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The National Children's Research Centre (NCRC), the single largest paediatric research centre in Ireland, has been in existence for over 50 years and is located on the grounds of the largest children's hospital in Ireland; Children's Health Ireland at Crumlin. Professor Puri was appointed as the Director of the Research in 1989 and became President of the NCRC in 2009, a position he held until 2016. Professor Puri is one of the most cited paediatric surgical researchers in the world. His research work has been cited over 23,500 times in peer-reviewed articles with an h-index of 76 and i10-index of 494. The aim of this review is to analyse the most relevant areas of academic research at the NCRC, Dublin, during the years when Prof. Puri was Director/President of the NCRC. In addition, the relevant factors essential to create a successful paediatric surgical research centre will be discussed. A literature search using PubMed/Medline was carried out using the search terms "Prem Puri" over a 40-year period (1980-2020). Articles were analysed to identify the most significant research areas in the field of paediatric surgical research and the relevant laboratory and clinical findings. In addition, a separate analysis of successful funding and human factors, such as research fellows working at the NCRC, was also carried out. During the period under review, Professor Puri's team published 750 articles in peer-reviewed journals. Three main areas of research were reviewed with a total number of 391 articles: congenital diaphragmatic hernia (CDH) was the topic with the largest number of publications (153), followed by Hirschsprung's disease (HSCR) (144) and Vesicoureteral reflux (VUR) (94). Eighty research fellows, all paediatric surgeons, from 18 different countries were trained in basic science under the supervision of Professor Puri at the NCRC. Over the last three decades, the NCRC has been one of the most successful centres for paediatric surgical research in the world. The three areas of research with the largest number of publications were identified as CDH, HSCR and VUR. Various factors can explain the success of the NCRC: (a) the constant presence, for over 30 years, of a very successful paediatric surgeon leading the centre, (b) a multicultural laboratory with research fellows from all over the world and (c) grants of more than 15 million raised over the years, which guaranteed a constant flow of resources for laboratory research., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aim of the Study: Potassium (K + ) channels with a two-pore domain (K2P) are a large family of hyperpolarising ion channels which play a key role in cell excitability. This family comprises three members: TREK-1, TREK-2 and TRAAK. TRAAK channels have previously been reported to be expressed in murine enteric ganglia. To date, no data exist regarding TRAAK channel expression in the human colon. Thus, we designed this study to investigate TRAAK gene expression in the normal human colon and in Hirschsprung's disease (HSCR)., Methods: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify TRAAK gene expression, and immunolabelling of TRAAK proteins was visualized using confocal microscopy., Main Results: Confocal microscopy revealed TRAAK protein expression within both neurons and interstitial cells of Cajal in the myenteric plexus, with a reduction in both ganglionic HSCR colon and aganglionic HSCR colon, compared to controls. qRT-PCR analysis revealed a significant downregulation of the TRAAK gene in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05)., Conclusions: TRAAK gene expression is significantly downregulated in HSCR colon, suggesting a role for these ion channels in colonic neurotransmission. TRAAK downregulation within ganglionic specimens highlights the dysfunctional nature of ganglia in this region.

Background/purpose: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is unclear. Caveolin-1 (Cav-1) regulates the functions of different nitric oxide synthase (NOS) isoforms, which play critical roles in inflammation and intestinal epithelial barrier function. We designed this study to investigate the hypothesis that Cav-1 expression is altered in the bowel of patients with Hirschsprung's disease (HSCR)., Methods: HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). qRT-PCR analysis was undertaken to quantify Cav-1 gene expression, and Western blot analysis was undertaken to determine Cav-1 protein quantification. Immunolabelling of Cav-1 proteins was visualized using confocal microscopy., Results: qRT-PCR and Western blot analysis revealed that Cav-1 was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls (p < 0.01). Confocal microscopy revealed a markedly decreased expression of Cav-1 in colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls., Conclusion: To our knowledge, this is the first report of significantly decreased Cav-1 expression in patients with HSCR. Decreased expression of Cav-1 in the bowel of HSCR may increase susceptibility to HAEC in HSCR.

Background/purpose: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare congenital and generally fatal cause of functional intestinal obstruction in the newborn. The cause of this syndrome is unknown. Familial occurrence and reports of consanguinity in MMIHS implies that genetic factors may have an important role in the pathogenesis of this syndrome. The aim of the study was to determine the consequence of consanguinity for the incidence of MMIHS., Methods: A literature search was performed using the keywords "megacystis microcolon intestinal hypoperistalsis" for studies published between 1976 and 2018. Retrieved articles, including additional studies from reference lists, were reviewed for consanguinity between parents and recurrence of MMIHS between siblings. Data were extracted for cases where familial MMIHS was present., Results: A total of 450 patients with the diagnosis of MMIHS have been reported in the literature. There were 56 (12%) cases in which familial MMIHS was confirmed, 25 families with multiple siblings and 3 families with single affected infant. Of the 25 families with multiple siblings, 22 families had 2 siblings with confirmed MMIHS and 3 families had 3 children each with MMIHS. Consanguinity between parents was confirmed in 30 cases (18 siblings and 12 individual cases). Female-to-male ratio in the 30 patients was 4.4:1., Conclusion: The occurrence of MMIHS in the offspring of consanguineous parents and recurrence in siblings of healthy parents suggest that MMIHS is an autosomal recessive disorder. Pre-marital and pre-conception counselling of consanguineous populations is recommended to prevent harmful consequences.

Purpose: Voltage-gated potassium ion channels have long been implicated in gastrointestinal motility. Recent studies have highlighted the role of voltage-gated channel subfamily G member 3 (KCNG3) and 4 (KCNG4) genes in the electrical functioning of interstitial cells of Cajal and PDGFRα + cells of the mouse colon. We designed this study to investigate KCNG3 and KCNG4 expression in the normal human colon and in Hirschsprung's disease (HSCR)., Methods: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify KCNG3 and KCNG4 gene expression, and immunolabelling of KCNG3 and KCNG4 proteins was visualized using confocal microscopy., Results: qRT-PCR analysis revealed significant downregulation of the KCNG3 and KCNG4 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed KCNG3 and KCNG4 expression within neurons, ICC and PDGFRα + cells of the myenteric plexus and smooth muscle layers, with a reduction in both proteins in aganglionic and ganglionic HSCR colon compared to controls., Conclusion: KCNG3 and KCNG4 gene expression is significantly downregulated in HSCR colon, suggesting a role for these genes in colonic motility. KCNG3 and KCNG4 downregulation within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients.

Purpose: Smooth muscle cells are electrically coupled to ICC and PDGFRα + cells, to regulate smooth muscle contraction. Recent studies have reported that the voltage-gated sodium channel type 1β (Scn1b), and the chloride channel subunit, Fxyd1, are highly expressed by both ICC and PDGFRα + cells in the mouse colon. We designed this study to investigate the expression of the Scn1b and Fxyd1 genes in the normal human colon and in HSCR., Methods: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Scn1b and Fxyd1 gene expression, and immunolabelling of Scn1b and Fxyd1 proteins were visualized using confocal microscopy., Results: qRT-PCR analysis revealed significant downregulation of Scn1b and Fxyd1 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a reduction in Scn1b and Fxyd1 protein expression in both aganglionic and ganglionic HSCR colon compared to controls., Conclusion: Scn1b and Fxyd1 expression was significantly downregulated in HSCR colon. These results add to mounting evidence suggesting that the pulled-through ganglionic segment of bowel in these patients is abnormal, despite the presence of ganglion cells.

Aim of the Study: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is poorly understood. Inflammasomes are a large family of multiprotein complexes that act to mediate host immune responses to microbial infection and have a regulatory or conditioning influence on the composition of the microbiota. Inflammasomes and the apoptosis-associated speck-like protein (ASC) lead to caspase-1 activation. The activated caspase-1 promotes secretion of pro-inflammatory cytokines (IL-1β and IL-18) from their precursors (pro-IL-1β and pro-IL-18). Inflammasomes have been implicated in a host of inflammatory disorders. Among the inflammasomes, NLRP3, NLRP12 and NLRC4 are the most widely investigated. Knock-out mice models of inflammasomes NLRP3, NLRP12, NLRC4, caspase-1 and ASC are reported to have higher susceptibility to experimental colitis. The purpose of this study was to investigate the expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1β and pro-IL-18 in the bowel specimens from patients with HSCR and controls., Methods: Pulled-through colonic specimens were collected from HSCR patients (n = 6) and healthy controls from the proximal colostomy of children with anorectal malformations (n = 6). The gene expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1β and pro-IL-18 was assessed using qPCR. The protein distribution was assessed using immunofluorescence and confocal microscopy., Main Results: qRT-PCR analysis revealed that NLRP3, NLRP12, NLRC4, ASC and pro-IL-1β gene expressions was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls. Confocal microscopy revealed a markedly decreased expression of NLRP3, NLRP12, NLRC4 and ASC protein in the colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls., Conclusions: To our knowledge, this is the first study analyzing NLRP3, NLRP12, NLRC4, ASC and pro-IL-1β gene expressions in patients with HSCR. Decreased expression of NLRP3, NLRP12, NLRC4, ASC and pro-IL-1β in the aganglionic and ganglionic bowel may increase susceptibility of HSCR patients to develop HAEC.

Purpose: Hirschsprung's disease associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's disease (HSCR). Altered intestinal epithelial barrier function and abnormal microbiota are implicated in the pathogenesis of HAEC. IL-36γ, a member of the IL-1 superfamily, is involved in host defense and contributes to proinflammatory responses and development of inflammatory diseases. The IL36 receptor (IL1RL2) is an important mediator molecule in the inflammatory response. Animal data suggests that IL1RL2 is involved in mucosal healing. We designed this study to investigate the hypothesis that the IL-36γ axis is altered in HSCR., Methods: We investigated IL-36γ and IL1RL2 expression in ganglionic and aganglionic bowel of HSCR patients (n = 10) and controls (n = 10). qPCR, Western blotting and confocal immunofluorescence were performed., Main Results: qPCR and Western blot analysis revealed that IL-36γ is strongly expressed in the aganglionic and ganglionic colon of patients with HSCR. ILR1L2 expression was significantly decreased in HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a markedly increased expression of IL36γ in the colonic epithelium of patients with HSCR compared to controls. IL1RL2 was localized in the colonic epithelium and showed a markedly decreased expression in all HSCR specimens., Conclusion: To our knowledge, we report for the first time the expression of IL36γ and ILRL2 in the colon of patients with HSCR. The increased expression of IL36γ and the markedly decreased expression of IL1RL2 in the aganglionic and ganglionic bowel in HSCR may result in an increased inflammatory response and altered mucosal response healing leading to the susceptibility to develop HAEC.

Purpose: Hirschsprung's disease -associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's disease (HSCR). Altered intestinal epithelial barrier function is implicated in the pathogenesis of HAEC. IL-17 is a proinflammatory cytokine that plays a crucial role in host defense against microbial organisms in the development of inflammatory diseases. Act1 is an essential adaptor molecule required for the IL-17-mediated inflammatory responses via interaction with IL-17 receptor (IL-17R). We designed this study to investigate the hypothesis that Act1/Il-17R expression is upregulated in HSCR., Methods: We investigated Act1 and IL17R expression in ganglionic andaganglionic bowel of HD patients (n = 10) and controls (n = 10). qPCR, Western blotting and confocal immunofluorescence were performed., Main Results: qPCR and Western blot analysis revealed that Act1 and IL17R are strongly expressed in the aganglionic and ganglionic colon of patients with HSCR. Act1 and IL17R expression was significantly increased in HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a markedly increased expression of Act1 and IL17R in the colonic epithelium of patients with HSCR compared to controls., Conclusion: To our knowledge, we report, for the first time, the expression of Act1 in the human colon. The increased expression of Act1 and Il-17 in the aganglionic and ganglionic bowel in HSCR may result in IL-17-mediated increased inflammatory response leading to the development of HAEC.

Purpose: Interstitial cells of Cajal (ICCs) are pacemaker cells involved in facilitating neurotransmission and the generation of slow electrical waves necessary for colonic peristalsis. Their distribution has been found to be abnormal in the aganglionic and ganglionic colon in Hirschsprung's disease (HSCR) using c-kit-labelling. Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel thought to be specifically expressed on ICCs. Unlike c-kit, it plays a key role in ICC pacemaker activity. We aimed to investigate the utility of ANO1 in evaluating the colonic ICC network in HSCR., Materials and Methods: We collected full-length pull-through specimens from children with HSCR (n = 10). Control colon specimens were collected at colostomy closure in children with anorectal malformation (n = 6). The distribution of ANO1 and c-kit expression was evaluated using immunofluorescence and confocal microscopy. ANO1 expression was quantified using Western blot analysis., Results: ANO1 was not expressed on 23 % of c-kit immuno-positive cells in the circular muscle; however, 100 % of ANO1-positive ICCs were c-kit positive. The distribution of ANO1-positive ICCs was sparse in aganglionic colon, with a modest reduction in ICCs seen in the ganglionic colon in HSCR compared to controls (p = 0.044). ANO1 protein expression was reduced in aganglionic colon but similar in ganglionic colon relative to controls., Conclusions: ANO1 is preferential to c-kit in evaluating the ICC network in HSCR due to its specificity and functional importance. Abnormal distribution of ANO1-positive ICCs in the ganglionic colon in HSCR may contribute to persistent bowel symptoms in some patients after pull-through surgery.

Purpose: The pathophysiology of Hirschsprung's disease (HSCR) is not entirely understood. There is no clear explanation for the occurrence of the spastic or tonically contracted aganglionic segment of bowel. Kv11.1 (hERG) channels play a critical role in the regulation of the resting membrane potential as well as affecting either the force or frequency of contraction of smooth muscles. We designed this study to investigate the expression and distribution of hERG channels in the normal colon and the colon of patients with HSCR., Methods: We investigated hERG protein expression in both the ganglionic and aganglionic regions of HSCR patients (n = 10) versus normal control colon (n = 10). Protein distribution was assessed using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using real-time polymerase chain reaction, western blot analysis and densitometry., Results: Confocal microscopy of the normal colon revealed strong hERG channel expression in interstitial cells of Cajal, platelet-derived growth factor-alpha receptor- (PDGFRα(+)) positive cells and enteric neurons. hERG expression was markedly decreased in aganglionic bowel, whereas colonic hERG gene expression levels were significantly decreased in aganglionic compared to ganglionic bowel and controls (p < 0.05). Western blotting revealed decreased colonic hERG protein expression in aganglionic HSCR specimens compared to controls., Conclusions: We demonstrate, for the first time, the expression and distribution of hERG channels in the human colon. The decreased expression of hERG in the aganglionic colon may be responsible for the increased tone in the aganglionic narrow spastic segment of bowel.

Background: Rho-kinase (ROCK) is the primary effector protein in the RhoA pathway, which regulates Ca(2+)-independent smooth muscle contraction in the human bowel. This pathway has been reported to be hyper-activated in the aganglionic bowel of EDNRB-null (-/-) rats compared to the ganglionic bowel from EDNRB (+/+) rats. We hypothesised that ROCK expression is up-regulated in human aganglionic bowel and designed this study to investigate ROCK 1 and ROCK 2 expression in Hirschsprung's disease (HSCR) and controls., Materials and Methods: Full-length specimens were collected following pull-through surgery for HSCR (n = 9). Colonic controls (n = 6) were obtained during colostomy closure from patients with anorectal malformations. Distribution of ROCK 1/2 expression was evaluated using double-labelled immunofluorescence and confocal microscopy. ROCK1/2 protein expression was assessed in mucosa and tunica muscularis using western blot analysis., Results: There was strong expression of both ROCK 1 and ROCK 2 in interstitial cells of Cajal (ICCs) and ganglia. ROCK 1 expression was reduced in aganglionic bowel compared to HSCR ganglionic bowel and controls in both mucosa and tunica muscularis. ROCK 2 expression was similar in the colon of children with HSCR and controls., Conclusions: This is the first report of strong ROCK expression in colonic ICCs. Although the rat model of aganglionic bowel suggests that Ca(2+)-independent smooth muscle contraction involving ROCK is hyper-activated, our data indicate ROCK 1 expression is decreased in aganglionic bowel and ROCK 2 expression is unaltered in children with HSCR.

Skip segment Hirschsprung's disease (SSHD) involves a skip area of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. We report a case of SSHD in infant with total colonic aganglionosis (TCA). Although SSHD is a rare and controversial condition, it should be considered a definite entity in the evaluation of Hirschsprung's disease.

Purpose: The enteric nervous system (ENS) is a network of neurons and glia that lies within the gut wall. It is responsible for the normal regulation of gut motility and secretory activities. Hirschsprung's disease (HD) is a congenital defect of the ENS, characterised by an absence of ganglia in the distal colon. Intestinal neuronal dysplasia (IND) is a condition that clinically resembles HD, characterised by hyperganglionosis, giant and ectopic ganglia, resulting in intestinal dysmotility. Intestinal ganglioneuromatosis is characterised by hyperplasia and hypertrophy of enteric neuronal cells and causes chronic intestinal pseudo-obstruction (CIPO). Phosphatase and tensin homolog deleted on chromosome 10 (Pten) is a phosphatase that is critical for controlling cell growth, proliferation and cell death. A recent study of Pten knockout mice showed evidence of ganglioneuromatosis in the ENS suggesting a role for this protein in ENS development. Ganglioneuromatosis patients have also been shown to have a decreased level of Pten expression in the colon. The aim of our study was to investigate Pten expression in the ENS of HD and IND patients compared to normal controls., Methods: Resected tissue from 10 HD and 10 IND type B patients was fixed and embedded in paraffin wax. Normal control colon tissue was obtained from ten patients who underwent a colostomy closure for imperforate anus. Sections were cut and immunohistochemistry was carried out using a Pten antibody. Results were analysed by light microscopy., Results: Staining showed that Pten was strongly expressed in ganglia of both the submucosal and myenteric plexus of normal and HD specimens from the ganglionic colon. Pten expression was significantly reduced in the giant ganglia in IND patients in both the myenteric and submucosal plexuses compared to the normal controls. Specimens from the aganglionic region of HD did not show Pten expression., Conclusion: To the best of our knowledge, this is the first study demonstrating a marked reduction of Pten expression in the myenteric and submucous plexuses of IND patients. Neuronal Pten deficiency in IND may disrupt the chemical pathway associated with the proliferation and development of neuronal cells forming mature ganglia and thus cause motility dysfunction.

Purpose: Hirschsprung's disease is characterised by the congenital absence of ganglion cells beginning in the distal rectum and extending proximally for varying distances. 'Zonal aganglionosis' is a phenomenon involving a zone of aganglionosis occurring within normally innervated intestine. 'Skip segment' Hirschsprung's disease (SSHD) involves a 'skip area' of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. While Hirschsprung's disease is believed to be the result of incomplete craniocaudal migration of neural crest-derived cells, the occurrence of SSHD has no clear embryological explanation. The aim of this study was to perform a systematic review of SSHD, reported in the literature between 1954 and 2009, in order to determine the clinical characteristics of this rare entity and its significance., Methods: The first reported case of SSHD was published in 1954. A systematic review of SSHD cases in the literature, from 1954 to 2009, was carried out using the electronic database 'Pubmed'. Detailed information was recorded regarding the age, gender, presenting symptoms and location of the skip segment in each patient., Results: 24 cases of SSHD have been reported in the literature to date. 18/24 (75%) of these cases were males and 6/24 (25%) were females. Of these, 22/24 (92%) were cases of total colonic aganglionosis (TCA), and 2/24 (8%) were rectosigmoid Hirschsprung's disease. Of the 22 TCA cases, 9 (41%) had a skip segment in the transverse colon, 6 (27%) in the ascending colon, 2 (9%) in the caecum and 5 (23%) had multiple skip segments. In both rectosigmoid Hirschsprung's disease cases, the skip segment was in the sigmoid colon. Overall, the length of the skip segment was variable, with the entire transverse colon ganglionated in some cases., Conclusion: SSHD occurs predominantly in patients with TCA. The existence of a skip area of normally innervated colon in TCA may influence surgical management, enabling surgeons to preserve and use the ganglionated skip area during pull-through operations.

Introduction: The enteric nervous system of the chick embryo hindgut is derived from the vagal and sacral neural tube. Nerve cells from these regions produce various neuronal phenotypes, including inhibitory and excitatory nerve cells, providing intrinsic innervation to the chick embryo cloaca. We hypothesised that the vagal and sacral neural tubes provide the cloaca, with phenotypically similar nerve cells. The aim of our study was to investigate the origin of excitatory neurotransmission in the developing cloaca., Materials and Methods: Chicken embryos were incubated until the 10-12 somite stage (ss). To study the vagal neural tube contribution to the cloaca, this region was microsurgically ablated in ovo and replaced with the corresponding region from age-matched quail embryos. To study the sacral neural tube contribution to the cloaca, the vagal neural tube was ablated at the 10-12 ss, but not replaced with quail neural tube, thus, only the sacral neural crest cells remained. All embryos were harvested at E12 and E14, embedded in paraffin wax and serially sectioned. Immunohistochemistry was carried out on all embryos using human natural killer-1, quail non-chick perinuclear, choline acetyltransferase (ChAT) and substance P (SubP) antibodies., Results: Choline acetyltransferase- and SubP-positive neurons were seen to originate in both the vagal and the sacral neural tube. The vagal neural tube provided the majority of the nerve cells to the chick embryo cloaca and expressed both ChAT and SubP in both the myenteric and submucosal plexus. The sacral neural tube contributed a lesser amount of nerve cells to the chick embryo cloaca, but was also seen to produce both ChAT- and SubP-positive nerve cells in both ganglionated plexus., Conclusion: This study shows, for the first time, that the excitatory ChAT- and SubP-expressing neurons in the developing cloaca originate in both the vagal and the sacral neural tube. These results highlight the origin of phenotypically similar nerve cells in both regions of the neural tube, providing new insights into the developmental origin of the intrinsic innervation of the persistent cloaca.

Introduction: The enteric nervous system is an intrinsic network of nerve cells and glia within the gastrointestinal wall, which originates in the vagal and sacral neural tube. The vagal neural tube is known to supply the colorectum with the majority of its nerve cells, and its ablation during early development produces a hypoganglionic colorectum. We hypothesized that the cholinergic nerve activity in the chick embryo hypoganglionic colorectum is decreased similar to the human situation and, therefore, this study is designed to investigate cholinergic innervations in the chick embryo hypoganglionic colorectum., Materials and Methods: Chicken eggs were incubated until embryos reached the 10-12 somite stage. The vagal neural tube was microsurgically ablated and eggs were returned to the incubator until embryos reached E12 and E14. Whole embryos were fixed and embedded in paraffin wax. Transverse sections were cut and immunohistochemistry was performed using a neural crest cell antibody, human natural killer-1 (HNK-1), and a choline acetyltransferase antibody (ChAT)., Results: The results showed that in normal embryos, the colorectum contained many nerve cells (HNK-1) and ChAT-positive nerve cells and fibres, while in embryos with a hypoganglionic colorectum, the number of nerve cells (HNK-1) and ChAT-positive nerve cells and fibres was decreased., Conclusion: Cholinergic nerve activity is decreased as a result of a reduction in nerve cell numbers in the chick embryo colorectum. These results suggest that the cholinergic activity in the hypoganglionic chick model resembles that of human hypoganglionosis.