Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: Their projections in the guinea-pig small intestine

Changes in the distribution of 5-hydroxytryptamine-like immunoreactivity have been examined in enteric neurons at various times after microsurgical lesions of the enteric plexuses. In the myenteric plexus, varicose immunoreactive nerve fibres disappeared or were reduced in number in ganglia anal to an interruption of the myenteric plexus. Up to about 2 mm on the anal side, all varicose immunoreactive fibres disappeared from the ganglia. At about 14–16 mm below an interruption, there were about 50% of the normal number of fibres in the myenteric ganglia and at about 24 mm the innervation was normal. In the submucosa, fibres immunoreactive for 5-hydroxytryptamine were absent from an area on the anal side following interruption of the myenteric plexus. From consideration of the pattern of disappearance, it is deduced that some myenteric nerve cell bodies send immunoreactive axons in an anal direction to supply submucous ganglia. The axons run for about 8 mm in the myenteric plexus, enter the submucosa and then run for a further 4 mm approximately.Thus, varicose fibres immunoreactive for 5-hydroxytryptamine, which occur around the enteric ganglion cells of both plexuses arise from nerve cell bodies in the myenteric ganglia that send their axons in an anal direction.     

The expression pattern of laminin isoforms in Hirschsprung disease reveals a distal peripheral nerve differentiation

Hirschsprung disease (HD), a developmental disorder, is associated with failure of enteric ganglia formation. Signaling molecules, including secreted basement membrane molecules, derived from the mesenchyme of the gut wall play an important role in the colonization and/or differentiation of the enteric nervous system. The current study aims to define the possible alterations of laminins involved in the pathogenesis of HD. Expression of the various laminin alpha, beta, and gamma chains, was assessed in the aganglionic, transitional, and ganglionic bowel segments of patients with HD or with other motor disorders. Cytoskeletal, neuronal, and glial markers were also included in this study. The major finding highlighted by the present work concerns the clear identification and location of myenteric aganglionic plexuses in HD with some of the laminin antibodies, which reveal a peripheral nerve type of differentiation. Furthermore, we could show an increase of laminin alpha5 chain immunostaining in the dilated muscle of the ganglionic bowel upstream the distal aganglionic region in a subgroup of patients with HD, as well as a relocalization of laminin alpha2 chain in the subepithelial basement membrane. Overall, these basement membrane molecules could provide useful markers for diagnosis of aganglionosis or hypoganglionosis.     

Glial cell line-derived neurotrophic factor family receptors are abnormally expressed in aganglionic bowel of a subpopulation of patients with Hirschsprung's disease

Hirschsprung's disease (HSCR), a congenital disease, is characterized by the absence of ganglion cells in the ganglion plexuses of the caudal most gut. In the aganglionic colon, the plexus remnants are replaced by aggregates of glial cells and hypertrophied nerve fibers. Signaling of glial cell line-derived neurotrophic factor (GDNF)-GFRAs-receptor tyrosine kinase (RET) is crucial for the development and maintenance of ganglion cells. Mutations of genes such as GDNF and RET lead to the perturbation of this signaling pathway, which causes HSCR. To understand the role of GFRAs in ganglion cells and the pathogenesis of HSCR, we intended to determine the specific cell lineages in the enteric nervous system that normally express GFRAs but are affected in HSCR. We studied colon biopsy specimens from 13 patients with HSCR (aged 1 day to 38 months) and 6 age-matched patients without HSCR as normal controls. RT-PCR, in situ hybridization, and immunohistochemistry were performed to examine the expression and cellular distributions of GFRAs in resected bowel segments of normal infants and those with HSCR. In normal infants and normoganglionic colon of patients with HSCR, the expression of GFRA1 was restricted to the glial cells and neurones of the ganglion plexuses. GFRAs expression was found to be markedly reduced in the aganglionic colons of 3 infants with HSCR but was unaffected in the aganglionic colons of 10 other infants with HSCR. Residual GFRA expression was restricted to enteric glial cells in the plexus remnants of the aganglionic colons. Hypertrophied nerve fibers were not found to express GFRA1. We provide the first evidence that abnormal expression of GFRAs in the enteric nervous system may be involved in the pathogenesis of HSCR in a subpopulation of patients.     

Nervous system development in normal and atresic chick embryo intestine: an immunohistochemical study

Intestinal motility disorders are a common complication after surgery for neonatal intestinal atresia. Although intestinal atresia causes alterations in the enteric nervous system, especially in its inner structures (nervous fibers in the mucosa, submucous and deep muscular plexuses), how these alterations develop is unclear. The chick model is a useful research tool for investigating the ontogenesis of the enteric nervous system and the pathogenesis of congenital bowel diseases. More information is needed on the overlap between the developing enteric nervous system and intestinal atresia. Because vasoactive intestinal polypeptide and substance P are typical intestinal neuropeptides, and vasoactive intestinal polypeptide acts as a modulator in neurodevelopment and an inhibitor of smooth muscle cell proliferation, our aim in this study was to investigate the distribution of their immunoreactivity in the developing enteric nervous system of normal and experimental chick models. We studied gut specimens excised from normal chick embryos (aged 12–20 days) and experimental chick embryos (aged 15–20 days) that underwent surgical intervention on day 12 to induce intestinal atresia (atresic embryos) or simply to grasp the bowel loop (sham-operated embryos). In normal chick embryos we showed vasoactive intestinal polypeptide and substance P immunoreactivity from day 12 in the submucous and myenteric plexuses. The distribution of peptide immunoreactivity differed markedly in atresic and normal or sham-operated gut embryos. These differences especially affected the inner structures of the enteric nervous system of specimens proximal to atresia and were related to the severity of dilation. Because nerve structures in the gut wall mucosa and submucous and deep muscular plexuses play a role in motility control and stretch sensation in the intestinal wall, our findings in the chick embryo may help to explain how gut motility disorders develop after surgery for neonatal intestinal atresia.     

Expression profile of some neuronal and glial cell markers in the ovine ileal enteric nervous system during prenatal development

The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer’s patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60–90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60–90 days), but later (90–150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60–90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120–150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120–150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons.     

Opioid-like immunoreactive neurons in secretomotor pathways of the guinea-pig ileum

In this study we sought to establish the distribution, projections and neurochemical coding of opioid immunoreactive neurons in secretomotor pathways of the guinea-pig ileum. Non-cholinergic secretomotor neurons in the submucous ganglia have been shown to be immunoreactive for dynorphin A 1-8, dynorphin A 1-17, dynorphin B and alpha neo-endorphin while cholinergic neurons have been shown to be immunoreactive for dynorphin A 1-8 only. Thus all submucous neurons in the guinea-pig ileum are immunoreactive for prodynorphin-derived peptides. Two major populations of opioid immunoreactive fibres projecting to the submucous ganglia have been established. Firstly, neurons immunoreactive for prodynorphin-derived peptides and vasoactive intestinal peptide project anally from the myenteric plexus to the submucous ganglia. Secondly, a substantial proportion of sympathetic postganglionic fibres immunoreactive for tyrosine hydroxylase, and projecting from the coeliac ganglion to submucous ganglia, have been shown to be immunoreactive for prodynorphin-derived peptides. Other smaller populations of opioid-immunoreactive neurons include fibres immunoreactive for substance P, enkephalin and dynorphin A 1-8 which project from the myenteric plexus to the non-ganglionated plexus of the submucosa. These fibres are probably excitatory motor neurons to the muscularis mucosae. The present paper has described several distinct populations of opioid immunoreactive neurons in secretomotor pathways of the guinea-pig ileum. Furthermore we have shown that these enteric or postganglionic sympathetic neurons contain opioid peptides in combination with other neurotransmitter substances. These results should provide a firmer basis on which to plan functional experiments to elucidate the physiological role of opioid peptides in the enteric nervous system.     

Immunohistochemical study of neuron specific enolase and S-100 protein in Hirschsprung's disease

Summary The distribution of whole differentiated neurons in the intestines from 15 children with Hirschsprung's disease was investigated using neuron specific enolase (NSE) and the perineuronal elements were studied using S-100 protein immunostaining.In aganglionic segments, NSE immunoreactive ganglion cells and S-100 positive satellite cells were absent, but the hypertrophic nerve trunks did show a markedly positive NSE and S-100 immunoreactivity.Two different forms of aganglionic segment were present. One was the middle aganglionic segment of long segment aganglionosis which was almost completely dennervated. In the other type, there were several NSE positive nerve fibers in the muscularis propria of both the aganglionic segment of short segment aganglionosis and the distal aganglionic segment of long segment aganglionosis. These latter two aganglionic segments seemed to be innervated by extrinsic nerves.     

P2X2受体在大鼠胃肠系统肌间神经节中的分布

目的:研究P2X2受体在大鼠胃肠系统肌间神经节中的分布,为研究ATP作为神经递质对胃肠道肌间神经节节细胞的作用提供形态学基础。方法:免疫组织化学。结果:从胃到结肠均有P2X2受体阳性神经元和神经纤维分布,胃、小肠和结肠分别约60％、70％和50％的肌间神经节节细胞为P2X2受体阳性。结论:胃肠道系统多数肌间神经节节细胞都能通过ATP受体特别是P2X2受体对ATP产生反应。     

Somatostatin neurons in the small intestine of the guinea pig: A light and electron microscopic immunocytochemical study combined with nerve lesion experiments by laser irradiation

Summary Somatostatin-like immunoreactive neurons are present in both the myenteric and the submucous plexuses of the small intestine of the guinea pig. Dense varicosities of immunopositive nerve fibres surround the ganglionic cells, some of which also display somatostatin-like immunoreactivity. Immunoelectron microscopy demonstrated axo-somatic synapse formation between the somatostatin immunoreactive neuronal elements. Nerve lesion experiments using argon laser irradiation showed that most of the somatostatin-like immunoreactive fibres of the myenteric plexus were directed anally, whereas those of the submucous plexus had no directional polarity.     

Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in lethal spotted mutant mice

In order to gain insight into the process of colonization of the bowel by the neural crest-derived precursors of enteric neurons, the development of the enteric nervous system was examined in lethal spotted mutant mice, a strain in which a segment of bowel is congenitally aganglionic. In addition, nerve fibers within the ganglionic and aganglionic zones of the gut of adult mutant mice were investigated with respect to their content of acetylcholinesterase, immunoreactive substance P, vasoactive intestinal polypeptide and serotonin, and their ability to take up [³Hserotonin. In both the fetal gut of developing mutant mice and in the mature bowel of adult animals abnormalities were limited to the terminal 2 mm of colon. The enteric nervous system in the proximal alimentary tract was indistinguishable from that of control animals for all of the parameters examined. In the terminal bowel, the normal plexiform pattern of the innervation and ganglion cell bodies were replaced by a coarse reticulum of nerve fibers that stained for acetylcholineserase and were continuous with extrinsic nerves running between the colon and the pelvic plexus. These coarse nerve bundles contained greatly reduced numbers of fibers that displayed substance P- and vasoactive intestinal polypeptide-like immunoreactivity, but a serotonergic innervation was totally missing from the aganglionic bowel. During development, acetylcholineserase and uptake of [³Hserotonin appeared in neural elements in the foregut of mutant mice on the 12th day of embryonic life (E12), about the same time these markers appeared in the forgut in normal mice. By day E14, neurons expressing one or the other marker were recognizable as far distally as about 2 mm from the anus. The appearance of neurons in segments of gut grown for 2 weeks as expiants in culture was used as an assay for the presence of neuronal progenitor cells in the segments of fetal bowel at the time of explantation. Both acetyl- cholinesterase activity and uptake of [³Hserotonin developed in neuronsin vitro in expiants of proximal bowel between days E10 and E17. At all times, however, the terminal 2mm of mutant but not normal fetal gut gave rise to aneuronal cultures. In some mutant mice rare, small, ectopically-situated pelvic ganglia were found just outside aganglionic segments of fetal colon. Uptake of [³Hserotonin, normally a marker for intrinsic enteric neurites, was found in these ganglia.The experiments suppport the hypothesis that the terminal 2 mm of the gut in lethal spotted mutant mice is intrinsically abnormal and thus cannot be colonized by the precursors of enteric neurons. The defect seems to be specific in that both cells and processes of intrinsic enteric neurons, including all serotonergic and most peptidergic neurites, seem to be excluded from the abnormal region while extrinsic nerve fibers, including sympathetic and sensory axons, are able to enter the aganglionic zones. Since examination of neural progenitor cells has failed to reveal a significant proximo-distal displacement of these cells through the enteric tube during development of the murine bowel, a defect in the migration of precursor cells down the alimentary tract to the terminal gut seems unlikely to be substantially involved in the pathogenesis of aganglionosis. This conclusion is supported by the normal enteric nervous system in proximal regions of the mutant gut and the presence of enteric type neurons outside of, but at the same level as the aganglionic region.     

Hu蛋白作为豚鼠小肠肌间丛神经元标志物的研究

比较5种神经元标志物在小肠肌间丛神经元中的特异性。应用新发现的神经元标志物Hu蛋白，对豚鼠肠神经系统（ENS）的小肠肌间丛神经元进行免疫荧光染色研究，并与其他神经元标志物MAP－2，PGP9.5,NSE,TUJ-1进行比较。结果表明：Hu蛋白主要分布于神经元胞浆中，神经元核也有较浅的染色，尤其在whole-mount上，每个神经元都清晰可见。MAP－2作为神经元标志物，也能显示完好的细胞形态，但由于粗大神经纤维的强染色，使神经元计数不准确。PGP9.5,NSE和TUJ－1对体外培养的小肠肌间丛神经元染色良好，但在whole-mount上，神经纤维强染色，无法计数分析。结果提示在豚鼠肠神经系统小肠肌间丛神经元的计数研究中，Hu蛋白是理想的神经元标志物。     

Selective demonstration of mural nerves in ganglionic and aganglionic colon by immunohistochemistry for glucose transporter-1: prominent extrinsic nerve pattern staining in Hirschsprung disease

OBJECTIVE: Hypertrophic nerves have long been considered a histopathologic feature of the aganglionic segment in Hirschsprung disease, but they remain incompletely explained. The purpose of this study was to define the nature and diagnostic importance of hypertrophic nerves in Hirschsprung disease and to clarify their relation to nearby smaller nerve fibers. METHODS: We used an immunoperoxidase staining technique to compare the distribution of 2 nerve markers-erythrocyte-type glucose transporter (GLUT-1), a marker of perineurium, and nerve growth factor receptor, a marker of both nerve fibers and perineurium-in aganglionic tissue (12 resected specimens and 4 rectal biopsies) and control tissue (6 autopsy specimens and 17 rectal biopsies) of children. RESULTS: In control ganglionic tissue, the myenteric and submucosal areas contained only occasional GLUT-1-positive nerves (usually less than 50 microm in diameter), but extramural extrinsic (serosal) nerves were invariably positive for GLUT-1. In aganglionic tissue, GLUT-1-positive nerves in the myenteric and submucosal areas were frequent and included both large (50-150 microm) and small (<50 microm) diameter nerves. Nerve growth factor receptor-positive fibers were frequent in all layers of all tissue studied. In aganglionic bowel, a distinct perineurium could be identified in the largest nerves, but nerve growth factor receptor had poor discrimination for small perineurium-sheathed nerves. CONCLUSION: Most nerves, of both large and small diameter, in the myenteric and submucosal plexus of aganglionic bowel are GLUT-1 positive. Serosal extrinsic nerves stain identically, supporting the interpretation that the mural nerves are of extrinsic origin. Mural GLUT-1-positive nerves, when they are multiple and especially when they are greater than 50 microm in diameter (a figure which may be used as a threshold for hypertrophic nerves), are suggestive of Hirschsprung disease.     

New roles of EDNRB and EDN3 in the pathogenesis of Hirschsprung disease

PurposeHirschsprung disease is characterized by the absence of intramural ganglion cells in the myenteric and submucosal plexuses within distal intestine, because of a fail in the enteric nervous system formations process. Endothelin-3-endothelin receptor B signaling pathway is known to play an essential role in this process. The aim of this study was to evaluate the implication of the EDN3 and EDNRB genes in a series of patients with Hirschsprung disease from Spain and determinate their mutational spectrum.MethodsWe performed the mutational screening of both genes in 196 patients with Hirschsprung disease using denaturing high-performance liquid chromatography technology. A case-control study using TaqMan Technology was also carried out to evaluate some common polymorphisms and haplotypes as susceptibility factors for Hirschsprung disease.ResultsBesides several novel mutations in both genes, we found a truncating mutation in an alternative isoform of EDNRB. Interestingly, we obtained an overrepresentation of a specific EDN3 haplotype in cases versus controls.ConclusionsOur results suggest that the isoform EDNRBΔ3 might be playing an essential role in the formation of enteric nervous system. In addition, based on the haplotype distribution, EDN3 might be considered as a common susceptibility gene for sporadic Hirschsprung disease in a low-penetrance fashion.     

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.     

Altered distribution of interstitial cells of Cajal in Hirschsprung disease

CONTEXT: Constipation or recurrent intestinal dysmotility problems are common after definitive surgical treatment in Hirschsprung disease (HD). c-Kit-positive interstitial cells of Cajal (ICCs) play a key role in the motility function and development of the gastrointestinal tract. Interstitial cells of Cajal that carry the tyrosine kinase receptor (c-Kit) develop as either myenteric ICCs or muscular ICCs under the influence of the kit ligand, which can be provided by neuronal and nonneuronal cells, for example, smooth muscle cells. OBJECTIVE: To investigate the distribution of myenteric and muscular ICCs in different parts of the colon in HD. METHODS: Resected bowel specimens from 8 patients with rectosigmoid HD were investigated using combined staining with c-Kit enzyme and fluorescence immunohistochemistry and acetylcholinesterase and nicotinamide adenine dinucleotide phosphate (NADPH) histochemistry in whole-mount preparations and conventional frozen sections. RESULTS: In the normal bowel, ICCs formed a dense network surrounding the myenteric plexus and at the innermost part of the circular muscle. Myenteric ICCs were absent or sparse in the aganglionic bowel and sparse in the transitional zone. The expression of myenteric ICCs in the ganglionic bowel in HD was reduced compared to that in the normal bowel, and they formed only sparse networks. Muscular ICCs were found in the aganglionic bowel, transitional zone, and normoganglionic bowel of HD in a reduced density compared to the normal bowel. CONCLUSION: This study demonstrates altered distribution of ICCs in the entire resected bowel of HD patients. This finding suggests that persistent dysmotility problems after pull-through operation in HD may be due to altered distribution and impaired function of ICCs.     

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease

Background  

Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach.     

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine.

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.