Absence Of Enteric Ganglion Cells Research Articles

Ernica Clinical Consensus Statements on Total Colonic and Intestinal Aganglionosis

BackgroundHirschsprung 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. MethodsA 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. ResultsThe 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. ConclusionsTotal 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 StudyClinical consensus statement. Level of Evidence3a.

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.

A new systematization of histological analysis for the diagnosis of Hirschsprung's disease

BackgroundHirschsprung's Disease (HD) is characterized by intestinal sub-occlusion and the absence of enteric ganglion cells. A rectal biopsy examination is performed to confirm the diagnosis. In a recent study, we demonstrated that the analysis of 60 sections of rectal mucosa and submucosa stained by H&E may ensure a 90% diagnostic accuracy. Although the need to analyze so many sections makes the process of reading the slides more time-consuming, this encouraged us to study their distribution in the healthy rectal submucosa, to simplify the diagnosis. ObjectivesTo develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus. MethodsUsing the calretinin technique, we studied the distribution of plexuses in 60 fragments of rectal submucosa from 19 cadavers. After the study, the reading method created was used for diagnosis in 47 cases of suspected HD, using H&E staining. The accuracy was verified by comparing the results obtained with H&E to those obtained with the acetylcholinesterase technique, the golden standard in our laboratory. ResultsThe study of submucosal plexus distribution showed that just by examining the submucosal region every 20 µm, approximately, it is possible to locate a ganglionic plexus, and we have already been able to diagnose HD with 93% accuracy. ConclusionThe study of ganglion cell distribution enabled the creation of a simplified method for reading the slides. The method applied achieved good accuracy and it can be used as an alternative method in HD diagnosis.

Hubungan Status Gizi Pasien Penyakit Hirschsprung dengan Infeksi Luka Operasi di RSPAL dr. Ramelan Surabaya Periode Waktu 2016-2020

Background: Hirschsprung's disease is a congenital intestinal motility disorder that causes functional intestinal obstruction due to the absence of enteric ganglion cells in the distal intestine. The management of each case of Hirschsprung's disease is surgery, where surgery on the digestive tract increases the occurrence of surgical site infections. Disturbances in the gut cause decreased resistance to infection, delayed cognitive development, and problems with nutritional status.
 Methods: This study was a descriptive and analytical observational study, where the primary data were taken from the registration data of Hirschprung's disease patients at RSPAL dr. Ramelan Surabaya in 2016-2020. The nutritional status of the patient was determined by plotting the weight per age curve according to WHO for children aged 0-5 years.
 Results: Observations of 38 patients who met the study requirements, obtained 4 patients who had poor nutritional status and 1 patient who had excess nutritional status, none of which experienced complications in the form of surgical wound infections. In 10 patients with poor nutritional status, 2 experienced complications in the form of surgical wound infection and 23 patients with good nutritional status, 1 experienced complications in the form of surgical wound infection. The results of the contingency coefficient correlation test showed P-value = 0.408 (p>α).
 Conclusion: There was no relationship between the nutritional status of Hirschprung's disease patients with surgical wound infections at RSPAL dr. Ramelan Surabaya
 Keywords: Hirschsprung's disease, nutritional status of children, surgical site infection.

Associations of Mucosal Nerve Fiber Innervation Density with Hirschsprung-Associated Enterocolitis: A Retrospective Three-Center Cohort Study.

Hirschsprung's disease (HSCR) is a congenital intestinal neurodevelopmental disorder characterized by the absence of enteric ganglion cells in the distal colon. Although Hirschsprung-associated enterocolitis (HAEC) is the most frequent life-threatening complication in HSCR, to date reliable biomarkers predicting the likelihood of HAEC are yet to be established. We established a three-center retrospective study including 104 HSCR patients surgically treated between 1998 and 2019. Patient-derived cryopreserved or paraffin-preserved colonic tissue at surgery was analyzed via βIII-tubulin immunohistochemistry. We subsequently determined extrinsic mucosal nerve fiber density in resected rectosigmoid specimens and classified HSCR patients accordingly into nerve fiber-high or fiber-low groups. We compared the distribution of clinical parameters obtained from medical records between the fiber-high (n = 36) and fiber-low (n = 68) patient groups. We assessed the association between fiber phenotype and enterocolitis using univariate and multivariate logistic regression adjusted for age at operation. Enterocolitis was more prevalent in patients with sparse mucosal nerve fiber innervation (fiber-low phenotype, 87%) compared with the fiber-high phenotype (13%; p = 0.002). In addition, patients developing enterocolitis had a younger age at surgery (3 vs. 7 months; p = 0.016). In the univariate analysis, the odds for enterocolitis development in the fiber-low phenotype was 5.26 (95% confidence interval [CI], 1.67-16.59; p = 0.005) and 4.01 (95% CI, 1.22-13.17; p = 0.022) when adjusted for age. Here, we showed that HSCR patients with a low mucosal nerve fiber innervation grade in the distal aganglionic colon have a higher risk of developing HAEC. Consequently, histopathologic analysis of the nerve fiber innervation grade could serve as a novel sensitive prognostic marker associated with the development of enterocolitis in HSCR patients.

Acetylcholinesterase staining for the pathological diagnosis of Hirschsprung's disease.

Hirschsprung's disease (HD) is a congenital disease manifesting various degrees of functional bowel obstruction caused by the absence of enteric ganglion cells, which are usually absent in the colonic segment of the HD patient. Because the aganglionic segment of HD always includes the rectum, pathological diagnosis can be made using a rectal sample. HD should be diagnosed as early as possible because serious complications, such as acute enterocolitis or toxic megacolon, can develop without a definitive diagnosis and appropriate treatment. In the mid-1900s, HD was diagnosed by HE staining of specimens obtained by full-thickness biopsy. Since then, the combination of rectal mucosal biopsy and rubeanic acid-amplificated AChE staining has been brought about by the following milestones: the discovery that the submucosal plexus and the intermuscular plexus had the same level of nerve migration; the findings of research on acetylcholine (ACh) and acetylcholinesterase (AChE) in the intestinal tract; and the establishment of a rubeanic acid amplification method. Consequently, the diagnostic rate of HD improved dramatically in the 1980s. This review outlines the history of diagnostic methods for HD, the roles of ACh and AChE in the intestine, and the method of AChE staining.

Altered expression of AKT1 and P38A in the colons of patients with Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a functional obstruction of the gastrointestinal tract due to the congenital absence of enteric ganglion cells. The proto-oncogene RET is one of the primary genes implicated in the aetiology of HSCR. We designed this study to investigate the expression of 10 RET regulatory network genes in the colons of patients with HSCR. HSCR tissue specimens (n = 28) were collected at the time of pull-through surgery. qPCR analysis was applied to compare the expression levels of 10 genes in the RET regulatory network. Western blot analysis was performed to quantify the protein expression. Immunohistochemistry was performed to determine the localization of AKT1 and P38A in HSCR colon tissue. AKT1 (p = 0.015) and P38A (p = 0.039) were both significantly downregulated in the aganglionic segment compared to those in the ganglionic segment in HSCR patients (n = 28). Western blot analysis revealed the decreasing protein expression of AKT1 and P38A in the aganglionic segment compared to ganglionic segment and control colon tissues (p < 0.05). Immunohistochemistry staining revealed that both AKT1 and P38A were localized in the colonic mucosa and were significantly decreased in the aganglionic segment. To our knowledge, we report for the first time the expression of RET regulatory network genes in the colons of patients with HSCR. The markedly decreased expression of AKT1 and P38A suggested a possible role in HSCR pathogenesis.

Current practice patterns of rectal suction biopsy in the diagnostic work-up of Hirschsprung's disease: results from an international survey.

The diagnosis of Hirschsprung's disease (HD) was revolutionized by the introduction of rectal suction biopsy (RSB), allowing specimens to be taken without general anesthesia on the ward or as an out-patient procedure. However, insufficient tissue samples are not uncommon, and subsequently histopathologists often remain reluctant to confirm the presence or absence of enteric ganglion cells merely on the basis of submucosal RSBs. The aim of this study was to evaluate the current usage of RSB in the diagnostic work-up of HD based on an international survey. A 15-item questionnaire was distributed among participants and faculty members at the 21st International Meeting of the Pediatric Colorectal Society. Eighty-seven pediatric surgeons from 30 countries completed the anonymous survey (response rate 70.2%), grouped into 68 (78.2%) staff surgeons and 19 (21.8%) trainees, with a median work experience of 18years (range 2-45years). Of these, 74 (85.1%) use RSB in the diagnostic work-up of patients with suspected HD, whereas 13 (14.9%) prefer open full-thickness biopsy under general anesthesia. In total, 47 (63.5%) respondents perform ≥20 RSBs (range 3-100 RSBs) per year. Five different RSB instruments were reported, the most common ones being rbi2 (65.0%), Solo-RBT (15.0%) and multipurpose suction biopsy kit (8.3%). Only 22 (29.7%) of the respondents use a defined negative suction pressure, with a median of 10mL air (range 6-25mL air). The most proximal reported biopsy site was located at a median of 2cm (range 1-15cm) above the pectinate line and a median of 2 (range 1-5) specimens are routinely taken, mainly from the posterior rectal wall. Insufficient tissue samples with need for repeat RSB were encountered in a median of 10% (range 0-40%). Most frequently used staining methods for rectal biopsies are hematoxylin/eosin (75.9%), acetylcholinesterase (73.6%), and calretinin (33.3%). Overall, 36 (48.6%) respondents had experienced RSB-related complications, including self-limiting rectal blood loss (n=28), persistent rectal bleeding requiring blood transfusion (n=9) and rectal perforation requiring surgical intervention (n=7). Although RSB is considered to be today's gold standard for the diagnosis of HD, many aspects of its current usage are lacking consensus. Therefore, a prospective multi-center study or larger global audit appears warranted to identify if the present survey reflects common surgical practice and to establish universal standards for RSB.

Fluorescence Visualization of the Enteric Nervous Network in a Chemically Induced Aganglionosis Model

Gastrointestinal motility disorders, severe variants in particular, remain a therapeutic challenge in pediatric surgery. Absence of enteric ganglion cells that originate from neural crest cells is a major cause of dysmotility. However, the limitations of currently available animal models of dysmotility continue to impede the development of new therapeutics. Indeed, the short lifespan and/or poor penetrance of existing genetic models of dysmotility prohibit the functional evaluation of promising approaches, such as stem cell replacement strategy. Here, we induced an aganglionosis model using topical benzalkonium chloride in a P0-Cre/GFP transgenic mouse in which the neural crest lineage is labeled by green fluorescence. Pathological abnormalities and functional changes in the gastrointestinal tract were evaluated 2–8 weeks after chemical injury. Laparotomy combined with fluorescence microscopy allowed direct visualization of the enteric neural network in vivo. Immunohistochemical evaluation further confirmed the irreversible disappearance of ganglion cells, glial cells, and interstitial cell of Cajal. Remaining stool weight and bead expulsion time in particular supported the pathophysiological relevance of this chemically-induced model of aganglionosis. Interestingly, we show that chemical ablation of enteric ganglion cells is associated with a long lifespan. By combining genetic labeling of neural crest derivatives and chemical ablation of enteric ganglion cells, we developed a newly customized model of aganglionosis. Our results indicate that this aganglionosis model exhibits decreased gastrointestinal motility and shows sufficient survival for functional evaluation. This model may prove useful for the development of future therapies against motility disorders.

Expression patterns of dishevelled-2 in different colon tissue segments in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a congenital disorder characterized by an absence of enteric ganglion cells in the terminal regions of the gut during development. To date, the cause of HSCR remains unclear, although the pathogenesis of this complex disease is hypothesized to be influenced by numerous genetic and environmental factors. Dishevelled‑2 (DVL‑2) is a subtype of the dishevelled protein, which is known to be involved in embryonic development. In the present study, the pathogenesis of HSCR was investigated by measuring the expression of the DVL‑2 gene and protein using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), western blotting and immunohistochemistry staining in the aganglionic and ganglionic segments of colonic tissues in patients with HSCR. The results showed that the level of DVL‑2 mRNA in the aganglionic segments was 0.28 fold that of the ganglionic segments. Similarly, the protein expression of DVL‑2 was lower (11.31±2.23) in the aganglionic segments than that of the ganglionic segments (35.21±2.66), as assessed by western blot analysis. Furthermore, immunohistochemical staining demonstrated that DVL‑2 expression was significantly higher in the mucosal and submucosal layers from ganglionic colon segments compared with that from the aganglionic segments. The data suggest that the expression of DVL‑2 in colon tissue segments may be important in the pathogenesis of HSCR.

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.

Electrical activity of the intestine of mice with hereditary megacolon and absence of enteric ganglion cells.

Mice with a recessive gene which reduces the number of ganglion cells of the large intestine and produces megacolon similar to Hirschsprung's disease were studied. Electrical activity of the small bowel consisted of electrical slow waves and action potentials and showed no difference between the mice with megacolon and their normal siblings. Electrical slow waves and action potentials occurred in the large intestine of both normal and abnormal mice. The principal difference between normal mice and their abnormal siblings was increased incidence of discharge of action potentials associated with uncoordinated phasic contractions superimposed upon tonic contracture of the circular muscle layer of the distal aganglionic segment in the abnormal mouse. The distended colon of the abnormal mouse and the entire length of the normal bowel showed bursts of action potentials which accompanied peristaltic waves of circular muscle contraction. During propulsive motility in the rectum, activation of the circular muscle was preceded by coordinated contraction of the longitudinal muscle only in the normal bowel. Symptoms of megacolon can be accounted for by the absence of spontaneously active inhibitory neurons from the enteric plexuses of the distal segment of the large bowel.