Diagnosis of Hirschsprung disease by analyzing acetylcholinesterase staining using artificial intelligence.

OBJECTIVES: Classical Hirschsprung disease (HD) is defined by the absence of ganglion cells in the rectosigmoid colon. The diagnosis is made from rectal biopsy, which reveals the aganglionosis and the presence of cholinergic hyperinnervation. However, depending on the method of rectal biopsy, the quality of the specimens and the related diagnostic accuracy varies substantially. To facilitate and objectify the diagnosis of HD, we investigated whether software-based identification of cholinergic hyperinnervation in digitalized histopathology slides is suitable for distinguishing healthy individuals from affected individuals. METHODS: N = 190 samples of 112 patients who underwent open surgical rectal biopsy at our pediatric surgery center between 2009 and 2019 were included in this study. Acetylcholinesterase (AChE) stained slides of these samples were collected and digitalized via slide scanning and analyzed using two digital imaging software programs (HALO, QuPath). The AChE-positive staining area in the mucosal layers of the intestinal wall was determined. In the next step machine learning was employed to identify patterns of cholinergic hyperinnervation. RESULTS: The area of AChE-positive staining was greater in HD patients compared to healthy individuals (p < 0.0001). Artificial intelligence-based assessment of parasympathetic hyperinnervation identified HD with a high precision (area under the curve [AUC] 0.96). The accuracy of the prediction model increased when nonrectal samples were excluded (AUC 0.993). CONCLUSIONS: Software-assisted machine-learning analysis of AChE staining is suitable to improve the diagnostic accuracy of HD.

Evaluation and Management of Total Colonic Hirschsprung Disease: A Comprehensive Review From the American Pediatric Surgical Association (APSA) Hirschsprung Disease Interest Group.

BACKGROUND: Children with total colonic Hirschsprung disease (TCHD) are a unique group of patients with pre- and postoperative management challenges. This review provides a rational, expert-based approach to diagnosing and managing TCHD. METHODS: The guidelines were developed by the Hirschsprung Disease Interest Group members established by the American Pediatric Surgical Association (APSA) Board of Governors. Group discussions, literature review, and expert consensus were used to summarize the current knowledge regarding diagnosis, staged approach, the timing of pull-through, and pre-and postoperative management in children with TCHD. RESULTS: This paper presents recommendations for managing TCHD before and after reconstruction, including diagnostic criteria, surgical approaches, bowel management, diet, antibiotic prophylaxis, colonic irrigations, and post-surgical considerations. CONCLUSIONS: A clear understanding of the unique challenges posed by TCHD and consensus on its treatment are lacking in the literature. This review standardizes this patient group's pre- and postoperative management. LEVEL OF EVIDENCE: V.

Surgical Strategies in Total Colonic Aganglionosis: Primary Pullthrough-Pathway of Care.

Background: Total colonic aganglionosis, as a rare variant of Hirschsprung Disease, still poses challenges to surgeons in terms of diagnosis and management. The optimal preparation for pullthrough is crucial for reconstructive surgery. This study aims to explore our surgical pathway for children with total colonic aganglionosis (TCA) and to describe the prehabilitation necessary to prepare for successful reconstructive pullthrough surgery. Methods: A prospective review of children with TCA receiving an abdominal surgical intervention between 1/22 and 4/24. The cohort included children receiving mapping +/- primary ileoanal pullthrough. An analysis of preoperative, perioperative, and postoperative data, and a short-term follow-up were performed. Results: A total of 18 children with TCA and no prior pullthrough received an abdominal intervention during the 29-month study period, and 5/18 (27.8%) were female. The children had a median of 4 (range 2-7) prior external surgeries; all had a stoma; 6 (33%) children received parental nutrition; 12 children underwent a mapping of the ganglia distribution and bowel length at a median age of 11 months (range 3-54), and in 10 of them, we relocated the stoma. There was a mean involvement of 15 (5-93) cm small bowel aganglionosis, with the remaining mean ganglionic small bowel having a length of 178 cm (110-254). A total of 11 children underwent straight primary ileoanal pullthrough of the stoma site at a median age of 16.7 months (10-133). Conclusions: The timely diagnosis of TCA still challenges the care team, and most children have a rough journey involving several surgeries until their diagnosis is established. The ensure bowel function with an adequate working stoma is the key to enabling enteral nutrition and growth, which are the baseline requirements to undertake a successful pullthrough procedure and restore continuity. Careful perioperative bowel management and parents' active involvement supports children with Hirschsprung Disease achieving the best possible quality of life.

The Diagnostic Pathway of Hirschsprung's Disease in Paediatric Patients: A Single-Centre Experience.

BACKGROUND: The presenting symptoms of patients with Hirschsprung's disease (HD) are a failure to pass meconium, abdominal distension, and bilious vomiting. The gold standard diagnosis is a rectal biopsy to confirm aganglionosis. The aim of our study was to describe the diagnostic pathway of Hirschsprung's disease at our institution and document the indication for a rectal biopsy. METHODS: We have performed a prospective collection of all patients who underwent a rectal biopsy to exclude HD from December 2022 until September 2023 including. The following data were collected: patient's age, presenting symptoms, type of biopsy, failure rate, complications, and histopathological results. RESULTS: We identified 33 patients who underwent 34 rectal biopsies at 0.6 years of age. A total of 17 patients had a rectal suction biopsy (RSB), and 17 patients underwent a partial thickness under general anaesthesia (GA). 1/17 (6%) patients had an inconclusive RSB and subsequently underwent a biopsy under GA. Constipation and chronic abdominal distension plus vomiting were the most common presenting symptoms throughout all ages. Five patients (15%) had a rectal biopsy that was positive for HD. CONCLUSION: A protocolised approach to the assessment of infants and children with suspected HD ensures the appropriate utilisation of invasive procedures such as biopsy.

Extended total colonic aganglionosis and total intestinal aganglionosis: Challenging enemies.

AIM: Extended total colonic aganglionosis (ETCA) represents uncommon forms of Hirschsprung disease (HD), with aganglionosis extending into the proximal small bowel. ETCA management is challenging and associated with poor outcomes and high mortality. This study compares management and outcomes of ETCA to more common HD forms. METHODS: A retrospective cohort of HD patients (2012-2023) from two institutions. Three HD forms were compared: short-segment HD (SSHD, n = 19), long-segment HD or total colonic aganglionosis (LS/TCA, n = 9) and ETCA (n = 7). RESULTS: Normally innervated segments in ETCA patients ranged 0-70 cm. Median times to first surgery were; ETCA = 3 days versus TCA = 21 days (p = 0.017) and SSHD = 95 days (p < 0.001), respectively. Median number of surgeries were; ETCA = 4, versus TCA = 2 (p = 0.17) and SSHD = 1 (p = 0.002), respectively. All the patients underwent a definitive pull-through procedure, except four ETCA patients with a permanent jejunostomy and residual aganglionic segment of 57-130 cm. ETCA patients had 92% lower odds of enterocolitis (14%) compared to TCA patients (67%, p = 0.054), and comparable odds to SSHD patients (16%, p = 0.92). ETCA mortality was 14%. CONCLUSION: Extended total colonic aganglionosis patients require earlier and multiple interventions. Leaving an aganglionic segment may be advantageous, without increasing risk for enterocolitis. Tailored surgical treatment and rehabilitation programmes may prevent mortality and need for transplantation.

Surgical maneuvers for long-segment Hirschsprung pull-through in unique patients.

PURPOSE: Patients with Hirschsprung disease affecting the splenic flexure or more proximal segments present a surgical challenge. Mobilizing the transverse colon to the pelvis during a pull-through may obstruct the distal ileum, or the length may be insufficient to reach the lower pelvis. This retrospective study aimed to describe two surgical techniques that facilitate mobilization of the transverse colon and their outcome. METHODS: We included patients operated on between April 2017 and April 2024 and analyzed sex, comorbidities, type of pull- through, age at pull-through, history of previous surgeries, cause of the proximal transverse colon pull-through, technique used (Deloyers or Turnbull), complications , postoperative outcome and follow-up. The first technique used was the maneuver described by Turnbull. This operation creates a mesenteric defect and mobilizes the colon into this mesenteric window at the distal ileum level. The second technique was described by Deloyers and involves a 180-degree rotation of the right colon by dissecting the right colon attachment and the hepatocolic ligament. The cecum and the ileocecal valve are placed in the right upper quadrant, and the distal colon is mobilized into the pelvis. RESULTS: We included 13 patients, 12 boys and 1 girl. Eight patients had previous surgeries in another hospital: five had an initial transverse colostomy, and three had an ileostomy. The remaining five had the initial operation in our hospital: two had an ileostomy, two had a colostomy, and one had a primary pull-through. The median age at pull-through was 16 months (4-59 months). We used the Turnbull technique in four patients whose aganglionosis was limited to the middle transverse colon. The Deloyers technique was used in the remaining patients, with ganglion cells in the proximal transverse colon. We left a protective ileostomy in five patients. The median follow-up was 4.5 years (3 months to 10 years). The stoma takedown is pending in one patient. CONCLUSION: The Turnbull and Deloyers techniques were helpful in patients with aganglionosis affecting the transverse colon.

Ernica Clinical Consensus Statements on Total Colonic and Intestinal Aganglionosis.

BACKGROUND: Hirschsprung disease is a congenital intestinal motility disorder characterized by an absence of enteric ganglion cells. Total colonic aganglionosis and near total or total intestinal aganglionosis, defined as absence of ganglion cells in the entire colon and with variable length of small bowel involved, are life-threatening conditions which affect less than 10 % of all patients with Hirschsprung disease. The aim of this project was to develop clinical consensus statements within ERNICA, the European Reference Network for rare congenital digestive diseases, on four major topics: Surgical treatment of total colonic aganglionosis, surgical treatment of total intestinal aganglionosis, management of poor bowel function in total colonic and/or intestinal aganglionosis and long-term management in total colonic and or intestinal aganglionosis. METHODS: A multidisciplinary panel of representatives from ERNICA centers was invited to participate. Literature was searched, using specified search terms, in Medline (ALL), Embase and Google Scholar. Abstracts were screened and full text publications were selected. The panel was divided in four groups that extracted data from the full text publications and suggested draft statements for each of the major topics. A modified Delphi process was used to refine and agree on the statements. RESULTS: The consensus statement was conducted by a multidisciplinary panel of 24 participants from 10 European countries, 45 statements reached consensus after 3 Delphi-rounds. The availability of high-quality clinical evidence was limited, and most statements were based on expert opinion. Another 25 statements did not reach consensus. CONCLUSIONS: Total colonic and total intestinal aganglionosis are rare variants of Hirschsprung disease, with very limited availability of high-quality clinical evidence. This consensus statement provides statements on the surgical treatment, management of poor bowel function and long-term management for these rare patients. The expert panel agreed that patients benefit from multidisciplinary and personalized care, preferably in an expert center. TYPE OF STUDY: Clinical consensus statement. LEVEL OF EVIDENCE: 3a.

High-Resolution Anorectal Manometry as a Screening Tool for Hirschsprung's Disease: A Comprehensive Retrospective Analysis.

Hirschsprung's disease (HD) is characterized by a congenital absence of enteric ganglion cells in the intestine, posing challenges in diagnosis, particularly in pediatric patients. The gold standard, rectal suction biopsy (RSB), carries risks, prompting an exploration of non-invasive alternatives such as high-resolution anorectal manometry (HR-ARM) for HD screening. We conducted a retrospective analysis of 136 patients suspected of HD between 2018 and 2022, which were stratified into three age groups: ≤12 months, ≤24 months, and >24 months. Criteria for suspicion included delayed meconium passage, unresponsive chronic constipation, and abnormal prior test results. HR-ARM, supplemented by additional tests, confirmed 16 HD cases. HR-ARM exhibited 93.75% sensitivity, 89.47% specificity, 99.03% negative predictive value (NPV), and 55.56% positive predictive value (PPV). Notably, HR-ARM consistently performed well in patients ≤ 2 years old but demonstrated reduced efficacy in older children, which was likely due to complications from chronic constipation. This study underscores HR-ARM's promise as a non-invasive HD screening tool, especially in younger patients. However, its limitations in older children warrant consideration. Establishing standardized protocols, particularly for assessing the recto-anal inhibitory reflex, is crucial. Further research is imperative to optimize HR-ARM's diagnostic role across varied age groups in HD assessment.

Sacral Neural Crest-Independent Origin of the Enteric Nervous System in Mouse.

BACKGROUND & AIMS: The enteric nervous system (ENS), the gut's intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases but has been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. METHODS: We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3-dimensional reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further used coculture assays to determine the specificity of cell migration from different neural tissues into the hindgut. RESULTS: Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but only pan-NCCs contributed to the ENS. Dual-lineage tracing combined with 3-dimensional reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Coculture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. CONCLUSIONS: sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung's disease.

Skipped Aganglionic Lengthening Transposition (SALT) is highly effective to achieve enteral autonomy in selected patients with intestinal failure secondary to total intestinal aganglionosis.

BACKGROUND: Intestinal Failure, parenteral nutrition (PN) dependence, and subsequent liver disease are the most challenging and life-threatening complications of short bowel syndrome experienced by patients with total intestinal aganglionosis. Skipped Aganglionic Lengthening Transposition (SALT) showed to be a promising procedure to overcome such problems. We herein report the results of two patients who underwent SALT at the Umberto Bosio Center for Digestive Diseases. PATIENTS AND METHODS: Between November 2019 and July 2022, 2 patients with total intestinal aganglionosis underwent SALT as autologous intestinal lengthening procedure. Perioperative data and long-term outcomes are reported. Patient #1-A 18 month-old male (PN dependant) with 30 cm of ganglionated bowel at birth experienced a 35% increase of intestinal length after SALT (from 43 to 58 cm) thanks to three 5 cm interposed aganglionic loops. Postoperative course was uneventful and he was totally weaned by PN after 28 months postoperatively. He is without PN only receiving enteric feeding 53 months after the procedure. Patient #2-A 11 year-old female (PN dependant) with 100 cm of ganglionated jejunum underwent SALT at 11 years and experienced a 19% increase of bowel length thanks to four 5 to 7 cm interposed aganglionic loops. Postoperatively she required excision of two out of the four loops due to severe strictures and inadequate perfusion with a subsequent overall 10% increase of length after SALT. Of note, she improved significantly with a progressive reduction of PN that has been stopped after 18 months. CONCLUSION: Skipped aganglionic lengthening transposition (SALT) seems to be very effective in improving nutrients absorption in patients with total intestinal aganglionosis by increasing absorptive bowel surface and decelerating intestinal flow for a longer and more effective contact of enteric material with ileal mucosa. Provided these impressive results are confirmed in the very long-term, SALT could become a valid alternative for the treatment of patients with total intestinal aganglionosis carrying at birth at least 20 to 30 cm of ganglionated jejunum.

Clinicopathologic evaluation of congenital idiopathic megaesophagus in a Gordon Setter puppy: a case report and development and application of peripherin immunohistochemistry for detection of ganglion cells.

We examined a case of congenital idiopathic megaesophagus (CIM) in a 5-wk-old female Gordon Setter puppy by means of contrast radiography, autopsy, histopathology, and immunohistochemistry. Clinical and radiologic findings included weight stagnation and marked generalized esophageal dilation with ventral displacement of the heart and lungs. These findings were confirmed at autopsy, and segments of the thoracic esophagus were sampled for histopathology. On histopathology, diffuse esophageal muscular atrophy, mucosal erosions, mononuclear inflammation, and a marked reduction in the number of myenteric plexus structures and number of ganglion cells were present (aganglionosis). The latter was determined immunohistochemically using an anti-peripherin antibody as the primary reagent, which provides a strong tool for the histologic confirmation of CIM. The histologic findings share some similarities to lesions associated with megaesophagus in Friesian foals, as well as esophageal achalasia and Hirschsprung disease in humans.

Diagnostic accuracy of barium enema versus full-thickness rectal biopsy in children with clinically suspected Hirschsprung's disease: A comparative cross-sectional study.

Background and Aims: Hirschsprung's disease (HSD) remains a common cause of pediatric intestinal obstruction. Barium contrast enema (BE) is the primary imaging modality for the evaluation of clinically suspected cases. Here, we aimed to assess the diagnostic accuracy of BE in children with clinically suspected HSD when compared to a gold standard full-thickness rectal biopsy (FTRB). Methods: We recruited and consecutively enrolled children with clinically suspected HSD at two tertiary teaching hospitals. Participants underwent BE imaging and two radiologists interpreted the findings independently. Participants further underwent FTRB by pediatric surgeons as the confirmatory test. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and receiver operating characteristics (ROC) with the area under the curve (AUC) were calculated on Stata version 14.2, taking FTRB as the standard. Results: We enrolled 55 cases, of which 49 completed the evaluation and were included in the final analysis. The median age was 9.4 months (interquartile range: 2-24], with a male-to-female ratio of 4.4:1. The sensitivity, specificity, PPV, and NPV of BE were 0.95 (95% confidence interval [CI] [0.81-0.99]), 0.73 (95% CI [0.39-0.94]), 0.92 (95% CI [0.82-0.97]), and 0.80 (95% CI [0.50-0.94]), respectively. On AUC, the diagnostic accuracy of BE compared to the confirmatory FTRB was 0.84 (95% CI [0.69-0.98]). The diagnostic accuracy was higher in neonates (ROC: 1.00) when compared to infants (ROC: 0.83) or those above 1 year of age (ROC: 0.798). HSD-suggestive BE findings were associated with absence of ganglion cells on FTRB (χ 2 = 23.301, p < 0.001). Inverted rectosigmoid ratio and transition zone were more sensitive in detecting HSD of 0.92 (95% CI [0.74-0.98]) and 0.81 (95% CI [0.63-0.92]), respectively. Conclusion: BE is sufficiently accurate in the diagnosis of children with HSD, suggesting BE would likely be used to inform surgical management in settings where confirmatory biopsy is lacking. However, clinical judgment is warranted in interpreting negative BE findings.

Rectal biopsy: is it really necessary in anorectal malformations?

AIM OF THE STUDY: The coexistence of Hirschsprung's disease (HD) with anorectal malformation (ARM) is rare but many surgeons still ask pathologists to look for ganglia in the terminal rectum or fistula. In this study, we aimed to highlight the rarity of this association and question the necessity of histological evaluation. METHODS: After obtaining board review approval, rectal specimens of ARM patients who underwent corrective surgery in the last 8 years were re-analyzed by two blinded pathologists for the presence and structure of ganglia. Clinical and radiological data of patients retrieved from center records and correlated with histopathologic findings. MAIN RESULTS: 67 patients with ARM were identified, distal rectal specimen was obtained in 47. The median age at the time of surgery was 11 months (2 days-59 months). A normal pattern of ganglia was present in 51.1% (24/47), 29.7% (14/47) had aganglionosis and 19.1% (9/47) were inconclusive due to insufficient material. None of the aganglionic specimens showed other histological findings of HD. Patients were followed up regularly in the outpatient colorectal clinic for a median of 87 months (42-117 m). Only 6 experienced severe constipation (3 ganglionic, 2 no biopsy, 1 aganglionic), all of whom responded to a bowel management program, and none developed other manifestations of HD (abdominal distension, failure to thrive or enterocolitis) or required additional surgery. CONCLUSIONS: Our results strongly suggest that the association of ARM and HD is extremely rare and the practice of searching for ganglia in the distal rectum or fistula of ARM patients should be discouraged to avoid potential misdiagnosis and overtreatment.

A unicentric cross-sectional observational study on chronic intestinal inflammation in total colonic aganglionosis: beware of an underestimated condition.

BACKGROUND: Inflammatory Bowel Diseases (IBD) are known to occur in association with Hirschsprung disease (HSCR). Most of cases are represented by Crohn Disease (CD) occurring in patients with Total Colonic Aganglionosis (TCSA) with an estimated prevalence of around 2%. Based on these considerations and on a number of provisional data belonging to our Center for Digestive Diseases, we developed a unicentric cross-sectional observational study aimed at describing phenotype, genotype, pathology and metagenomics of all patients with TCSA and Crohn-like lesions. RESULTS: Out of a series of 62 eligible TCSA patients, 48 fulfilled inclusion criteria and were enrolled in the study. Ten patients did not complete the study due to non-compliance or withdrawal of consent and were subsequently dropped out. A total of 38 patients completed the study. All patients were tested for chronic intestinal inflammation by a combination of fecal calprotectine (FC) or occult fecal blood (OFB) and underwent fecal metagenomics. Nineteen (50%) tested positive for FC, OFB, or both and subsequently underwent retrograde ileoscopy. Fourteen patients (36.8%) presented Crohn-like lesions, occurring after a median of 11.5 years after surgery (range 8 months - 21.5 years). No statistically significant differences regarding demographic, phenotype and genotype were observed comparing patients with and without lesions, except for need for blood transfusion that was more frequent in those with lesions. Faecal microbiome of patients with lesions (not that of caregivers) was less biodiverse and characterized by a reduction of Bacteroidetes, and an overabundance of Proteobacteria. FC tested negative in 3/14 patients with lesions (21%). CONCLUSIONS: Our study demonstrated an impressive 10-folds higher incidence of chronic inflammation in TCSA. Up to 50% of patients may develop IBD-like lesions postoperatively. Nonetheless, we failed in identifying specific risk factors to be used to implement prevention strategies. Based on the results of our study, we suggest screening all TCSA patients with retrograde ileoscopy regardless of FC/OFB values. The frequency of endoscopic assessments and the role of FC/OFB screening in prompting endoscopy is yet to be determined.

State of the Art Bowel Management for Pediatric Colorectal Problems: Hirschsprung Disease.

After an initial pull-though, patients with Hirschsprung disease (HD) can present with obstructive symptoms, Hirschsprung-associated enterocolitis (HAEC), failure to thrive, or fecal soiling. This current review focuses on algorithms for evaluation and treatment in children with HD as a part of a manuscript series on updates in bowel management. In constipated patients, anatomic causes of obstruction should be excluded. Once anatomy is confirmed to be normal, laxatives, fiber, osmotic laxatives, or mechanical management can be utilized. Botulinum toxin injections are performed in all patients with HD before age five because of the nonrelaxing sphincters that they learn to overcome with increased age. Children with a patulous anus due to iatrogenic damage of the anal sphincters are offered sphincter reconstruction. Hypermotility is managed with antidiarrheals and small-volume enemas. Family education is crucial for the early detection of HAEC and for performing at-home rectal irrigations.

Impact of cryopreservation on viability, gene expression and function of enteric nervous system derived neurospheres.

Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children's quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality. Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function. Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing. Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.

Rare presentation and retrograde diagnosis of total colonic aganglionosis in a female infant: a case report.

BACKGROUND: Total colonic aganglionosis is an extremely rare variant of Hirschsprung's disease, which is predominant in males and can be seen in 1:50,000 live births. The presented case not only depicts a rare case, but also unusual clinical, laboratory, and instrumental data. CASE PRESENTATION: A 2-day-old Caucasian female newborn was transferred to our hospital from maternity. The initial presentation was reverse peristalsis, abdominal distention, and inability to pass stool. Fever had started before the patient was transferred. Hirschsprung's disease was suspected, and tests such as contrast enema and rectal suction biopsy were done. Before enterostomy, the management of the disease included fluid resuscitation, colonic irrigation, antibiotic administration, enteral feeding, and supportive therapy. During ileostomy operation, no transition zone was visualized and full-thickness biopsy samples were retrieved from the rectum and descending colon. After surgical intervention, status significantly improved-defervescence and weight gain most importantly improved. CONCLUSION: It is well known that diagnosis of total colonic aganglionosis may be delayed for months or even years since the transition zone may not be visible and rectal suction biopsy, unlike full-thickness biopsy, is not always reliable. It might be more prudent not to be derailed because of negative radiography and rectal suction biopsy. Also, doctors should be more suspicious of the disease if signs and symptoms are starting to be consistent with Hirschsprung-associated enterocolitis, despite biopsy and radiology results.

Ileal atresia and total colonic hirschsprung disease in a 36-week neonate: A case report.

Intestinal atresia and hirschsprung disease are two common causes of bowel obstruction in neonates; simultaneous occurrence is rare. This report delineates a 36-week newborn with ileal atresia and total colonic hirschsprung who was referred to our unit due to failure of meconium passage during the first 48 h after birth.

Long-term Bowel function and pediatric health-related quality of life after transanal rectal mucosectomy and partial internal anal sphincterectomy pull-through for Hirschsprung Disease.

Objective: The aim of this study was to define controlled outcomes for bowel function and quality of life (QoL) after transanal rectal mucosectomy and partial internal anal sphincterectomy pull-through (TRM-PIAS, A modified Swenson procedure) for Hirschsprung disease (HD). Background: We have previously shown that a novel modification of transanal rectal mucosectomy and partial internal anal sphincterectomy (TRM-PIAS, A modified procedure) for Hirschsprung disease have the advantage of low postoperative Hirschsprung associated enterocolitis. The controlled long-term follow-up studies evaluating Bowel Function Score (BFS) and Pediatric Quality of Life Inventory (PedsQoL, age <18 years) remain unclear. Methods: Between Jan 2006 and Jan 2016, 243 Patients underwent TRM-PIAS older than 4 years were included, while experienced redo surgery because of complication were excluded. Patients were compared with age- and gender-matched 244 healthy children each randomly selected from the 405 general population. The enrollee was investigated for questionnaires on BFS and PedsQoL. Results: One hundred and ninety-nine (81.9%) patients' representatives for the entire study population responded. The mean age of patients was 84.4 months (48-214 months). Compared with controls, patients reported impairment of hold back defecation, fecal soiling, and the urge to defecate (P < 0.05), and no significantly different in fecal accidents, constipation and social problems. With advancing age, the total BFS of HD patients improved, with a tendency close to the normal level beyond 10 years old. But, after grouped according to presence or absence of HAEC, the non-HAEC group experienced more dramatic improvement with age increasing. Conclusions: Compared with matched peers, significant impairment of fecal control prevails after TRM-PIAS in HD patients, but bowel function improve with age and recovery faster than conventional procedure. It should be emphasized that post-enterocolitis is a high-risk factor for delayed recovery.

Genetics of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a classical model of enteric neuropathy, occurring in approximately 2-2.8 in 10,000 newborns. It is the commonest form of congenital bowel obstruction and is characterized by the absence of enteric ganglia in distal colon. Recent advances in genome-wide association analysis (GWAS) and next generation sequencing (NGS) studies have led to the discovery of a number of new HSCR candidate genes, thereby providing new insights into the genetic architecture and molecular mechanisms of the disease. Altogether, these findings indicated that genetic heterogeneity, variable penetrance and expressivity, and genetic interaction are the pervasive characteristics of HSCR genetics. In this review, we will provide an update on the genetic landscape of HSCR and discuss how the common and rare variants may act together to modulate the phenotypic manifestation. Translating the genetic findings to genetic risk prediction and to optimize clinical outcomes are undoubtedly the ultimate goals for genetic studies on HSCR. From this perspective, we will further discuss the major obstacles in the clinical translation of these latest genetic findings. Lastly, new measures to address these clinical challenges are suggested to advance precision medicine and to develop novel alternative therapies.