The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Thyroid Hormone in the Pathogenesis of Congenital Intestinal Dysganglionosis.

INTRODUCTION: The objective of this study is to investigate the role of thyroid hormone (TH) in the pathogenesis of intestinal dysganglionosis (ID). METHODS: A zebrafish model of congenital hypothyroidism (CH) was created by exposing the larvae to the 6-propyl-2-thiouracil (PTU). The enteric neurons were labeled with anti-HuC/D antibodies. The number of enteric neurons was counted. The larval intestine was dissociated and stained with anti-p75 and anti-α4 integrin antibodies. Mitosis and apoptosis of the p75+ α4 integrin+ enteric neural crest cells (ENCCs) were studied using flow cytometry. Intestinal motility was studied by analyzing the transit of fluorescent tracers. RESULTS: PTU (25 mg/L) significantly reduced TH production at 6- and 9-days post fertilization without changing the body length, body weight, and intestinal length of the larvae. Furthermore, PTU inhibited mitosis of ENCCs and reduced the number of enteric neurons throughout the larval zebrafish intestine. Importantly, PTU inhibited intestinal transit of fluorescent tracers. Finally, thyroxine supplementation restored ENCC mitosis, increased the number of enteric neurons, and recovered intestinal motility in the PTU-treated larvae. CONCLUSIONS: PTU inhibited TH production, reduced the number of enteric neurons, impaired intestinal motility, and impeded ENCC mitosis in zebrafish, suggesting a possible role of CH in the pathogenesis of ID.

Effects of Dachengqi Decoction () on morphological changes in enteric nerve system of rats with multiple organ dysfunction syndrome.

OBJECTIVE: To observe the morphological changes in enteric nerve system (ENS) of rats with multiple organ dysfunction syndrome (MODS) treated by Dachengqi Decoction (, DCQD). METHODS: Fifty Wistar rats were randomly assigned to the control group, MODS model group and DCQD treated group. The rats in MODS model group and DCQD treated group were injected Escherichia coli (E. coli) suspension into abdominal cavity under sterile condition. The DCQD treated group was gavaged with DCQD 2 days before the E. coli suspension was injected. Twenty-four hours after injection, the proximal segment of intestine was resected and studied by immunohistofluorescence using vesicular acetylcholine transporter, vasoactive intestinal polypeptide (VIP), substance P (SP) and neuronal nitric oxide synthase (nNOS) antibodies. The whole-mount preparations were observed by laser scanning confocal microscope to detect the changes of quantity and fluorescence integral optical density (IOD) value of intestine enteric nerves. RESULTS: Compared with the control group, the quantity and IOD value of acetylcholine (ACh), VIP, SP and nitric oxide (NO) nerves of intestine in the MODS group were significantly decreased (P<0.01), and the network of enteric nerves was remarkably disrupted. Compared with the MODS group, the quantity and fluorescence IOD value of ACh, VIP, SP and NO nerves in the DCQD group were significantly increased (P<0.01), and the network of enteric nerves was remarkably recovered. CONCLUSION: DCQD can protect and repair damage in the network of ACh, SP, NO and VIP nerves in rats with MODS, which may be one of mechanisms involved in promoting gastrointestinal motility by DCQD.

Mesenchymal stem cells and conditioned medium avert enteric neuropathy and colon dysfunction in guinea pig TNBS-induced colitis.

Damage to the enteric nervous system (ENS) associated with intestinal inflammation may underlie persistent alterations to gut functions, suggesting that enteric neurons are viable targets for novel therapies. Mesenchymal stem cells (MSCs) offer therapeutic benefits for attenuation of neurodegenerative diseases by homing to areas of inflammation and exhibiting neuroprotective, anti-inflammatory, and immunomodulatory properties. In culture, MSCs release soluble bioactive factors promoting neuronal survival and suppressing inflammation suggesting that MSC-conditioned medium (CM) provides essential factors to repair damaged tissues. We investigated whether MSC and CM treatments administered by enema attenuate 2,4,6-trinitrobenzene-sulfonic acid (TNBS)-induced enteric neuropathy and motility dysfunction in the guinea pig colon. Guinea pigs were randomly assigned to experimental groups and received a single application of TNBS (30 mg/kg) followed by 1 × 10(6) human bone marrow-derived MSCs, 300 µl CM, or 300 µl unconditioned medium 3 h later. After 7 days, the effect of these treatments on enteric neurons was assessed by histological, immunohistochemical, and motility analyses. MSC and CM treatments prevented inflammation-associated weight loss and gross morphological damage in the colon; decreased the quantity of immune infiltrate in the colonic wall (P < 0.01) and at the level of the myenteric ganglia (P < 0.001); prevented loss of myenteric neurons (P < 0.05) and damage to nerve processes, changes in ChAT, and nNOS immunoreactivity (P < 0.05); and alleviated inflammation-induced colonic dysmotility (contraction speed; P < 0.001, contractions/min; P < 0.05). These results provide strong evidence that both MSC and CM treatments can effectively prevent damage to the ENS and alleviate gut dysfunction caused by TNBS-induced colitis.

Enteric neuropathy can be induced by high fat diet in vivo and palmitic acid exposure in vitro.

OBJECTIVE: Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model. DESIGN: Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 - 10(-3) M) and / or oleic acid (OA) (0 - 10(-3) M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry. RESULTS: HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival. CONCLUSIONS: HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.

Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is a congenital condition in which enteric ganglia, formed from neural crest cells (NCC), are absent from the terminal bowel. Dysmotility and constipation are common features of HSCR that persist following surgical intervention. This persistence suggests that the portion of the colon that remains postoperatively is not able to support normal bowel function. To elucidate the defects that underlie this condition, we utilized a murine model of HSCR. METHODS: Mice with NCC-specific deletion of Ednrb were used to measure the neuronal density and neurotransmitter expression in ganglia. KEY RESULTS: At the site located proximal to the aganglionic region of P21 Ednrb null mice, the neuronal density is significantly decreased and the expression of neurotransmitters is altered compared with het animals. The ganglia in this colonic region are smaller and more isolated while the size of neuronal cell bodies is increased. The percentage of neurons expressing neuronal nNOS and VIP is significantly increased in Ednrb nulls. Conversely, the percentage of choline acetyltransferase (ChAT) expressing neurons is decreased, while Substance P is unchanged between the two genotypes. These changes are limited to the colon and are not detected in the ileum. CONCLUSIONS & INFERENCES: We demonstrate changes in neuronal density and alterations in the balance of expression of neurotransmitters in the colon proximal to the aganglionic region in Ednrb null mice. The reduced neuronal density and complementary changes in nNOS and ChAT expression may account for the dysmotility seen in HSCR.

Clinical application of entire gastrointestinal barium meal combined with multi-temporal abdominal films in patients with intestinal neuronal dysplasia type B.

OBJECTIVE: To report and evaluate the application of entire gastrointestinal barium meal combined with multi-temporal abdominal films in the diagnosis of patients with intestinal neuronal dysplasia type B (IND type B). METHODS: Thirty-six patients with symptoms of long-standing constipation were enrolled in this study. The study took place at the Department of General Surgery, Xiangyang Central Hospital, Hubei Province, China from July 2007 to October 2012. All of them had already been subjected to the tests of barium enema and anorectal manometry and were suspected to be IND type B, but were not confirmed by mucous membrane acetylcholinesterase determination. All underwent the entire gastrointestinal barium meal combined with multi-temporal abdominal films. The data was collected and then analyzed retrospectively. RESULTS: After entire gastrointestinal barium meal combined with multi-temporal abdominal films, 30 out of 36 cases in this group were diagnosed with intestinal neuronal diseases, and then were treated with appropriate surgical treatment. The postoperative pathological diagnosis was IND type B. The other 6 patients in this group still could not be diagnosed explicitly after the test; thus, we treated them with conservative treatment. CONCLUSION: Entire gastrointestinal barium meal combined with multi-temporal abdominal films has the advantage of being able to test the gastrointestinal transfer capabilities and to find physiological and pathological changes simultaneously. It could provide important proof for the diagnosis of patients with intestinal neuronal dysplasia type B.

Postnatal administration of 2-oxoglutaric acid improves the intestinal barrier affected by the prenatal action of dexamethasone in pigs.

OBJECTIVE: The potential effects of the prenatal administration of dexamethasone and the postnatal treatment with 2-oxoglutaric acid on postnatal development of the small intestine of farm animals have not been examined experimentally. The aim of this study was to establish the changes in morphologic parameters of the small intestine damaged by the prenatal action of dexamethasone in piglets supplemented with 2-oxoglutaric acid. METHODS: Three milligrams dexamethasone was administered intramuscularly every second day from day 70 of pregnancy to parturition and then piglets were supplemented with 2-oxoglutaric acid for 35 d of postnatal life (0.4 g/kg of body weight). The histomorphometry of the pig duodenum and jejunum was determined. Immunohistochemical staining with anti-Ki-67, CD3, null T cells, cadherin, claudin, and neurofilament antibodies was performed. RESULTS: Maternal treatment with dexamethasone decreased and limited the expression of claudin and cadherin in the epithelium. Dexamethasone led to thinning of the myenteron of the duodenum and the middle part of the jejunum in weaned piglets and influenced duodenal glands that became more elongated compared with control glands. Moreover, 2-oxoglutaric acid increased cell proliferation and the amount and maturity of peripheral blood lymphocytes in the duodenum and jejunum. It supported epithelial integrity and changed the circularity of the nerve plexuses. CONCLUSION: The 2-oxoglutaric acid administered to piglets while suckling induced a complete recovery from intestinal damage caused by the prenatal action of dexamethasone.

Knowledge of ancient Hindu surgeons on Hirschsprung disease: evidence from Sushruta Samhita of circa 1200-600 BC.

BACKGROUND: Congenital megacolon is eponymously named after Harold Hirschsprung, who accurately described the clinical features in 1886. Recent research revealed that this condition is perhaps well known for centuries before him. AIM: This article is intended to examine if ancient Hindu surgeons knew about congenital megacolon. METHODS AND MATERIALS: Sushruta Samhita is an ancient tome of Ayurvedic surgery compiled by Sushruta (circa 1200-600 bc). Passages of interest were identified by browsing the authentic English translation of the compendium. Accuracy of translation was verified by comparing to the original Sanskrit verses with the help of a Sanskrit scholar. RESULTS: A condition called Baddha Gudodaram, described in the Samhita, closely resembles Hirschsprung disease. There are indications that ancient Indians even deciphered the etiology as defective vayu alias vata (nerves). Although the ailment was considered incurable, a palliative operation has been discussed. Descriptive details of the operation match with that of sigmoid colostomy. CONCLUSION: Evidence from Sushruta Samhita indicates that Hindu surgeons of prehistoric India probably had considerable knowledge about Hirschsprung disease. Further research, corroborating other sources of evidence, is required to confirm this claim.

Neuroprotective action of Ginkgo biloba on the enteric nervous system of diabetic rats.

AIM: To investigate the effect of Ginkgo biloba extract on the enteric neurons in the small intestine of diabetic rats. METHODS: Fifteen Wistar rats were divided into three groups: control group (C), diabetic group (D) and diabetic-treated (DT) daily with EGb 761 extract (50 mg/kg body weight) for 120 d. The enteric neurons were identified by the myosin-V immunohistochemical technique. The neuronal density and the cell body area were also analyzed. RESULTS: There was a significant decrease in the neuronal population (myenteric plexus P = 0.0351; submucous plexus P = 0.0217) in both plexuses of the jejunum in group D when compared to group C. With regard to the ileum, there was a significant decrease (P = 0.0117) only in the myenteric plexus. The DT group showed preservation of the neuronal population in the jejunum submucous plexus and in the myenteric plexus in the ileum. The cell body area in group D increased significantly (P = 0.0001) in the myenteric plexus of both segments studied as well as in the ileum submucosal plexus, when compared to C. The treatment reduced (P = 0.0001) the cell body area of the submucosal neurons of both segments and the jejunum myenteric neurons. CONCLUSION: The purified Ginkgo biloba extract has a neuroprotective effect on the jejunum submucous plexus and the myenteric plexus of the ileum of diabetic rats.

Immunohistochemical study of vasoactive intestinal peptide (VIP) enteric neurons in diabetic rats supplemented with L-glutamine.

The purpose of this work was to study the area of the varicosities of nerve fibers of myenteric neurons immunoreactive to vasoactive intestinal peptide (VIP-IR) and of the cell bodies of VIP-IR submucosal neurons of the jejunum of diabetic rats supplemented with 2% L-glutamine. Twenty male rats were divided into the following groups: normoglycemic (N), normoglycemic supplemented with L-glutamine (NG), diabetic (D) and diabetic supplemented with L-glutamine (DG). Whole-mounts of the muscle tunica and the submucosal layer were subjected to the immunohistochemical technique for neurotransmitter VIP identification. Morphometric analyses were carried out in 500 VIP-IR cell bodies of submucosal neurons and 2000 VIP-IR varicosities from each group. L-Glutamine supplementation to the normoglycemic animals caused an increase in the areas of the cell bodies (8.49%) and varicosities (21.3%) relative to the controls (P < 0.05). On the other hand, there was a decrease in the areas of the cell bodies (4.55%) and varicosities (28.9%) of group DG compared to those of group D (P < 0.05). It is concluded that L-glutamine supplementation was positive both to normoglycemic and diabetic animals.

Neuroinvasion in sheep transmissible spongiform encephalopathies: the role of the haematogenous route.

BACKGROUND: It is generally believed that after oral exposure to transmissible spongiform encephalopathy (TSE) agents, neuroinvasion occurs via the enteric nervous system (ENS) and the autonomic nervous system. As a result, the dorsal motor nucleus of the vagus nerve is the initial point of disease-associated prion protein (PrP(d)) accumulation in the brain. HYPOTHESIS AND AIM: If direct ENS invasion following oral infection results in an early and specific brain targeting for PrP(d) accumulation, such topographical distribution could be different when other routes of infection were used, highlighting distinct routes for neuroinvasion. METHODS: An immunohistochemical study has been conducted on the brain of 67 preclinically infected sheep exposed to natural scrapie or to experimental TSE infection by various routes. RESULTS: Initial PrP(d) accumulation consistently occurred in the dorsal motor nucleus of the vagus nerve followed by the hypothalamus, regardless of the breed of sheep, PrP genotype, TSE source and, notably, route of infection; these factors did not appear to affect the topographical progression of PrP(d) deposition in the brain either. Moreover, the early and consistent appearance of PrP(d) aggregates in the circumventricular organs, where the blood-brain barrier is absent, suggests that these organs can provide a portal for entry of prions when infectivity is present in blood. CONCLUSIONS: The haematogenous route, therefore, can represent a parallel or alternative pathway of neuroinvasion to ascending infection via the ENS/autonomic nervous system.

Anorectal electromanometrical patterns in children with isolated neuronal intestinal dysplasia.

BACKGROUND: The aim of this study was to report our new findings on anorectal electromanometrical patterns in patients with isolated neuronal intestinal dysplasia (IND) type B. METHODS: We reviewed and analyzed the records of preoperative anorectal electromanometric examinations in 17 patients (10 male and 7 female) with IND. The diagnosis of IND was made based on a pathological examination. RESULTS: Mean age of the patients was 6.3 years (range 4 months to 16 years). In the preoperative barium enema study, a narrowed distal segment with proximal dilatation was noted in 8 patients, dilatation of the sigmoid or rectum without a narrowed distal segment was observed in 4 patients, and no specific findings were found in 5 patients. Out of 17 patients, only 5 patients showed positive staining for AChE in the rectal suction specimens. The electromanometric examination showed no significant difference in anal resting pressure and the length of the high pressure zone between the IND and the functional constipation (FC) groups. The frequency of anal peristalsis in the IND group, however, was significantly lower than that in the FC group. Sixteen IND patients showed an internal relaxation. However, the threshold value to evoke the relaxation in the IND group was significantly higher than that in the FC group. Moreover, the latent period of reflex and the duration of a relaxation in the IND group were longer than those in the FC group. Finally, two specific shapes of reflex wave ("W" or "U" shape) were observed in 10 patients with isolated IND type B, while no such shapes were noted in the FC group. CONCLUSION: These new findings support the notion that anorectal electromanometry is a safe and useful screening examination for IND patients.

Allergic proctitis and abdominal distention mimicking Hirschsprung's disease in infants.

AIM: To determine the incidence and clinical aspects of allergic proctitis (AP) in infants with symptoms that mimic Hirschsprung's disease (HD). METHODS: One hundred and five patients less than 6 months of age, who underwent barium enema, anorectal manometry and rectal suction biopsy due to suspicion of HD, were enrolled. Comparison of the patient characteristics associated with each disease was based on the results of the triple testing. The sensitivity and specificity of the three tests, for the diagnosis of HD, were evaluated. RESULTS: The mean age of enrolled patients was 2.1+/-0.9 months. Based on the three tests, 39 patients (37.1%) were diagnosed with HD, seven patients (6.7%) with AP, and 53 (50.5%) had normal results. Of the 54 patients with transitional zone and a reversed rectosigmoid index on the barium enema, four (7.4%) were patients with AP. The mean age of the AP patients (3.1+/-1.5 months old) was older than the HD children (1.4+/-0.9 months old). The sensitivity of the three tests for HD was 97.4%, 87.2% and 92.3% and the specificity was: 74.2%, 78.8% and 100%, respectively. CONCLUSIONS: In the infants with severe abdominal distention, the incidence of AP mimicking HD was relatively high. Therefore, consideration of AP should be part of the differential diagnosis in infants with severe abdominal distention or findings that mimic HD. For differentiation of these disorders, a rectal suction biopsy is very useful.

Histologic criteria for the diagnosis of allied diseases of Hirschsprung's disease in adults.

A few reports in the literature have discussed the histologic criteria for the diagnosis of allied diseases of Hirschsprung's disease in adults, and studies report that intestinal neuronal dysplasia (IND) in adults may develop from IND in infants. The aim of this study was to examine the differences between the histological findings of IND in infants and those in adults, and to assess whether allied diseases of Hirschsprung's disease (HD) in adults should be considered as congenital or acquired diseases. For these purposes, we studied nine adult patients with severe constipation, and an adult patient with acute intestinal obstruction. We routinely examined the patients using barium enema, anorectal manometry and rectal mucosal biopsy. However, in patients suspected of allied diseases, we carried out full-thickness rectal biopsies. In seven operated cases, we also examined resected intestines. The tissue samples were examined using AChE-staining, NADPH-diaphorase staining, HE-staining, and silver impregnation. Histologically, we diagnosed two males as having HD, two males as having IND, five patients (two males and three females) as having hypoganglionosis, and one female as having a degeneration of the intramural plexus. The following conclusions were drawn: 1) Inflammations such as ulcerative colitis or ischemic colitis may cause IND TYPE B in adults whose histological findings are similar to those generally seen in infants; 2) It is suggested that IND is closely related to hypoganglionosis; 3) In hypoganglionosis, a patient with findings of elevated AChE-positive nerve fibers in the mucosa and AChE-positive nerve fibers in an arterial wall may belong to a subtype of IND; 4) Most of the allied diseases of HD in adults may occur as an acquired disease, not as a congenital disease.

A new rapid acetylcholinesterase histochemical method for the intraoperative diagnosis of Hirschsprung's disease and intestinal neuronal dysplasia.

The most commonly used acetylcholinesterase (AChE) method for the diagnosis of Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) was first introduced in 1964 by Morris Karnovsky and Logan Roots. This technique requires about 80 - 120 minutes incubation time and cannot be used for the intraoperative diagnosis of HD and IND. To avoid these limitations, in 1994 Kobayashi et al first proposed an accelerated modified method in two different versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propose a new rapid variation of AChE staining which avoids the use of DAB and naphthol, notably toxic reagents, but follows the same acceleration principle of Kobayashi's technique. Our modified rapid AChE requires a total incubation time of only 8 minutes, which is compatible with intraoperative histochemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obtained from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 microm cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as chromogenic substance. The two complementary histochemical techniques alpha-naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intraoperatively for the staining of ganglion cells. The diagnosis was confirmed postoperatively with conventional AChE Karnovsky technique, comparing the extensions of hyperganglionic, hypoganglionic and aganglionic segments in each studied case. The new rapid AChE modified method can identify ganglion cells and fibers using a dark brown precipitate. In all the cases studied, the intestinal innervation pattern identified with this modified technique was similar to that obtained with Karnovsky AchE. Seventy-eight HD, 8 isolated IND and 6 HD associated with an evident IND segment were diagnosed. This new rapid AChE histochemical technique avoids the use of DAB and naphthol, and can thus be considered safe for operators. Rapid AChE is a valid tool for both the evaluation of aganglionosis extension and for the identification of IND pattern during surgery. We recommend this very reliable method for the intraoperative diagnosis of HD and IND, in association with other enzymatic markers of ganglion cells (ANE or LDH). We propose the following diagnostic protocols: a) for preoperative histochemical study: conventional AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AChE plus ANE.