Aganglionic Bowel Research Articles

Involvement of down-regulated E2F3 in Hirschsprung's disease

Background/PurposeHirschsprung's disease (HSCR) is a common cause of neonatal bowel obstruction characterized by the absence of ganglion cells in the colon. Impaired migration of the neural crest cells (NCCs) has been implicated as one of the main causes of HSCR. E2F3, a member in the E2F family, which plays a crucial role in the control of the cell cycle is correlated with neuron migration. However, the function of E2F3 in the development of the enteric nervous system still remains unknown. This study aims to reveal the correlation of E2F3 in the progress of HSCR. MethodsBy using reverse transcriptase polymerase chain reaction (RT-PCR) and western blot assay, we investigated levels of E2F3 expression in 58 HSCR patients, both in the aganglionic bowel segment and the normal ganglionic segment, and in 39 unrelated controls. By in vitro assays, we used the siRNA method to knock-down the level of E2F3 expression in 293T cell lines. Furthermore, transwell assay was used to detect cell migration ability. ResultsAberrant lower expression level of E2F3 was detected in the HSCR-S segment compared with the control group by RT-PCR and western blot assay. Besides, down-regulated E2F3 could suppress the cell migration. ConclusionsThis is the first study showing the down-regulation of E2F3 in HSCR, bringing new insight to the mechanism of the impaired migration of neural crest cells.

Totally Transanal LESS Pull-Through Colectomy: A Novel Approach for Avoiding Abdominal Wall Incision in Children with Long-Segment Intestinal Aganglionosis

Abstract Introduction: Minimally invasive surgery in children with long-segment intestinal aganglionosis aims to reduce the number of abdominal wall incisions. Conventional laparoscopic and laparoendoscopic single-site (LESS) surgeries fulfill this goal. In children, natural orifice translumenal endoscopic surgery (NOTES™; American Society for Gastrointestinal Endoscopy [Oak Brook, IL] and Society for American Gastrointestinal and Endoscopic Surgeons [Los Angeles, CA]) has been limited because of fear of access site complications. We present a novel technique of totally transanal LESS pull-through colectomy (TLPC), avoiding abdominal wall incision, which combines LESS technology and the NOTES approach. Two boys and one girl (2.5 months, 6 months, and 5 years of age, respectively) with sigmoid and transverse colon aganglionosis underwent surgery. The TLPC procedure consisted of an endorectal technique with submucosal dissection starting 1 cm orally from the dentate line to above the peritoneal reflection, where the rectal muscle was divided circumferentially. After ligation of the rectal mucosa, the proximal bowel was replaced into the abdominal cavity, and a TriPort(®) (Olympus Surgical Technologies Europe, Hamburg, Germany) was introduced transanally. Mesenterial resection of the aganglionic bowel was accomplished via transanal LESS until the normoganglionic colon segment was reached and pulled down to the site of anastomosis. After removal of the port, a conventional pull-through procedure was performed. All children displayed normal bowel movements and were complication-free during the follow-up period of up to 7 months. TLPC combines the minimally invasive LESS surgery with the scarless concept of NOTES and allows resection of long-segment aganglionosis without abdominal incision. TLPC is a safe, effective, and feasible surgical procedure in children with long-segment intestinal aganglionosis.

Mast Cells Connections with Nerve Fibers in Hirschsprung’s Disease

Introduction: Hirschsprung’s disease (HD) is rare (1/5000 newborns) congenital condition of the bowel nervous system, clinically characterized by dilatation and hypertrophy of colon. There are an increased number of mast cells (MC) in aganglionic bowel segments and in those with intestinal neuronal dysplasia (IND). The exact role of MC in HD is still not known. The aim of this study was to examine the colonic distribution of mast cells (MC) and the relationship between MC and nerve fibers and to estimate connections of these cells with gut nervous system. Material and Methods: Distal colon wall specimens were harvested from patients with normal bowel function and with normal ganglion cells, patients with constipations and with normal ganglion cells and a group of patients with Hirschsprung’s disease. The age of the patients ranged from 2nd day of live to 12 years. The density of the mast cells was calculated using a light microscope with digital camera. The connections of localization of a MC with nerve fibers and ganglions were taken under consideration and observed under light microscope. Test for the significance of differences between groups were performed using the Kruskal-Wallis test. Results: We revealed that there is a greater number of the MC in mucosa, lamina propria, submucosa and muscle layer of large bowel in patients with HD. There was no difference in density of the MC in serosa. Some of the colon MC in HD was located near or they were even attached to the hypertrophic nerve trunks. We found a correlation of MC number with the number of nerve fibers in HD patients. Conclusions: Higher number of MC and their localization in close connection with hypertrophied nerve trunks in patients with HD than in patients with constipation or normal bowel function, is a proof of the influence of MC on defected nervous system in aganglionic bowel. MC probably affect hypertrophied nerve trunks. Mobilization and activation of the mast cells which produce and store NGF can be directed to repair defective nervous system. Obviously, MC repairing reaction is not effective in restoring nervous system function of the gut.

Soave transanal one-stage endorectal pull-through in the treatment of Hirschsprung′s disease of the child above two-year-old: A report of 20 cases

The definitive treatment of Hirschsprung's disease is the removal of the aganglionic bowel by a pull-through surgery. In most cases, this surgery is performed in infancy or in the neonatal period as presentation in older children and adulthood is uncommon. It is a retrospective study of 20 patients above two-year-old who underwent a transanal Soave one-stage endorectal pull-through procedure for Hirschsprung's disease between January 2002 and December 2010. Twenty patients were recruited in this study. Fourteen were males and six were females. Patient ages ranged from 2 to 14 years (median age: five years and three months). All patients presented with persistent constipation and abdominal distension. Two of them had an intestinal obstruction that required colostomy. Ten patients (50%) had a recto-sigmoid Hirschsprung's disease. All patients were operated on using a Soave one-stage endorectal pull-through procedure. The laparoscopy was necessary during the pull-through in three cases. The average duration of the intervention was 240 minutes. That represents almost the double of the duration of the same procedure in newborns and infants in our department (130 minutes). Early postoperative complications included one case of anastomosis leakage and one case of intussusception. Late postoperative complications were perineum irritation in five cases (25%), anal stenosis in four cases (20%) and enterocolitis in one case (5%). None of our patients developed fecal incontinence. Soiling was reported in four cases (20%). There was no death. Soave transanal one-stage endorectal pull-through is safely feasible in children of more than two years of age. Laparoscopy may be necessary whenever there are difficulties in the pull-through.

Transitional zone pull through: surgical pathology considerations

Incomplete resection of the transitional zone (TZ) between histologically normal and aganglionic bowel in Hirschsprung disease is a putative cause of postoperative dysmotility. A review of literature indicates that diverse histopathological indexes have been used to define the TZ, and validated and reproducible diagnostic criteria have not been established. As a consequence, the proximal margin of the TZ is difficult to delimit, and the length of the TZ in a given patient depends on the diagnostic criteria used. Seromuscular biopsies are inadequate to exclude TZ, as diagnostic indexes may involve only a portion of the bowel circumference or the submucosa. Most published investigations of postoperative outcome after a TZ pull through (TZPT) conclude that the latter can cause persistent obstructive symptoms, which necessitate reoperation. However, the results of these studies are difficult to translate into clinical practice because most lack appropriate controls, and the overwhelming majority provide inadequate histopathological descriptions for reference at the time of intraoperative frozen section analysis. At present, a conservative approach based on frozen section examination of the entire proximal margin of the resection to exclude obvious subcircumferential aganglionosis (contiguous gap between ganglia of more than one-eighth of the circumference), hypoganglionosis (continuous string of myenteric ganglia comprised of 1 or 2 ganglion cells without surrounding neuropil), or hypertrophic submucosal nerves (>2 nerves with widths ≥40 μm per high-power field) seems prudent. Well-controlled studies to correlate proximal margin histology, especially subtle anatomic or immunohistochemical changes, with postoperative outcome are needed.

Investigation of L‐type Ca2+ current in the aganglionic bowel segment in Hirschsprung’s disease

Studies on animal models of Hirschsprung's disease (HD) suggest that L-type Ca(2+) channels are down-regulated in the aganglionic bowel segment, however, this has yet to be confirmed in HD patients. The objective of this study was to test the hypothesis that L-type Ca(2+) current density is decreased in smooth muscle cells (SMC) obtained from the aganglionic bowel segment of patients with HD in comparison with those from the ganglionic segment. Smooth muscle cells were freshly isolated from colon samples obtained from HD patients undergoing pull-through surgery. L-type Ca(2+) currents were recorded using the perforated patch configuration of the whole cell voltage clamp technique and the expression levels of CACNA1C transcripts (which encode L-type Ca(2+) channels) in the ganglionic and aganglionic bowel segments were compared using real-time quantitative PCR. All SMC displayed robust currents that had activation/inactivation kinetics typical of L-type Ca(2+) current, were inhibited by nifedipine and enhanced by the L-type Ca(2+) channel agonists FPL 64176 and Bay K 8644. Moreover, FPL 64176 activated currents were also inhibited by nifedipine. However, there was no significant difference in L-type Ca(2+) current density, CACNA1C subunit expression or sensitivity to the pharmacological agents noted above, between SMC isolated from the ganglionic and aganglionic regions of the HD colon. In contrast to studies on genetic animal models of HD, L-type Ca(2+) currents are not down-regulated in the aganglionic bowel segment of HD patients and are therefore unlikely to account for the impaired colonic peristalsis observed in these patients.

Hirschsprung Disease of the Adult Mimicked by Chronic Constipation or Vice Versa

Hirschsprung disease is a rare congenital disorder characterized by a functional obstruction of the distal bowel due to aganglionic bowel segments with impaired motility. It is a rare condition in the adult, and usually mimicked by a long standing history of constipation, requiring great astuteness to diagnose. We present a case with all the clinical and radiologic characteristics of adult Hirschsprung disease, which had a final diagnosis of chronic constipation in a mentally impaired individual. doi:10.4021/jmc731w

Is there a link between the calcium sensing receptor and Hirschsprung's disease? A mutational analysis

IntroductionHirschsprung's disease (HD) is a congenital malformation of the hindgut characterized by the absence of enteric ganglion cells in the submucosal and myenteric plexuses. Hirschsprung's disease is routinely treated by resection of the aganglionic bowel and pull-through procedure of the proximal ganglionated bowel to the anorectal region. Occasionally, after resection of the aganglionic bowel, HD patients still experience persistent disturbances in gut motility. The etiology of HD as well as the underlying pathomechanism for postoperative disturbances in gut motility is unclear. Molecular analysis of putative candidate genes may help to clarify the pathophysiology of these conditions. In the present study, we investigated the correlation between mutations and polymorphisms in the calcium sensing receptor (CaSR) and HD patients. MethodsMutational screening of the CaSR coding sequence was performed via polymerase chain reaction and direct sequencing of the amplified samples from 63 HD patients and 100 controls. ResultsWe identified 3 common polymorphisms (p.A986S, p.G990R, and p.Q1011E) residing in exon 7 and 1 polymorphism in intron 5 (IVS 5-88 T>C) of the CaSR gene. Overall, 16 patients (25%) and 25 controls (25%) were carriers of the p.A986S polymorphism (P = 1). The incidence of p.R990G polymorphism of patients was twice as high as in the control group (P = .17). Furthermore, we verified a 4 times higher incidence of p.Q1011E polymorphism carriers in patients compared to the control group (P = .1). ConclusionWe found a higher incidence of R990G and Q1011E polymorphisms in HD patients compared to controls. However, there was no statistically significant association between HD and the 3 polymorphic variants in the intracellular signaling region of CaSR. It will be important to further investigate genetic variations in CaSR in more patient cohorts to better characterize the function of this gene and to establish a correlation between CaSR variants and HD.

The microenvironment in the Hirschsprung's disease gut supports myenteric plexus growth

The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects. Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-β-1 (TGFβ-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated. The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.

Reoperation for Hirschsprung Disease: Pathology of the Resected Problematic Distal Pull-Through

Hirschsprung disease, which consists of aganglionosis of the rectum and sometimes more proximal bowel, requires surgical removal of the aganglionic bowel and creation of ganglionated neorectum using proximal normally innervated bowel. The border between aganglionic and ganglionic bowel is irregular; the transition zone features variable quantities of ganglion cells and numerous large nerves. We report the histopathology of pull-through bowel segments resected because of poor postoperative outcome from 30 patients (22 boys, 8 girls). The most common indication for reoperation was severe constipation/obstruction. Transition zone (bowel with at least two nerves ≥40 µm diameter per 400× high-power field, and ganglion cells) or aganglionic bowel (bowel with at least two nerves ≥40 µm per high-power field diameter, but without ganglion cells) was found in 19/30 (63%) resections. In colons resected because of familial adenomatous polyposis, rare high-power fields showed two enlarged nerves; the mean age of those patients (135 ± 49.4 months) was significantly higher than that of the patients undergoing redo pull-through surgery (67.9 ± 42.8 months). Additional pathology included stricture and enterocolitis. Although there are multiple causes for poor outcomes following surgical therapy for Hirschsprung disease, abnormal innervation of the bowel used for pull-through is common. We recommend that intraoperative consultation at primary pull-through procedure include frozen section evaluation of the circumference of the bowel to be used for pull-through to confirm histologically the presence of both ganglion cells and normal-caliber nerves. The criteria used in this study are most suitable for infants and young children.

Hirschsprung Disease of the Adult Mimicked by Chronic Constipation or Vice Versa?

Hirschsprung disease is a rare congenital disorder characterized by a functional obstruction of the distal bowel due to aganglionic bowel segments with impaired motility. It is a rare condition in the adult, and usually mimicked by a long standing history of constipation, requiring great astuteness to diagnose. We present a case with all the clinical and radiologic characteristics of adult Hirschsprung disease, which had a final diagnosis of chronic constipation in a mentally impaired individual. J Med Cases. 2012;3(5):277-279 doi: https://doi.org/10.4021/jmc731w

NONRUMINANT NUTRITION SYMPOSIUM: Involvement of gut neural and endocrine systems in pathological disorders of the digestive tract1,2

The functioning of the gastrointestinal tract is under the control of the most extensive system of peripheral neurons in the body, the enteric nervous system, and the largest endocrine system of the body, the GEP endocrine system. The enteric nervous system in large mammals contains 500 million neurons, and the GEP endocrine system produces more than 30 hormones. Numerous enteric neuropathies affecting both humans and animals have been described and digestive disorders affect commercially important species, such as horses and cattle. The most severe enteric neuropathies (e.g., lethal white syndrome in horses or Hirschsprung's disease in humans) can be fatal. Also, horses with ileus or other digestive disorders are commonly euthanized. In this review we discuss examples of enteric neuropathies that affect agricultural animals and humans: prion disease, postoperative ileus, distal enteric aganglionosis, and infective diarrhea. Enteric neurons and glia are a location of prion proteins and are involved in transmission of the infection from gut to brain and brain to gut. Postoperative ileus is a complex disorder involving the local inhibitory effects of sympathetic nervous system activation and the release of opioids, presumably from enteric neurons. Intestinal inflammation, especially of the external muscle that includes enteric ganglia, also occurs in ileus. Congenital distal bowel aganglionosis, responsible for lethal white syndrome in horses, Hirschsprung's disease in humans, and similar conditions in mice and rats, is a fatal condition if untreated. Mutations of the same genes can cause the condition in each of these species. The only effective current treatment is surgical removal of the aganglionic bowel. Infectious diarrheas involve activation of enteric secretomotor neurons by pathogens and the toxins they produce, which causes substantial fluid loss. Strategies to target enteric neurons in the treatment of secretory diarrheas have not been developed. Disorders of enteroendocrine cells, other than GEP endocrine tumors, are less well documented. However, evidence for the involvement of gut endocrine cells in a subset of patients with irritable bowel syndrome, and in the symptomology of celiac disease, has been demonstrated. Further investigation of the involvement of enteric neural and endocrine signaling systems in digestive disorders, especially in agricultural and companion animals, may lead to diagnostic and therapeutic advances.

Maternal Microchimerism in Hirschsprung’s Disease

Hirschsprung's disease (HD) presents with severe constipation due to absent ganglion cells in the distal rectum. We sought to determine whether maternal chimeric cells are present in aganglionic bowel. We hypothesize that chimeric cells are part of the unfavorable microenvironment that leads to the destruction of enteric neurons in HD. Intestinal biopsies and resections from seven male patients with HD were compared with four male patients with chronic constipation and six with bowel atresia. Fluorescence in situ hybridization was used to identify chimeric cells based on male/female (XX/XY) differences. The location and immunophenotype of chimeric cells were also studied. Chimeric cells were present more often in the small intestine and rectum, compared with the appendix and colon. Patients with HD had a greater number of chimeric cells per 10× magnification field than patients with chronic constipation or congenital atresia. Chimeric cells were predominantly in the submucosa and outer longitudinal muscle layer in HD. Immunophenotyping identified over 40% of chimeric cells as inflammatory. Chimeric cells are present in greater numbers in aganglionic bowel than in other disorders. Clustering of chimeric cells in areas of absent ganglia lends support to the proposed role of maternal microchimerism in allo-autoimmune responses.

Smooth muscle proteins from Hirschsprung’s disease facilitates stem cell differentiation

The transplantation of neural crest derived stem cells (NCSC's) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC's were studied in vitro. Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC's were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated. The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation. Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC's and is so an appropriate target for neural stem cell treatment.

Is complete resection of the aganglionic bowel in extensive total aganglionosis up to the middle ileum always necessary?

Background/Purpose Total colonic aganglionosis is an unusual form of Hirschsprung disease, reflected by the small number of published case studies. The goal of this study was to analyze our cases of the last 22 years and report on our experience with 3 cases of extended total aganglionosis proximal to the middle ileum in which remaining aganglionic bowel was left in situ. Materials and Methods In a period of 22 years (from January 1988 to April 2010), we operated on 15 children with total aganglionosis. These children were among 163 children with Hirschsprung disease. In 3 patients with extended aganglionosis proximal to the middle ileum, remaining aganglionic bowel was left in situ. Data were collected from the medical records, including each patient's demographics, medical history, primary treatment, complications, and follow-up. Results Eleven of the 15 children received an ileostomy as first treatment, and 4 children underwent a primary Duhamel procedure. Early postoperative complications included an abscess after an open procedure in a patient with trisomy 21 syndrome and an incorrect leveling resection that required an ileostomy. There were 2 deaths at 6 and 16 months postoperatively. The mortality rate was 13.3%. Of the 15 patients, 7 (46%) presented with at least 1 episode of postoperative enterocolitis, and only 2 of these patients had more than 3 episodes. We treated 3 patients with extended aganglionosis up to midileum (20%) in which the proximal resection level was in the aganglionic segment of ileum at the level of the ileostomy. The ileostomies were shown to be functioning satisfactorily before the pull through, so going more proximal to the ileostomy might have meant that the patient would present short bowel problems. These 3 patients had good bowel function postoperatively. Conclusion In principle, resection of the aganglionic bowel is mandatory to relieve obstruction and subsequent complications. There are situations, however, where the complications of a short bowel begin to outweigh the benefits of resection. In these cases, leaving remaining aganglionic bowel in situ may be an advantageous option for the patients with total aganglionosis extending to the midileum whose ileostomy functions well.

The Problematic Duhamel Pouch in Hirschsprung’s Disease: Manifestations and Treatment

The Duhamel operation is commonly employed to treat Hirschsprung's disease (HD). We have seen a number of patients referred to our center with problems following a Duhamel procedure performed elsewhere, and have analyzed our experience with these children. We reviewed 17 patients with Hirschsprung's disease who underwent a Duhamel procedure elsewhere, in whom we performed a redo pull-through for persistent symptoms of constipation, impaction, and enterocolitis. All patients (n=17) had constipation/impaction or enterocolitis, 9 of whom were soiling due to overflow incontinence. Biopsies of the pulled-through bowel found 6 patients with persistent aganglionic bowel and 2 patients with ganglion cells present but hypertrophic nerves, a finding we interpreted as "transition zone bowel". The remaining 9 patients without a pathological indication for reoperation had a mega Duhamel pouch. All patients underwent a redo operation: 8 via a posterior sagittal approach (7 with a laparotomy, 1 without) and 9 by a transanal, Swenson-type resection with a laparotomy. The posterior sagittal approach was used in cases with severe pelvic fibrosis considered unsuitable for a trans-anal operation. 15 patients were followed up postoperatively for longer than 2 months, 13 of whom now have voluntary bowel movements, including 8 who need a small dose of laxatives. 2 patients are still diverted. Although perhaps successful for many patients around the world, the Duhamel pull-through can leave patients with significant symptoms, including impaction and overflow incontinence. It is unclear why some patients with a Duhamel pouch do not empty well. Clearly, those patients with a mega Duhamel pouch suffer from impaction. These patients need to be detected, because reoperation with resection of the Duhamel pouch can dramatically improve their quality of life.

Total colonic aganglionosis: a surgical challenge. How to avoid complications?

IntroductionTotal colonic aganglionosis represents a significant challenge for pediatric surgeons. Long-term results are suboptimal and complications are very common. We analyzed our experience to formulate recommendations to achieve better results and avoid complications and sequelae.MethodsThe medical records of patients with total colonic aganglionosis that were operated on by us primarily or secondarily were reviewed. We evaluated: number of operations performed, preventable complications, bowel control or presence of stomas, and clinical follow-up. Based on this experience we describe our current approach for this condition. IRB approval was obtained.Results27 patients were identified (19 males, 8 females). 12 patients had the primary pullthrough performed by us and 15 were operated on elsewhere before coming to us for reoperation. The average number of operations per patient was 6.8 (1–40). We identified several preventable complications: ileostomy prolapse or stricture (21), severe diaper rash (10), obstructive symptoms following a pouch or patch-type of pullthrough (9), infection, abscess, and fistula after the pullthrough (5); wrong histologic diagnosis leading to colostomy opening in aganglionic bowel (4) with consequent pullthrough of aganglionic intestine in two of them; anastomotic stricture/acquired atresia (3); and destroyed anal canal and permanent fecal incontinence (2). 15 patients have bowel control; 11 have an ileostomy: temporary (7) and permanent (4); and one is less than 3 years of age. Length of follow-up ranged from 1 to 17 years. Based on this experience, our approach for this condition consists of: colectomy with straight ileoanal anastomosis and ileostomy at presentation, followed by ileostomy closure only when the child is toilet trained for urine and is willing to tolerate rectal irrigations.ConclusionTotal colonic aganglionosis remains a serious surgical challenge. Patients suffering from the condition, have multiple complications, sequelae, and often require reoperations. We found that it is possible to prevent many of these by properly fixing the stoma, avoiding pouch or patch procedures, delaying ileostomy closure, having pathology expertise, and with meticulous surgical technique starting the dissection/anastomosis well above the dentate line.

Enteric neurons synthesize netrins and are essential for the development of the vagal sensory innervation of the fetal gut

During fetal life, vagal sensory fibers establish a reproducible distribution in the gut that includes an association with myenteric ganglia. Previous work has shown that netrin is expressed in the bowel wall and, by acting on its receptor, deleted in colorectal cancer (DCC), mediates the guidance of vagal sensory axons to the developing gut. Because the highest concentration of netrins in fetal bowel is in the endoderm, we tested the hypothesis that the ingrowth of vagal afferents to the gut would be independent of the presence of enteric neurons, although enteric neurons might influence the internal distribution of these fibers. Surprisingly, experiments indicated that the vagal sensory innervation is intrinsic neuron-dependent. To examine the vagal innervation in the absence of enteric ganglia, fetal Ret -/- mice were labeled by applying DiI bilaterally to nodose ganglia. In Ret -/- mice, DiI-labeled vagal sensory axons descended in paraesophageal trunks as far as the proximal stomach, which contains neurons, but did not enter the aganglionic bowel. To determine whether neurons produce netrins, enteric neural-crest-derived cells (ENCDCs) were immunoselected from E15 rat gut. Transcripts encoding netrin-1 and -3 were not detected in the ENCDCs, but appeared after they had given rise to neurons. When these neurons were cocultured with cells expressing c-Myc-tagged netrin-1, the neurons displayed netrin-1, but not c-Myc, immunoreactivity. Enteric neurons thus synthesize netrins. The extent to which neuronal netrin accounts for the dependence of the vagal sensory innervation on intrinsic neurons, remains to be determined.

Sa1600 Colonoscopy As a New Tool in Determining the Transition Zone in Endoanal Pull-Through in Hirschsprung's Disease - Preliminary Results

Colonoscopy As a New Tool in Determining the Transition Zone in Endoanal Pull-Through in Hirschsprung’s Disease Preliminary Results Erika P. Ortolan, Pedro L. Lourencao, Gisele O. Orsi, Bonifacio K. Takegawa Surgery, Botucatu Medical School UNESP Univ Estadual Paulista, Botucatu, Brazil Background: Surgical treatment of Hirschsprung’s disease (HD) has changed in recent decades, with attempts to reduce extensive surgical dissections. Independent of the technique used, the surgery consists in colectomy of the aganglionic region, beyond the transition zone. Since De la Torre & Ortega first described the exclusive transanal endorectal pull-through (TEPT) technique in 1998, it has become the technique of choice, especially in short aganglionic segments. However, recognizing the transition zone during TEPT is very difficult, leading to a procedure involving several frozen section biopsies and that requires an experienced pathologist. Kohno et al (2005) described the use of colonoscopy in determining a landmark for the location of pull-through with relatively accuracy, denominated the “shorebreak” finding, based on the principle that while normal bowel exhibits peristaltic movements, aganglionic bowel does not. He developed a retrospective study comparing the shorebreak finding in colonoscopy marked with India ink with pathological findings in frozen section biopsies. Based on this single report, our group decided to test this new approach, in a prospective study. The purpose of this work was to report our preliminary results concerning the prospective use of colonoscopy as a new tool to determine the transition zone in HD. Patients and Methods: Colonoscopy was prospectively performed on four patients with HD diagnosis, previously confirmed by anorectal manometry, contrast enema and suction biopsies. The exam was performed just prior to TEPT, using the same anesthetic and bowel prep procedure. The area marked with India ink was chosen following observation of colon motility during colonoscopy, in which the absence of motility identified the beginning of the aganglionic area. For endoscopic tattooing, physiological saline was injected locally submucosally to raise the mucosa, followed by the injection of India ink. All the exams were performed by the same pediatric surgery team member (main author), who was unaware of the contrast enema results. Informed consent to perform the exam was obtained. Results: In all patients, the shorebreak finding marked with India ink was equivalent to the aganglionic segment length and transition zone in contrast enemas. The patients were submitted to TEPT beyond this marked area. The excised colon was opened and the transition zone was macroscopically equivalent to the marked area. These results were confirmed by anatomopathological findings using HE, acetylcholinesterase and calretinin stains. Conclusion: Given these preliminary results, it is our conclusion that endoscopic marking of the shorebreak finding provides an important new tool for determining the point of TEPT in the treatment of HD, especially in hospitals that cannot depend on intraoperative frozen sections or experienced pathologists.

Histochemical staining for intestinal dysganglionosis: over 30 years experience with more than 1,500 biopsies

Intestinal dysganglionosis are a group of anomalies of the enteric nervous system that constitute infrequent but severe forms of constipation. Histochemical stainings are the gold standard diagnostic procedure for intestinal dysganglionosis. This study describes our experience with histochemistry in a large series of patients. Between 1977 and 2010, 1,589 biopsies from children with persistent chronic constipation were studied. The specimens were snap frozen, sectioned and stained with acetylcholinesterase (AChE), acetylcholinesterase counterstained with hematoxilin and succinic dehydrogenase (SDH) histochemical stainings. Among the 1,589 biopsies, 946 (59.5%) were rectal biopsies, 242 (15.2%) were internal sphincter biopsies, 346 (21.8%) were intestinal mapping studies and 42 (2.7%) of them were colon specimens from surgical resections. From the rectal biopsy group, 544 (57.5%) patients were reported as normal. Hirschsprung disease was found in 163 (17.2%) patients with a median age at diagnosis of 8 months and a male to female ratio of 3:1. Intestinal neuronal dysplasia was found in 162 (17.2%) patients, hypoganglionosis in 3 (0.3%) of them and ganglioneuromatosis in 1 (0.1%). In 73 (7.7%) patients, the biopsy was not conclusive for different reasons. 34 out of the 42 resected colon specimens were Hirschsprung disease. Intestinal neuronal dysplasia was found in the proximal segment of the aganglionic bowel in 15 out of 34 (44%) patients. All the aganglionic resected colon specimens had a previous aganglionic rectal biopsy. There were no false positive results in this group. Histochemical stainings continue to be the gold standard in the diagnosis of intestinal dysganglionosis. The combination of two histochemical staining techniques provides a high level of accuracy in the diagnosis of intestinal dysganglionosis.