Oligoneuronal Hypoganglionosis in Patients with Idiopathic Slow-Transit Constipation

PURPOSE: Several alterations of the enteric nervous system have been described as an underlying neuropathologic correlate in patients with idiopathic slow-transit constipation. To obtain comprehensive data on the structural components of the intramural nerve plexus, the colonic enteric nervous system was investigated in patients with slow-transit constipation and compared with controls by means of a quantitative morphometric analysis. METHODS: Resected specimens were obtained from ten patients with slow-transit constipation and ten controls (nonobstructive neoplasias) and processed for immunohistochemistry with the neuronal marker Protein Gene Product 9.5. The morphometric analysis was performed separately for the myenteric plexus and submucous plexus compartments and included the quantification of ganglia, neurons, glial cells, and nerve fibers. RESULTS: In patients with slow-transit constipation, the total ganglionic area and neuronal number per intestinal length as well as the mean neuron count per ganglion were significantly decreased within the myenteric plexus and external submucous plexus. The ratio of glial cells to neurons was significantly increased in myenteric ganglia but not in submucous ganglia. On statistical analysis, the histopathologic criteria (submucous giant ganglia and hypertrophic nerve fibers) of intestinal neuronal dysplasia previously described in patients with slow-transit constipation were not completely fulfilled. CONCLUSION: The colonic motor dysfunction in slow-transit constipation is associated with quantitative alterations of the enteric nervous system. The underlying defect is characterized morphologically by oligoneuronal hypoganglionosis. Because the neuropathologic alterations primarily affect the myenteric plexus and external submucous plexus, superficial submucous biopsies are not suitable to detect these innervational disorders.     

Analysis of the RET, GDNF, EDN3, and EDNRB genes in patients with intestinal neuronal dysplasia and Hirschsprung disease

BACKGROUND: Hirschsprung disease (HSCR) is a frequent congenital disorder with an incidence of 1 in 5000 live births, characterised by the absence of parasympathetic intramural ganglion cells in the hindgut resulting in intestinal obstruction in neonates and severe constipation in infants and adults. Intestinal neuronal dysplasia (IND) shares clinical features with HSCR but the submucosal parasympathetic plexus is affected. IND has been proposed as one of the most frequent causes of chronic constipation and is often associated with HSCR. METHODS: We examined 29 patients diagnosed with sporadic HSCR, 20 patients with IND, and 12 patients with mixed HSCR/IND for mutations in the coding regions of the RET, GDNF, EDNRB, and EDN3 genes. The entire coding regions were analysed by single strand conformational polymorphism and DNA sequencing. RESULTS: Only three RET mutations were detected in patients with HSCR. In patients with IND or a mixed HSCR/IND phenotype, no mutations in these genes were observed. While HSCR and HSCR/IND showed over representation of a specific RET polymorphism in exon 2, IND exhibited a significantly lower frequency comparable with that of controls. CONCLUSIONS: The mutation frequency found in our sporadic HSCR patients (10%) and the allelic distribution of RET polymorphisms are comparable with earlier published data. A significantly different allelic distribution in an established HSCR associated polymorphism argues against common genetic pathways for HSCR and IND.     

Differential changes in intrinsic innervation and interstitial cells of Cajal in small bowel atresia in newborns

AIM: To investigate morphological changes of the enteric nervous system (ENS) and the interstitial cells of Cajal (ICCs) in small bowel atresia.METHODS: Resected small bowel specimens from affected patients (n = 7) were divided into three parts (proximal, atretic, distal). Standard histology and enzyme immunohistochemistry anti-S100, anti-protein gene product (PGP) 9.5, anti-neurofilament (NF), antic-kit-receptor (CD117) was carried out on conventional paraffin sections of the proximal and distal part. RESULTS: The neuronal and glial markers (PGP 9.5, NF, S-100) were expressed in hypertrophied ganglia and nerve fibres within the myenteric and submucosal plexuses. Furthermore, the submucous plexus contained typical giant ganglia. The innervation pattern of the proximal bowel resembled intestinal neuronal dysplasia. The density of myenteric ICCs was clearly reduced in the proximal bowel, whereas a moderate number of muscular ICCs were found. The anti-CD117 immunore- action revealed additional numerous mast cells. The distal bowel demonstrated normal morphology and density of the ENS, the ICCs and the mast cells.CONCLUSION: The proximal and distal bowel in small bowel atresia revealed clear changes in morphology and density of the ENS and ICCs.     

Morphometric quantification of normal submucous plexus in the distal rectum of adult healthy volunteers

OBJECTIVE: Inadequate morphometric characterization of the normal adult submucous plexus has precluded the diagnosis of colonic dysganglionoses associated with constipation, such as intestinal neuronal dysplasia type B (IND B). The internal submucous plexus (Meissner plexus) was morphometrically quantified in adult healthy volunteers. DESIGN: Open, prospective morphometric study in balanced groups of female and male volunteers. PARTICIPANTS: Thirty-seven adult healthy male and female volunteers with normal bowel function and no history of gastrointestinal disease. METHODS: Four jumbo rectal biopsies (3-5 mm3) were taken 5 and 10 cm above the pectinate line. Two expert gastrointestinal pathologists assessed biopsy sections after specific nerve cell staining for lactic dehydrogenase, nitric oxide synthase and acetylcholinesterase, mainly for characteristics of ganglia and nerve cells in the submucous plexus. RESULTS: No healthy individual demonstrated over 20% of submucosal ganglia as giant ganglia or more than four giant ganglia per 30 sections (the morphometric criteria for IND B). Single submucosal nerve cells and ganglion numbers halved between 10 and 5 cm above the pectinate line, but there were no age or gender differences. The biological variability of nerve cell and ganglion density in the submucous plexus was large. CONCLUSIONS: Healthy adults show less than 20% of submucosal ganglia as giant ganglia and no more than four giant ganglia per 30 rectal biopsy sections. There is therefore no overlap with the histomorphological criteria of IND B. These data therefore support the specificity of the previously defined criteria for IND B in adults.     

突触素单克隆抗体免疫组化实验诊断肠神经发育不良症的价值

为探讨肠神经发育不良症（IND）的有效诊断方法，对45例术前诊断为先天性巨结肠（HD）的慢性肠梗阻患儿行巨结肠根治手术，并对其结肠痉挛段标本进行突触素单克隆抗体免疫组化实验。结果显示，16例结肠标本可见粘膜下神经丛过渡增生并有巨神经节（诊断为IND），与正常对照组比较，P＜0．01，其中14例有异位神经节（9例合并HD，下称HAIND），其病理切片中发现有异位的神经节细胞，且部分神经节细胞有变形，空泡化；10例IND（其中8例HAIND）可固有层和粘膜肌染色增强，与正常对照组比较，P＜0．01。认为突触素单克隆抗体免疫组化实验显示粘膜一神经丛过度增生，巨神经节可作为诊断IND的硬性指标；异位神经节，固有层和粘膜肌染色增强可作为诊断IND的辅助指标。在病理切片中应用突触素单克隆抗体免疫组化实验，可清晰显示肠壁神经丛的发育情况，是诊断IND的可靠方法。     

Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color.

Mutations in the gene encoding the endothelin receptor type B (EDNRB) produce congenital aganglionic megacolon and pigment abnormalities in mice and humans. Here we report a naturally occurring null mutation of the EDNRB gene in spotting lethal (sl) rats, which exhibit aganglionic megacolon associated with white coat color. We found a 301-bp deletion spanning the exon 1-intron 1 junction of the EDNRB gene in sl rats. A restriction fragment length polymorphism caused by this deletion perfectly cosegregates with the sl phenotype. The deletion leads to production of an aberrantly spliced EDNRB mRNA that lacks the coding sequence for the first and second putative transmembrane domains of the G-protein-coupled receptor. Radioligand binding assays revealed undetectable levels of functional EDNRB in tissues from homozygous sl/sl rats. We conclude that EDNRB plays an essential role in the normal development of two neural crest-derived cell lineages, epidermal melanocytes and enteric neurons, in three mammalian species--humans, mice, and rats. The EDNRB-deficient rat may also prove valuable in defining the postnatal physiologic role of this receptor.     

Altered tryptophan hydroxylase 2 expression in enteric serotonergic nerves in Hirschsprung’s-associated enterocolitis

AIM: To determine if expression of colonic tryptophan hydroxylase-2 (TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung’s-associated enterocolitis. METHODS: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung’s disease (HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung’s-associated enterocolitis (HAEC). Controls were collected at colostomy closure in children with anorectal malformation (n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers. RESULTS: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα⁺ cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zone bowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC. CONCLUSION: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC.     

Submucosal hypoganglionosis causing chronic idiopathic intestinal pseudo-obstruction.

A 39-year-old woman presented with recurrent symptoms suggestive of intestinal obstruction. She was put on total parenteral nutrition (TPN) and consequently developed sepsis and endocarditis. TPN was stopped and a venting enterostomy was performed. Biopsies of mucosa and submucosa were taken at surgery; immunohistochemistry for neuronal proteins, protein gene product 9.5 (PGP 9.5) and the glial S-100-protein was done. Many enlarged nerve fiber strands were found in the submucosa. Few small ganglia containing a small number of nerve cells could be observed, suggesting hypoganglionosis. This patient with chronic idiopathic intestinal pseudoobstruction of neurogenic type had a defect in the submucous plexus, whereas visceral neuropathies are usually characterized by defects of the myenteric plexus with normal submucous plexus.     

Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes

The enteric nervous system (ENS) is composed of neurons and glial cells, organized as interconnected ganglia within the gut wall, which controls peristalsis of the gut wall and secretions from its glands. The Ret receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development; humans with mutations in the RET locus have Hirschsprung disease (HSCR, an absence of ganglia in the colon), and mice lacking Ret have total intestinal aganglionosis. The Ret mutant mouse provides a tool for identifying genes implicated in development of the ENS. By using RNA from WT and Ret mutant (aganglionic) gut tissue and DNA microarrays, we have conducted a differential screen for ENS-expressed genes and have identified hundreds of candidate ENS-expressed genes. Forty-seven genes were selected for further analysis, representing diverse functional classes. We show that all of the analyzed genes are expressed in the ENS and that the screen was sensitive enough to identify genes marking only subpopulations of ENS cells. Our screen, therefore, was reliable and sensitive and has identified many previously undescribed genes for studying ENS development. Moreover, two of the genes identified in our screen Arhgef3 and Ctnnal1, have human homologues that map to previously identified HSCR susceptibility loci, thus representing excellent candidates for HSCR genes. This comprehensive profile of ENS gene expression refines our understanding of ENS development and serves as a resource for future developmental, biochemical, and human genetic studies.     

Changes in adrenergic and peptidergic nerves in the submucous plexus of streptozocin-diabetic rat ileum.

The effect of streptozocin diabetes on the distribution of adrenergic and peptidergic nerves in the submucous plexus of rat ileum was investigated and compared with the changes in the myenteric plexus of the same region of ileum. There was an increase in the intensity of immunoreactivity in vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactive nerve fibers and neurons and a decrease in calcitonin gene-related peptide-like immunoreactivity but no change in substance P- and dopamine beta-hydroxylase-like immunoreactivity in the nerve fibers and neurons of the submucous plexus of both 8- and 16-wk streptozocin-diabetic rat ileum. However, in the myenteric plexus of the diabetic rat ileum, there was enlargement of varicosities and an increase followed by a slight decrease in the intensity of immunoreactivity of vasoactive intestinal polypeptide- and dopamine beta-hydroxylase-like immunoreactive nerve fibers and neurons, increased substance P-like immunoreactivity in diabetes at 16 wk, and an initial decrease (at 8 wk) followed by a recovery of calcitonin gene-related peptide-like immunoreactivity at 16 wk, but no change in neuropeptide Y-like immunoreactivity. The markedly different changes in peptidergic and adrenergic nerves between the two enteric plexuses show that diabetic neuropathy induced by streptozocin is not selective and involves factors other than neurotransmitter types.     

应激对大鼠结肠神经系统nNOS表达的影响

目的:探讨应激对大鼠结肠神经系统nNOS表达的影响. 方法:SD大鼠30只随机分为对照组,应激组和L-NAME 组,采用水浸-束缚应激(WRS)动物模型,用免疫组织化学ABC法检测nNOS在大鼠结肠黏膜下神经丛和肌间神经丛的表达,应用计算机图像分析系统对其表达进行定量分析．结果:与对照组比较,应激组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值明显减少(P=0．02或P =0．005),阳性神经元细胞数的平均密度增加(P=0．04 或P=0．01),表达增强,且在黏膜上皮细胞、固有层淋巴细胞也有nNOS表达．L-NAME组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的灰度值较应激组增加 (P=0．04),平均密度下降(P=0．04或P=0．03),表达减弱,而与对照组比较均无明显差异(P>0．05)．结论:应激可引起大鼠结肠神经系统nNOS表达增强, 提示一氧化氮(NO)在应激所致的结肠功能失调中可能起重要作用．     

Rectal biopsy for diagnosis of intestinal neuronal dysplasia in children: a prospective multicentre study on interobserver variation and clinical outcome

BACKGROUND: Intestinal neuronal dysplasia (IND) of the colonic submucous plexus is considered to be a congenital malformation of the enteric nervous system causing symptoms resembling those of Hirschsprung's disease. In contrast with the established diagnosis of aganglionosis using enzyme histochemistry, controversy exists over the diagnostic criteria of IND on rectal biopsies previously defined by a consensus report and the causal relation between morphological findings and clinical symptoms. AIMS: The interobserver variability was prospectively investigated with respect to final diagnoses and several histological features in rectal biopsy specimens from children suspected of having colonic motility disturbances. METHODS: 377 biopsy specimens from 108 children aged 4 days to 15 years were independently coded without knowledge of clinical symptoms by three experienced pathologists for 20 histological features, and a final diagnosis was given for every case. Interobserver variation for the different items and the final diagnosis were analysed using Cohen's kappa statistic. Clinical data at biopsy and outcome after 12 months were related to morphological findings. RESULTS: The three pathologists agreed completely with respect to the diagnosis Hirschsprung's disease (kappa = 1), but in only 14% of the children without aganglionosis. In 15 (17%) of the 87 children without aganglionosis, at least one pathologist judged the case as normal, while another diagnosed IND. kappa values were close to the zero value expected by chance for the diagnoses normal and IND. Young age was related to the presence of several morphological features-for example, acetylcholine esterase staining and presence of giant ganglia. Children with chronic constipation diagnosed as having IND, given no other specific diagnosis by any of the pathologists, were significantly younger (median 8.8 months) and had a higher cure rate after one year (60%) than constipated patients considered by all observers to have no histological abnormalities (median 6.1 years, cure rate 23%). CONCLUSIONS: In contrast with Hirschsprung's disease, there is a high interobserver variation with regard to the different morphological features and final diagnosis of IND, based on the criteria and conditions of the previous consensus report. The high frequency of histological "abnormalities" in young infants suggests that some of the features may represent a normal variant of postnatal development rather than a pathological process. Investigations using more refined and morphometric methods in rectal specimens from infants and children without bowel disease are needed to define the normal range of morphological appearance at different ages. These preliminary data indicate that, with current knowledge, rectal biopsy for diagnostic purposes should only be performed in constipated children for diagnosis of Hirschsprung's disease.     

Localization of endothelinlike immunoreactivity and endothelin binding sites in human colon

The potent vasoconstrictor endothelin was originally isolated from vascular endothelial cells but has since been found in several other tissues. The aim of this study was to establish whether endothelinlike immunoreactivity occurs in human enteric nerves and to identify endothelin binding sites using immunocytochemical and in vitro autoradiographic techniques. Endothelinlike immunoreactivity was localized to nerve bundles throughout the colon and to most of the ganglion cells of the two major plexuses, many of which costored vasoactive intestinal polypeptide. High-affinity (dissociation constant = 0.35 +/- 0.014 nmol/L; mean +/- SEM) binding sites for endothelin 1, with an apparent binding capacity of 92 +/- 6.3 amol/mm2 (mean +/- SEM), were demonstrated in the myenteric plexus, with less dense binding being found in the submucous plexus, mucosa, muscle layers, and blood vessel walls. Competition data suggested two populations of binding sites, both showing high affinities for endothelins 1 and 2, vasoactive intestinal constrictor, and sarafatoxin b but differentiated by their affinity for endothelin 3 and sarafatoxin c. This study provides evidence that endothelin is a neuropeptide in the human intestine with binding sites on neural plexuses and mucosa, suggesting a role in the modulation of intestinal motility and secretion.     

Notable postnatal alterations in the myenteric plexus of normal human bowel

BACKGROUND: Nitric oxide is the most important transmitter in non-adrenergic non-cholinergic nerves in the human gastrointestinal tract. Impaired nitrergic innervation has been described in Hirschsprung's disease, hypertrophic pyloric stenosis, and intestinal neuronal dysplasia (IND). Recent findings indicate that hyperganglionosis, one of the major criteria of IND, is age dependent. However, information is scanty regarding the neurone density in normal human bowel in the paediatric age group. AIMS: To determine neurone density, morphology, and nitric oxide synthase distribution of the normal myenteric plexus at different ages during infancy and childhood. METHODS: Specimens were obtained from small bowel and colon in 20 children, aged one day to 15 years, at postmortem examination. Whole mount preparations were made of the myenteric plexus, which were subsequently stained using NADPH diaphorase histochemistry (identical to nitric oxide synthase) and cuprolinic blue (a general neuronal marker). The morphology of the myenteric plexus was described and the neurone density estimated. RESULTS: The myenteric plexus meshwork becomes less dense during the first years of life. The density of ganglion cells in the myenteric plexus decreases significantly with age during the first three to four years of life. The NADPH diaphorase positive (nitrergic) subpopulation represents about 34% of all neurones in the myenteric plexus. CONCLUSIONS: The notable decrease in neurone density in the myenteric plexus during the first years of life indicates that development is still an ongoing process in the postnatal enteric nervous system. Applied to the clinical situation, this implies that interpretation of enteric nervous system pathology is dependent on the age of the patient.     

Origins of peptide and norepinephrine nerves in the mucosa of the guinea pig small intestine

Norepinephrine, acetylcholine, and certain peptides are contained in mucosal nerves and have potent effects on transepithelial water and electrolyte fluxes. It is difficult to ascribe roles for these nerves as their sources are unknown. The present studies were undertaken to determine the origins of nerve fibers that are found in the mucosa of the guinea pig small intestine and which contain one of the following substances: vasoactive intestinal peptide, substance P, somatostatin, neuropeptide Y, cholecystokinin, or norepinephrine. Nerve fiber origins were ascertained by making lesions to sever pathways through which the nerves could reach the mucosa. The lesioning operations were homotopic autotransplants of short (2 cm) segments of intestine; myectomies, in which a 5-10-mm length of intestine was stripped of longitudinal muscle and myenteric plexus; and extrinsic denervation, in which nerves reaching the intestine through the mesentery were severed. The results of these studies, considered along with previously published work, led to the upcoming conclusions. Nerve fibers in the mucosa showing immunoreactivity for vasoactive intestinal peptide, somatostatin, cholecystokinin, and neuropeptide Y arise from cell bodies in the overlying submucous plexus. Substance P fibers arise in part from the overlying submucous plexus and in part from the overlying myenteric plexus. Mucosal norepinephrine fibers arise from extrinsic sympathetic ganglia. Enkephalin, gastrin-releasing peptide, and 5-hydroxytryptamine, which are in some enteric nerves, are not found in submucous nerve cells and few, if any, fibers containing these substances supply the mucosa. Thus, the mucosa receives a dense nerve supply, much of which arises locally from submucous ganglia.     

Intestinale Innervationsstörungen: Anatomische Grundlagen und Diagnostik

Histopathological insights into intestinal innervation disorders require the knowledge of the anatomical topography and structure of the enteric nervous system (ENS). The ENS of the human colon is composed of intramural nerve meshes located in different layers of the intestinal wall (plexus musculares, plexus myentericus, plexus submucosi, plexus mucosi). A differentiated visualization of the components of the ENS is achieved-by enzyme- and immunohistochemical methods. In comparison to cross-sections, wholemount preparations of the intestinal wall allow a two dimensional demonstration of the network-character of the nerve plexus and preserve the anatomical in-situ conditions. A complete histopathological diagnosis of the ENS requires full-thickness biopsies or resected specimens, as an assessment of the entire plexus layers is achieved only by this procedure. The establishment of reference values by means of morphometrical studies of ENS contributes to an objective and standardized histopathological diagnosis. Aganglionosis, hypoganglionosis, intestinal neuronal dysplasia and heterotopias are considered to be the most acknowledged forms of intestinal innervation disorders. In addition, degenerative changes of enteric nerve plexus, visceral neuropathies and a disturbed homeostasis of enteric neurotransmitters are described. Recently, a pathogenetical significance for functional intestinal motility disorders has been attributed to interstitial cells of Cajal (ICC). The ENS and ICC play a major role in mediating and coordinating gastrointestinal motility and therefore shoud be taken into account in clinical disorders of the intestinal motor function.     

Serotoninergic neurons in human fetal intestine: an immunohistochemical study

The distribution of 5-hydroxytryptamine-like immunoreactivity was studied in whole-mount preparations of intestine from human fetuses. Immunoreactive nerve cell bodies were located in the myenteric plexus and were occasionally found in the submucous plexus; they were often seen to have long processes. Varicose fibers were found in the ganglia and internodal strands of the myenteric and submucous plexuses, in the deep muscular plexus of the circular muscle, and in the walls of some small mesenteric blood vessels immediately outside the intestine. This study provides evidence for the presence of serotoninergic nerves in the human intestine.     

Jejunal diverticulosis with perforation as a complication of Fabry's disease

This study presents the case of a patient who had jejunal diverticulosis with perforation and abscess formation as a complication of Fabry's disease. Light microscopy disclosed glycolipid deposition in the neurons and nerve fibers of the intestinal nerve plexuses and smooth muscle. Silver stains of the myenteric plexus in the involved segment of the bowel showed enlarged, granular argyrophobic neurons and a marked decrease in the number of argyrophilic neurons, with those remaining being enlarged and distorted by the cytoplasmic glycolipid accumulation. These abnormalities of the myenteric plexus suggest that jejunal diverticulosis may be the result of a variety of disorders of the smooth muscle or myenteric plexus, or both. We propose that jejunal diverticulosis in our patient was a consequence of uncoordinated smooth muscle activity resulting from Fabry's involvement of myenteric plexus neurons, with mucosal protrusion through the smooth muscle.     

Decreased expression of hyperpolarisation-activated cyclic nucleotide-gated channel 3 in Hirschsprung's disease

AIM: To determine if hyperpolarisation-activated nucleotide-gated(HCN) channels exist in human colon, and to investigate the expression of HCN channels in Hirschsprung's disease.METHODS:We investigated HCN1,HCN2,HCN3 and HCN4 protein expression in pull-through specimens from patients with Hirschsprung’s disease(HSCR,n=10)using the proximal-most ganglionic segment and distalmost aganglionic segment,as well as in healthy control specimens obtained at the time of sigmoid colostomy closure in children who had undergone anorectoplasty for imperforate anus(n=10).Fluorescent immunohistochemistry was performed to assess protein distribution,which was then visualized using confocal microscopy.RESULTS:No HCN1 channel expression was observed in any of the tissues studied.Both HCN2 and HCN4proteins were found to be equally expressed in the aganglionic and ganglionic bowel in HSCR and controls.HCN3 channel expression was found to be markedly decreased in the aganglionic colon vs ganglionic colon and controls.HCN2-4 channels were seen to be expressed within neurons of the myenteric and submucosal plexus of the ganglionic bowel and normal controls,and also co-localised to interstitial cells of Cajal in all tissues studied.CONCLUSION:We demonstrate HCN channel expression in human colon for the first time.Reduced HCN3expression in aganglionic bowel suggests its potential role in HSCR pathophysiology.     

Reaggregation of Rat Dissociated Myenteric Plexus in Extracellular Matrix Gels

The aim of this study was to investigate the growth behavior of freshly dissociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurotrophic factor (GDNF). Therefore, cell suspensions of the dissected myenteric plexus of newborn rats were cultured in freshly prepared gels of commercially available mixtures of collagen, laminin, and hepatoglycans as a first step towards mimicking the natural environment of the myenteric plexus. The cultures were kept either in chemically defined serum-free medium alone or supplemented with GDNF. Cultures on polylysine-coated glass cover slips served as controls. Dissociated myenteric plexus grown on polylysine formed dense clusters of neurons with radially outgrowing nerve fibers, while the neurons cultured in the gel reaggregated to much smaller clusters. These contained, depending on the culture conditions, 2–10 neurons. The morphology of the network that was seen in the gels after a few days in vitro resembled very closely the in situ situation of the submucous plexus and the myenteric plexus in hypoganglionic children. Electron microscope investigations showed a high degree of organization with fiber bundles and vesicle-containing varicosities and growth cones. Independent of the method of culturing, GDNF obviously influenced the growth behavior of the dissociated plexus. The size of the ganglia was larger, and the secondary network denser when GDNF was supplemented. Moreover, the enteric neurons in the gel cultures tended to be larger in size when treated with GDNF. Three-dimensional cultures of dissociated myenteric plexus in an ECM gel might be a valuable tool towards the understanding of the formation of the enteric nervous system during development, especially considering pathological conditions such as Hirschsprungs disease or other dysganglionic diseases.