Intestinal Peptide; Vipoma

Impaired function of the internal anal sphincter (IAS) may be implicated in postoperative obstructed defecation (POD) that may complicate Hirschsprung's disease (HD) patients. While innervation of part of the IAS in HD has been reported, accurate details based on anatomic landmarks that can explain the clinical morbidity seen in POD are lacking, and there appear to be no studies that specifically document the innervation of the "entire" IAS in HD. We used endothelin receptor-B knockout mice to represent HD (HD-mice) and C57B6 wild mice as controls (C-mice) to investigate the innervation of the entire IAS to assess the pathophysiology of POD experimentally.. The end-point of the longitudinal muscle layer was used to define the border between the IAS and the circular muscle layer (CML). Specimens of anorectum from HD- and C-mice were immunostained with PGP 9.5 and S100 as general nerve markers, nNOS and VIP as parasympathetic nerve markers, TH as a sympathetic nerve marker, and calretinin as a reliable diagnostic marker for HD. Immunostained cells/fibers were quantified using ImageJ.. On fluorescence microscopy, PGP 9.5, nNOS, and calretinin were significantly lower in the IAS of HD-mice than in C-mice (p < 0.05, respectively), while there were no significant differences between HD-mice and C-mice for S100, VIP, or TH.. We are the first to confirm that the expression of histochemical markers of innervation is abnormal throughout the "entire" IAS in HD-mice. Application of this finding may be beneficial for preventing POD and requires further research.

Topics: Anal Canal; Animals; Biomarkers; Calbindin 2; Disease Models, Animal; Hirschsprung Disease; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Muscle, Smooth; Nitric Oxide Synthase Type I; S100 Proteins; Tyrosine 3-Monooxygenase; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung's disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung's disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung's patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.

Topics: Adult Stem Cells; Animals; Biomarkers; Cell Culture Techniques; Cell Differentiation; Cell Separation; Enteric Nervous System; Flow Cytometry; Glial Fibrillary Acidic Protein; Heterografts; Hirschsprung Disease; Humans; Intestine, Large; Mice; Neural Stem Cells; Nitric Oxide Synthase Type I; Vasoactive Intestinal Peptide

Identification of neuronal progenitor/stem cells in the postnatal gut suggests the development of transplantation approaches to enteric nervous system (ENS) diseases. Many clinical applications would require engrafting large segments of postnatal gut in vivo. We investigated the ability of unselected gut cells vs selected enteric neural crest stem cells (eNCSCs) to engraft and differentiate in the postnatal gut in the Hirschsprung disease (HD, ednrb(sl/sl)) rat.. Total intestinal cells or eNCSCs (α(4) integrin(+), p75(++)) from embryonic day (E)14.5 rats carrying a marker transgene (human placental alkaline phosphatase, hPAP) were injected intraperitoneally (i.p.) into neonatal HD rats and their healthy littermates. The entire gut was systematically analyzed 3 weeks later for hPAP(+) cells between the serosal surface and the muscularis mucosae. Engrafted cells were examined for HuC/D, S-100B, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) expression.. No rats (0/33) injected with unselected cells had hPAP(+) cells in the ENS that expressed neuronal or glial markers. 5/11 healthy and 4/5 HD rats injected with eNCSCs showed widespread but low density engraftment in the ENS with cells expressing neuronal or glial markers. Neurons expressed nNOS and VIP. There was no engraftment in the colon of either HD or wildtype rats.. Enteric neural crest stem cells will engraft diffusely throughout the postnatal gut of HD rats and differentiate into neurons and glia. Engraftment is not uniform, likely related to age-dependent changes in the gut mesenchyme. Intraperitoneal injection is easily performed in sick neonates and may be developed as a technique to supply exogenous ENS cells to the diseased postnatal gut.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Hirschsprung Disease; Injections, Intraperitoneal; Intestines; Neural Crest; Neurons; Nitric Oxide Synthase Type I; Rats; Rats, Inbred WKY; Stem Cell Transplantation; Vasoactive Intestinal Peptide

In 1970, Drs. Said and Mutt isolated a novel peptide from porcine intestinal extracts with powerful vasoactive properties, and named it vasoactive intestinal peptide (VIP). Since then, the biological actions of VIP in the gut as well as its signal transduction pathways have been extensively studied. A variety of in vitro and in vivo studies have indicated that VIP, expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons, is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP actions are believed to be mediated mainly by interactions with highly expressed VPAC(1) receptors and the production of nitric oxide (NO). Furthermore, VIP has been implicated in numerous physiopathological conditions affecting the human gut, including pancreatic endocrine tumors secreting VIP (VIPomas), insulin-dependent diabetes, Hirschsprung's disease, and inflammatory bowel syndromes such as Crohn's disease and ulcerative colitis. To further understand the physiological roles of VIP on the GI tract, we have begun to analyze the anatomical and physiological phenotype of C57BL/6 mice lacking the VIP gene. Herein, we demonstrate that the overall intestinal morphology and light microscopic structure is significantly altered in VIP(-/-) mice. Macroscopically there is an overall increase in weight, and decrease in length of the bowel compared to wild type (WT) controls. Microscopically, the phenotype was characterized by thickening of smooth muscle layers, increased villi length, and higher abundance of goblet cells. Alcian blue staining indicated that the latter cells were deficient in mucus secretion in VIP(-/-) mice. The differences became more pronounced from the duodenum to the distal jejunum or ileum of the small bowel but, became much less apparent or absent in the colon with the exception of mucus secretion defects. Further examination of the small intestine revealed larger axonal trunks and unusual unstained patches in myenteric plexus. Physiologically, the VIP(-/-) mice showed an impairment in intestinal transit. Moreover, unlike WT C57BL/6 mice, a significant percentage of VIP(-/-) mice died in the first postnatal year with overt stenosis of the gut.

Topics: Animals; Gastrointestinal Motility; Gastrointestinal Tract; Hirschsprung Disease; Ileus; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Vasoactive Intestinal Peptide

Cocaine- and amphetamine-regulated transcript (CART)-peptide is found in the brain and participates in the control of feeding behavior. It is also expressed in the peripheral nervous system and is suggested to have neuromodulatory and/or neurotrophic effects in rat intestine. The aims of this study were to investigate the presence of CART-peptide in the normal ganglionic as well as aganglionic intestine from patients with Hirschsprung's disease and the peptide's possible coexistence with other neurotransmitters.. Intestinal specimens from nine patients with Hirschsprung's disease were examined using immunohistochemistry. A double immunostaining technique was used in order to elucidate the presence of CART-peptide in NOS and VIP-containing enteric neurons.. In ganglionic intestine, CART-peptide was found in numerous nerve fibers, predominantly within the smooth muscle layers and in myenteric nerve cell bodies. A high degree of co-localization of CART with NOS and VIP was seen. Only very few CART immunoreactive nerve fibers and no nerve cell bodies were found in the aganglionic intestine.. This is the first report on the presence of CART-peptide in the human intestine. In the ganglionic intestine CART was detected mainly in myenteric neurons, while only very few CART-IR nerve fibers were found in the aganglionic intestine. This, together with the coexistence of CART with NOS and VIP, indicates an intrinsic origin of the CART-containing neurons and suggests that CART may influence NO and VIP-induced effects.

Topics: Antibodies, Anti-Idiotypic; Biomarkers; Child, Preschool; Colon; Enteric Nervous System; Female; Follow-Up Studies; Hirschsprung Disease; Humans; Immunoglobulin G; Immunohistochemistry; Infant; Male; Muscle, Smooth; Nerve Fibers; Nerve Tissue Proteins; Neurotransmitter Agents; Nitric Oxide Synthase; Prognosis; Retrospective Studies; Severity of Illness Index; Vasoactive Intestinal Peptide

It is not clear what contribution the internal anal sphincter makes to the impaired motility observed in patients with Hirschsprung's disease (HD). Neuropeptides have recently been shown to be neurotransmitters in the nonadrenergic, noncholinergic inhibitory and excitatory nerves in the human gut. To clarify the physiologic significance of vasoactive intestinal polypeptide and substance P in the internal anal sphincter of HD (aganglionosis), we investigated the enteric nerve responses on lesional and normal internal anal sphincter muscle strips above the dentate line.. The lesional and normal internal anal sphincter muscle strips above the dentate line were derived from patient with HD (9 cases) and patients who underwent rectal amputation for low rectal cancers (8 cases). A mechanographic technique was used to evaluate in vitro muscle responses to these peptides of adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers.. Nonadrenergic, noncholinergic inhibitory nerves were found to act on the normal internal anal sphincter but had no effect on the enteric nerves in aganglionosis. Peptidergic (vasoactive intestinal polypeptide and substance P) nerves were found to act on normal colon, but no effect was observed in the aganglionic internal anal sphincter.. These findings suggest that peptidergic nerves play an important role in the impaired motility observed in the internal anal sphincter with HD.

Topics: Aged; Anal Canal; Atropine; Electric Stimulation; Female; Ganglia, Autonomic; Gastrointestinal Motility; Hirschsprung Disease; Humans; In Vitro Techniques; Infant; Male; Middle Aged; Muscle Contraction; Muscle, Smooth; Phenoxybenzamine; Propranolol; Rectal Neoplasms; Substance P; Tetrodotoxin; Vasoactive Intestinal Peptide

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.

Topics: Child; Child, Preschool; Colon; Enteric Nervous System; Hirschsprung Disease; Humans; Immunohistochemistry; Infant; NADPH Dehydrogenase; Nitric Oxide Synthase; Vasoactive Intestinal Peptide

In order to investigate the causes of abnormal peristalsis of the colon in intestinal neuronal dysplasia (IND), we studied the structure of the myenteric plexus of IND colon using silver-impregnation (Suzuki's method) as well as the innervation of both IND colons and normal colons using immunofluorescence technique with monoclonal antibodies to synaptic vesicles, and antisera to vasoactive intestinal polypeptide (VIP), substance P (SP), methionine-enkephalin (Met-Enk), and gastrin-releasing peptide (GRP). The following results were obtained. 1) In the IND colon, the number of identifiable myenteric ganglia was decreased. In a few cases of IND, irreversible neuron degeneration can be involved in the pathogenesis of IND. 2) In the IND colon, the distribution and fluorescence intensity of synaptic vesicles coincided with those of peptidergic nerve fibers. In the normal colon, synaptic vesicles were much more numerous in the circular muscle layers than in the longitudinal muscle layers, and the fluorescence intensity of those in the circular muscle layers was stronger than that of those in the longitudinal muscle layers. On the other hand, in IND colon, there were fewer synaptic vesicles in the circular muscle layers, and their fluorescence intensity was weak, while there were many synaptic vesicles in the longitudinal muscle layers, and their fluorescence intensity was strong. 3) Morphological abnormalities may exist in synaptic vesicles in the circular muscle layers of the IND colon. 4) Regarding the peptidergic nerve fibers, in the IND colon, innervation of circular muscle layers by Met-Enk-, GRP- and SP-immunoreactive fibers was reduced, and longitudinal muscles were more strongly innervated by immunoreactive fibers than those in the normal colon. 5) Disturbed innervation of non-adrenergic non-cholinergic excitatory nerves may cause the disturbance of muscle contractions in the IND colon. In addition, an imbalance of peptidergic and synaptic vesicle's innervations in both muscle layers may be related to the abnormal peristalsis of IND colon. Also, morphological abnormalities of synaptic vesicles may be concerned with its abnormal peristalsis.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Child; Child, Preschool; Colon; Enkephalin, Methionine; Female; Fluorescent Antibody Technique; Ganglia, Autonomic; Gastrin-Releasing Peptide; Hirschsprung Disease; Humans; Infant; Infant, Newborn; Male; Myenteric Plexus; Nerve Fibers; Peptides; Reference Values; Substance P; Synaptic Vesicles; Vasoactive Intestinal Peptide

The pathogenesis of Hirschsprung's disease is not well understood. The suitability of the animal model for the unknown pathogenesis of inhibitory neurotransmission in Hirschsprung's disease was investigated.. Circular smooth muscle strips from the internal anal sphincter (IAS) and distal colon (2, 6, 8, 16, and 24 mm from the anal verge) from normal and Ls/Ls mice (mice homozygous for the lethal spotting mutation that develop fetal megacolon after aganglionosis of the terminal colon) were prepared to record changes in isometric tensions in response to different agents and nonadrenergic, noncholinergic nerve stimulation by electrical field stimulation.. Bethanechol was used to produce contraction of the smooth muscle strips of distal colon to record a decrease in the tension. Conversely, the IAS smooth muscle strips developed spontaneous tone. In the normal homozygous mice, electrical field stimulation caused a biphasic response, an initial decrease followed by an after-contraction, whereas in Ls/Ls mice, the predominant response was contraction. All smooth muscle strips from normal and Ls/Ls mice produced relaxation in response to sodium nitroprusside and vasoactive intestinal polypeptide.. Ls/Ls mice may serve as an appropriate animal model to investigate the pathogenesis of the inhibitory neurotransmission in Hirschsprung's disease in the distal colon and IAS.

Topics: Anal Canal; Animals; Colon; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Hirschsprung Disease; Mice; Mice, Mutant Strains; Muscle Relaxation; Muscle, Smooth; Neural Inhibition; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Reference Values; Rodent Diseases; Synaptic Transmission; Vasoactive Intestinal Peptide

After Hirschsprung's disease was ruled out for 25 children who had severe chronic constipation, the authors studied the distribution of immunoreactivity for substance P (SP) and vasoactive intestinal peptide (VIP) in the intestinal wall, using immunofluorescence. SP and VIP immunoreactivity identify excitatory and inhibitory nerve fibres, respectively. Full-thickness rectal biopsy specimens were unsatisfactory, so seromuscular biopsies of the caecum, transverse colon, and sigmoid colon were obtained (by laparoscopy and laparotomy; n = 10 patients). SP-immunoreactive fibres were markedly reduced in seven, with concomitant reduction of VIP-immunoreactive fibres in four. In two other patients, there was no obvious reduction in SP- or VIP-immunoreactive fibres. In a patient who subsequently was found to have multiple endocrine neoplasia type 2b, the myenteric plexus was markedly hyperplastic, with an increase in nerve cells and nerve fibres. VIP-immunoreactive fibres were increased, but SP-immunoreactive fibres were markedly decreased. Surgical options included proximal stoma, Malone operation, and subtotal colectomy with preservation of the rectum. Three children with subtotal colectomy have had improvement over short-term follow-up. The combination of seromuscular laparoscopic biopsies and immunofluorescence demonstration of neuropeptides may identify new variants of intestinal neuronal dysplasia than can be treated successfully with surgery.

Topics: Adenomatous Polyposis Coli; Biopsy; Child; Child, Preschool; Colectomy; Colon; Colonic Pseudo-Obstruction; Constipation; Female; Fluorescent Antibody Technique; Hirschsprung Disease; Humans; Male; Nerve Fibers; Substance P; Vasoactive Intestinal Peptide

To clarify the significance of peptidergic nerves in Hirschsprung's disease (aganglionosis), hypoganglionosis, and neuronal intestinal dysplasia (NID), we investigated enteric nerve responses in colonic tissues obtained from patients with these diseases. Colonic tissue specimens were obtained from 12 patients with aganglionosis, 8 patients with hypoganglionosis, and 6 patients with NID. Colon specimens from 20 patients without constipation were used as controls. A mechanograph was used to evaluate in vitro colonic responses to electrical field stimulation (EFS) of the adrenergic and cholinergic nerve blockers and gastrointestinal hormones. The following results were obtained: (1) Non-adrenergic inhibitory nerves were found to act on the normal human colon and to a lesser extent in colons with hypoganglionosis or NID, but had no effect on the enteric nerves in colons with aganglionosis. (2) Peptidergic neurotransmitters such as VIP, substance P, and neurotensin apparently act in the normal human colon, and to a lesser extent in the colons with hypoganglionosis or NID, but their effect was almost absent in aganglionosis. (3) VIP acts via neural mechanisms, while substance P and neurotensin may act both via nerves and also directly on the bowel smooth muscle. The diminution of action of non-adrenergic inhibitory nerves and peptidergic nerves may be largely related to the impaired motility observed in hypoganglionosis, NID and aganglionosis.

Topics: Adolescent; Adrenergic Fibers; Adult; Aged; Child; Child, Preschool; Cholinergic Fibers; Colon; Electric Stimulation; Female; Gastrointestinal Motility; Hirschsprung Disease; Humans; Infant; Male; Middle Aged; Muscle, Smooth; Neural Inhibition; Neuropeptides; Neurotensin; Substance P; Synaptic Transmission; Vasoactive Intestinal Peptide

The distribution of nerve fibers containing enkephalin (ENK)-like immunoreactivity was examined in the rectum of aganglionosis rats (AGRs) which completely lack the intramural ganglion cells in the large intestine, and was compared with that of their normal littermates. Furthermore, Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive neurons projecting to the rectum were examined using retrograde tracing combined with immunohistochemistry in the major pelvic ganglion of normal male rats. In the intermuscular space of the aganglionic rectum of AGRs, unlike the pattern of the normal intermuscular plexus, moderate numbers of ENK-like-immunoreactive fibers were arranged in an irregular, coarse network; greatly diminished numbers of immunoreactive fibers were found in the submucosa. No ENK-like-immunoreactive fibers were seen in the circular muscle layer and mucosa. In the normal rat rectum, ENK-like-immunoreactive fibers were seen throughout all layers, and immunoreactive nerve cells were found predominantly in the myenteric plexus of colchicine-treated animals. Fluoro-Gold injected into the upper rectum labelled numerous principal ganglion neurons in the major pelvic and inferior mesenteric ganglia. Less than 10% of tracer-labelled neurons were positive for fluorescein immunolabelling of MEAGL in the major pelvic ganglion; no immunoreactive neurons were found in the inferior mesenteric ganglion. In the major pelvic ganglion of the colchicine-treated normal rats, about 5% of principal ganglion neurons were immunoreactive for MEAGL. Comparison of serial paraffin sections of the major pelvic ganglion stained for tyrosine hydroxylase (TH), MEAGL and vasoactive intestinal polypeptide (VIP), respectively, revealed that more than half of MEAGL-like immunoreactive neurons were also positive for TH; there was no case showing co-existence of MEAGL with VIP in the principal neurons. These results indicate that a small number of enkephalin-containing neurons in the major pelvic ganglion project to the rectum, and that more than half of these neurons are postganglionic sympathetic. They may terminate mainly in the myenteric ganglia in the rectum.

Topics: Animals; Colchicine; Enkephalin, Methionine; Enkephalins; Fluorescent Antibody Technique; Fluorescent Dyes; Ganglia, Autonomic; Hirschsprung Disease; Immunohistochemistry; Male; Nerve Fibers; Neural Pathways; Rats; Rectum; Stilbamidines; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

The significance of dysplastic features in the surgical pullthrough segment of bowel in patients with Hirschsprung's disease (HD) has not yet been clarified. The aim of this study was to evaluate prospectively the ganglionated proximal bowel in 26 patients with HD (January 1988 through January 1991). The significance of dysplastic features and their influence on post operative outcome were evaluated by means of a newly devised histological scoring system based on the morphological features. Functional outcome was assessed clinically at follow-up interview. Comparison was with control specimens from 22 patients undergoing unrelated bowel surgery and a further 5 patients with neuronal intestinal dysplasia (NID). Results indicated a wide spectrum of histologically identified dysplastic features in patients with NID, the ganglionated bowel of HD and controls. Although individual abnormal features were noted in the control group, significant degrees of dysplasia were absent. The overall degree of dysplasia was less striking than that observed in NID and in the 5 patients in whom NID co-existed with HD. Dysplasia of the ENS in residual bowel could be correlated with postoperative dysfunction in 4 out of 5 patients (80%) with HD and features of co-existing NID. In addition, milder symptoms were noted in 50% of patients having a borderline score (5-6/12). This study emphasizes the relationship between clinical obstructive symptoms and a high degree of dysplasia within the ENS. A histological grading system is of value in evaluating the spectrum of abnormal findings and prospectively identifying those with functional significance in patients with NID co-existing with HD.

Topics: Acetylcholinesterase; Child; Child, Preschool; Female; Follow-Up Studies; Hirschsprung Disease; Humans; Immunoenzyme Techniques; Infant; Intestines; Male; Myenteric Plexus; Neuroglia; Neurons; Phosphopyruvate Hydratase; Postoperative Complications; Prognosis; Prospective Studies; Submucous Plexus; Vasoactive Intestinal Peptide

Despite technically satisfactory operations, at least 20% of children with Hirschsprung's disease have an unsatisfactory postoperative result. A possible explanation for their symptoms is the retention of ganglionic intestine which has demonstrable abnormalities of the enteric nervous system. The distribution of intestinal neural proteins and peptides in resected colons from patients with Hirschsprung's disease (n = 10) was compared with that in normal controls (n = 5). Immunocytochemistry was performed using antisera against general markers of the enteric nervous system (PGP 9.5, NSE, NFILs, and S-100 protein) and colonic neuropeptides (VIP, GAL, SP, NPY, CGRP, and Met-ENK). The distribution and density of peptide-containing nerve fibers varied greatly from one patient to another and no consistent pattern of neural disturbances could be discerned in aganglionic colon. At the proximal limit of resection, abnormalities of enteric innervation were detected in 8 of 10 studied specimens. Although ganglion cells staining positively for general neuronal markers were present in all cases, normal populations of neural cell bodies immunoreactive for neuropeptides could be found in only 2 specimens. Enlarged submucosal nerve trunks found in the most proximal area of most specimens, displayed immunoreactivity for general nerve markers and VIP, GAL, NPY, and CGRP. The widely practised conventional histopathological assessment of the proximal limit of colonic neural abnormalities may be inadequate.

Topics: Calcitonin Gene-Related Peptide; Child; Child, Preschool; Colon; Colostomy; Enkephalin, Methionine; Female; Galanin; Hirschsprung Disease; Humans; Immunoenzyme Techniques; Infant; Male; Nerve Fibers; Neuropeptide Y; Neuropeptides; Peptides; Submucous Plexus; Substance P; Vasoactive Intestinal Peptide

A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exhibits sequence homology with vasoactive intestinal polypeptide (VIP) and occurs in the mammalian brain, lung and gut. The distribution of PACAP in ganglionic and aganglionic portions of the large intestine of patients with Hirschsprung's disease was examined by immunohistochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were distributed in all layers of the ganglionic and aganglionic segments of the intestine, although they were less numerous in the latter, and PACAP-immunoreactive nerve cell bodies were seen in the ganglionic portion of the intestine. The concentration of immunoreactive PACAP was lower in the aganglionic than in the ganglionic segment of the intestinal wall. PACAP and VIP were found to coexist in both ganglionic and aganglionic segments of the intestine. Apparently, PACAP participates in the regulation of gut motility. The scarcer PACAP innervation of the aganglionic segment may contribute to the defect in intestinal relaxation seen in patients with Hirschsprung's disease.

Topics: Ganglia; Hirschsprung Disease; Humans; Immunohistochemistry; Intestine, Large; Nerve Fibers; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Radioimmunoassay; Vasoactive Intestinal Peptide

In Hirschsprung's disease, the aganglionic bowel is characterized by an absence of ganglion cells and an increased number of adrenergic and presumed cholinergic nerve fibers. In addition, a severe derangement of peptide-containing nerve fibers is encountered including a hyperinnervation of neuropeptide Y (NPY)-containing fibers. Using immunochemical and immunocytochemical methods, we examined the nature of the NPY-containing nerve fibers contributing to the hyperinnervation. The concentration of NPY was markedly increased in the aganglionic segment. Coexistence of NPY, vasoactive intestinal peptide (VIP), and the adrenergic enzyme tyrosine hydroxylase (TH) showed small populations of nerve fibers containing NPY/TH, NPY/VIP, or TH alone in ganglionic intestine. Numerous nerve fibers stored VIP but lacked NPY. These fibers did not contain TH, indicating that all VIP-containing fibers are nonadrenergic. In the aganglionic intestine there was a marked increase in the number of nerve fibers storing NPY/TH and NPY/VIP, whereas the fibers storing VIP alone were reduced in number. A small number of nerve fibers storing NPY alone occurred in the hypertrophic nerve bundles. NPY/VIP-containing nerve fibers were particularly numerous in the mucosa in aganglionic intestine, which may be of interest in the diagnosis of Hirschsprung's disease allowing the use of mucosal biopsy specimens. Thus, the proliferating NPY-containing nerve fibers in the aganglionic intestine seem to comprise three different populations, one adrenergic and two nonadrenergic, one of which contains in addition VIP.

Topics: Adrenergic Fibers; Child; Child, Preschool; Hirschsprung Disease; Humans; Immunohistochemistry; Infant; Intestine, Large; Nerve Fibers; Neuropeptide Y; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers.

Topics: Bombesin; Child; Child, Preschool; Colon; Enkephalin, Methionine; Female; Hirschsprung Disease; Humans; Infant; Intestinal Mucosa; Male; Muscle, Smooth; Neuropeptides; Peptide PHI; Substance P; Vasoactive Intestinal Peptide

Accurate delineation of the intramural pathway of abnormal enteric nerve fibres in Hirschsprung's disease has previously proved impossible because the neural network is invariably transected in conventional histological sections. With the technique of wholemount immunohistochemistry (WI), the bowel segment is converted into a rectangular sheet and the serosa, long muscle (LM), circular muscle (CM), submucosa, and mucosa are separated into layers to allow each nerve plexus to be examined intact and neural pathways traced. The entire resected bowel specimens of nine HD infants and five infants serving as controls were investigated, using neuron-specific enolase and vasoactive intestinal peptide (VIP) for WI. The major new findings are (1) More VIP fibres were observed in aganglionic bowel with WI than with conventional sections; (2) Thick nerve trunks in aganglionic bowel do not descend from intrinsic neurons of oligoganglionic bowel as previously suggested, but have an extrinsic origin, accompanying blood vessels as small nerves initially, expanding subsequently, and ending blindly in submucosa; (3) CM nerve fibres follow muscle fibres concentrically for long distances in aganglionic bowel; and (4) LM nerve fibres meander in spirals in aganglionic bowel instead of running straight. This study shows that (1) WI is highly sensitive; (2) nerve fibres in aganglionic bowel have an extrinsic origin; and (3) innervation abnormalities in Hirschsprung's disease are not only quantitative but qualitative.

Topics: Colon; Hirschsprung Disease; Humans; Immunohistochemistry; Infant; Neural Pathways; Phosphopyruvate Hydratase; Vasoactive Intestinal Peptide

The reduction of vasoactive intestinal peptide-(VIP) containing nerve fibres in the aganglionic segment in Hirschsprung's disease is thought to contribute to the sustained contraction of this intestinal segment. In order to study the significance of VIP in the pathogenesis of Hirschsprung's disease we used immunohistochemistry to evaluate the reduction of VIP-immunoreactive nerve fibres in aganglionic intestine compared to the ganglionic one. The VIP nerve fiber density was compared with the type of onset of disease (e.g. neonatal ileus or obstipation) and with the length of the aganglionic segment. No statistically significant correlation between these factors could be registered. This indicates a high complexity of the neuronal derangement in aganglionic intestine and that the degree of VIP deficiency alone does not correlate with the severity of the disease.

Topics: Colon; Female; Hirschsprung Disease; Humans; Immunohistochemistry; Infant; Male; Nerve Fibers; Vasoactive Intestinal Peptide

The purpose of this study is to examine the role of peptidergic nerves (VIP, Substance P, Neurotensin) in Hirschsprung's disease (aganglionosis) and hypoganglionosis in relation to the normoganlionic state of the colon using a mechanographic technique in vitro. The following results were obtained. 1) Normoganglionic muscle strips demonstrated the presence of intact non-adrenergic inhibitory nerve. The activities of such nerves, however, were reduced in hypoganglionic muscle strips, and were absent in aganglionic muscle strips. 2) Peptidergic nerve activities by VIP, substance P, and Neurotensin were present in normoganglionic muscle strips, while they were reduced in hypoganglionic muscle strips, and absent in aganglionic muscle strips. 3) VIP may act as a neurotransmitter-neuromodulator of non-adrenergic inhibitory nerve, while SP and Neurotensin may act as that of non-cholinergic excitatory nerve with some direct effect on the intestinal muscle.

Topics: Hirschsprung Disease; Humans; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Neurotensin; Neurotransmitter Agents; Substance P; Vasoactive Intestinal Peptide

The fine structure of neuronal perikarya and processes containing VIP-like immunoreactive material in the colon of patients with Hirschsprung's disease was investigated by immunoelectron microscopy. No VIP-like immunoreactive terminals were found in Auerbach's plexus of the ganglionic segment. However, VIP-like immunoreactive preterminal axons were frequently found to make synaptic contact with both immunoreactive and non-immunoreactive elements within Meissner's plexus. Therefore, the function of the VIP neurons in Auerbach's plexus seems to differ from that in Meissner's plexus. In the oligoganglionic segment, there were a few VIP-like immunoreactive processes, but no VIP-like immunoreactive synaptic formations. VIP-like immunoreactive processes were rarely encountered in the aganglionic segment. In both the oligo- and aganglionic segments, bowel relaxation is considered to be disturbed due to the lack of synaptic contacts of VIP-like immunoreactive neurons with other neuronal components.

Topics: Child; Colon; Hirschsprung Disease; Humans; Neurons; Vasoactive Intestinal Peptide

The distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity was examined immunohistochemically in whole-mount specimens of the colons of mutant rats, which completely lacked intramural nerve cells in the colon, and of their normal littermates. In the aganglionic colon, greatly diminished numbers of vasoactive intestinal polypeptide-like immunoreactive nerve fibers were found in the circular muscle layer, lamina propria of the mucosa, and in the submucosa. In the intermuscular space of the aganglionic colon, unlike the pattern of the normal Auerbach's plexus, vasoactive intestinal polypeptide-like immunoreactive nerve fibers were arranged in an irregular, coarse network. These findings suggest the existence of extrinsic nerves containing vasoactive intestinal polypeptide in the aganglionic colon of the hereditary aganglionic rat.

Topics: Animals; Colon; Disease Models, Animal; Hirschsprung Disease; Neurons; Peripheral Nerves; Rats; Vasoactive Intestinal Peptide

Diminished concentrations of the gut neuropeptide, vasoactive intestinal peptide (VIP), have been measured by radioimmunoassay in man and mouse models of Hirschsprung's disease. This in vitro study was designed to ascertain the functional response to VIP in aganglionic colon. Seven piebald lethal (PLM) mice with histologically verified aganglionosis and seven normal littermates (NLM) were sacrificed. Distal colonic segments were placed in standard oxygenated tissue baths and responses to electrical field stimulation (EFS), acetylcholine (ACh), and VIP recorded and analyzed by a motility index (MI). Aganglionic colonic tissues from PLM exhibited marked basal contractile activity in contrast to NLM (MI = 19.5 +/- 2.0 SEM v 6.5 +/- 3.6 SEM, P less than .01). In NLM tissues, VIP reduced the MI to ACh challenge by 49% (P less than .01), while in PLM tissues, a nonsignificant 22% reduction was observed. VIP blocked the response to EFS in NLM tissues, while no response was elicited to EFS in PLM tissues. An in vitro deficit in the VIP inhibitory response to ACh challenge is apparent in PLM with distal colonic aganglionosis. The increased basal activity and reduction in responsiveness to VIP, observed in the PLM tissues, support a generalized reduction in the function of the inhibitory innervation of the aganglionic colon.

Topics: Acetylcholine; Animals; Colon; Disease Models, Animal; Hirschsprung Disease; In Vitro Techniques; Mice; Mice, Inbred Strains; Muscle Contraction; Vasoactive Intestinal Peptide

The distributions of gut hormones in the colon of Hirschsprung's disease were investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments (ganglionic, oligoganglionic, and aganglionic) were stained by the unlabeled antibody enzyme method. The immunoreactivity of vasoactive intestinal polypeptide (VIP) was found to be reduced in the oligoganglionic and aganglionic segments. Antisera to substance P and met-enkephalin demonstrated immunoreactive cells and fibers in the ganglionic segment, whereas these cells and fibers were almost completely absent in the oligoganglionic and aganglionic segments. A similar distribution was seen for the mucosal endocrine cells with somatostatin immunoreactivity. Antisera to neurotensin, motilin, bombesin, and cholecystokinin revealed no immunoreactivity in the normal colon or the three segments. The differences in these peptides between normal and impaired colonal segments may be one of the causes of colon constriction in Hirschsprung's disease.

Topics: Adult; Child; Colon; Enkephalin, Methionine; Ganglia; Hirschsprung Disease; Humans; Immunochemistry; Mucous Membrane; Myenteric Plexus; Somatostatin; Submucous Plexus; Substance P; Vasoactive Intestinal Peptide

Topics: Animals; Colon; Female; Hirschsprung Disease; Histocytochemistry; Rats; Vasoactive Intestinal Peptide

The distribution of vasoactive intestinal polypeptide (VIP)-containing nerves and the contents of both VIP and substance P (S-P) in the intestines from 12 children with Hirschsprung's disease were examined using immunohistochemical methods and radioimmunoassay. VIP-containing nerve fibers were markedly decreased in number in the true muscle coats of aganglionic segments, while extrinsic hypertrophic nerve bundles in these segments showed positive VIP-immunoreactivities. This finding suggests the existence of extrinsic origins of VIP-containing nerves in the human gut. The contents of VIP were 44.5 +/- 8.2 in aganglionic segments and 130 +/- 17.1 pg/mg wet tissue weight in normoganglionic segments. The contents of S-P were 0.42 +/- 0.18 in aganglionic segments and 6.38 +/- 2.3 pg/mg wet tissue weight in normoganglionic segments. Both VIP and S-P contents in aganglionic segments were significantly reduced as assessed by the use of radioimmunoassay (p less than 0.001 and p less than 0.05). These abnormal peptidergic patterns of innervation might relate to the non-peristaltic state in Hirschsprung's disease.

Topics: Adolescent; Child; Child, Preschool; Hirschsprung Disease; Histocytochemistry; Humans; Infant; Intestines; Peripheral Nerves; Radioimmunoassay; Substance P; Vasoactive Intestinal Peptide

An increasing amount of evidence concerning the existence of non-adrenergic, non-cholinergic autonomous nerves has been presented during the past decade. These nerves contain different peptides which may act as neurotransmitters. The pathophysiology in Hirschsprung's disease is not yet fully explained. To throw further light upon it, the distribution and occurrence of different peptide-containing (peptidergic) nerves was studied. A semiquantitative immuno-histochemical method was used to assess the distribution and occurrence of nerves containing encephalin, GRP (gastrin-releasing peptide), VIP (vasoactive intestinal peptide) or substance P in four patients operated by Duhamel's procedure. The results indicate a total absence of encephalin and GRP containing nerves in the aganglionic segment. Such nerves could, however, be found in the normally ganglionated part of colon. The nerves containing VIP and substance P were fewer in the aganglionic segment than in the rest of the colon. The result is related to what is hitherto known about the specific effects of the different peptides.

Topics: Colon; Female; Gastrin-Releasing Peptide; Hirschsprung Disease; Humans; Infant; Male; Nerve Fibers; Peptides; Vasoactive Intestinal Peptide

The distribution of vasoactive intestinal polypeptide (VIP) in the colon of patients with Hirschsprung's disease was investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments, ganglionic, oligoganglionic and aganglionic, were stained by the unlabeled antibody enzyme method. VIP immunoreactive nerve cell bodies, nerve fibers and nerve endings were distributed throughout the ganglionic and oligoganglionic segments. In contrast, the aganglionic segment contained no VIP nerve endings and the number of fibers was reduced. These differences are thought to be a cause of constriction of the colon in Hirschsprung's disease and VIP neurons are therefore believed to participate in the relaxation of smooth muscle.

Topics: Child; Colon; Gastrointestinal Hormones; Hirschsprung Disease; Humans; Immunoenzyme Techniques; Myenteric Plexus; Nerve Fibers; Neurons; Submucous Plexus; Vasoactive Intestinal Peptide