Enough is enough: how many rectal suction biopsies do you need to diagnose Hirschsprung's disease?

PURPOSE: Rectal suction biopsy (RSB) is the gold standard for diagnosing Hirschsprung's disease (HD) in infants. Despite being a common procedure, no standard exists on the number of biopsy specimens and their respective level within the rectum. METHODS: We conducted a retrospective review of epidemiological and pathological data of patients who underwent RSB at our institution between January 2011 and May 2022. During RSB we obtain 4 specimens: at 1 cm, 3 cm and 5 cm above the dentate line, besides one specimen at the dentate line. We used a logistic regression model for statistical analysis and included control variables (e.g. underlying disease, weight at first biopsy, gestational age). RESULTS: A total of 92 patients underwent 115 biopsies, with an average of 3.77 specimens per session. Of the specimens taken at 1 cm above the dentate line 73.9% were conclusive, at 3 cm 75.9% and at 5 cm 79.2%. Specimens taken at the dentate line were squamous or transitional epithelia in 31.5% and therefore of no use for HD diagnostics. The specimen at 3 cm shows the highest discriminative power whether the biopsy session was diagnostic (p-value < 1%). CONCLUSIONS: We propose that a total of three specimens, namely one at 1 cm, one at 3 cm and one at 5 cm above the dentate line, is enough to diagnose or exclude HD.

Identification of signaling pathways that specify a subset of migrating enteric neural crest cells at the wavefront in mouse embryos.

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders.

Novel Alu insertion in the ZEB2 gene causing Mowat-Wilson syndrome.

Alu elements are short, interspersed elements located throughout the genome, playing a role in human diversity, and occasionally causing genetic diseases. Here, we report a novel Alu insertion causing Mowat-Wilson syndrome, a rare neurodevelopmental disorder, in an 8-year-old boy displaying the typical clinical features for Mowat-Wilson syndrome. The variant was not initially detected in genome sequencing data, but through deep phenotyping, which pointed to only one plausible candidate gene, manual inspection of genome sequencing alignment data enabled us to identify a de novo heterozygous Alu insertion in exon 8 of the ZEB2 gene. Nanopore long-read sequencing confirmed the Alu insertion, leading to the formation of a premature stop codon and likely haploinsufficiency of ZEB2. This underscores the importance of deep phenotyping and mobile element insertion analysis in uncovering genetic causes of monogenic disorders as these elements might be overlooked in standard next-generation sequencing protocols.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Enteric Nervous System Striped Patterning and Disease: Unexplored Pathophysiology.

The enteric nervous system (ENS) controls gastrointestinal (GI) motility, and defects in ENS development underlie pediatric GI motility disorders. In disorders such as Hirschsprung's disease (HSCR), pediatric intestinal pseudo-obstruction (PIPO), and intestinal neuronal dysplasia type B (INDB), ENS structure is altered with noted decreased neuronal density in HSCR and reports of increased neuronal density in PIPO and INDB. The developmental origin of these structural deficits is not fully understood. Here, we review the current understanding of ENS development and pediatric GI motility disorders incorporating new data on ENS structure. In particular, emerging evidence demonstrates that enteric neurons are patterned into circumferential stripes along the longitudinal axis of the intestine during mouse and human development. This novel understanding of ENS structure proposes new questions about the pathophysiology of pediatric GI motility disorders. If the ENS is organized into stripes, could the observed changes in enteric neuron density in HSCR, PIPO, and INDB represent differences in the distribution of enteric neuronal stripes? We review mechanisms of striped patterning from other biological systems and propose how defects in striped ENS patterning could explain structural deficits observed in pediatric GI motility disorders.

Mesenchymal Stem Cells Attenuates Hirschsprung diseases - Associated Enterocolitis by Reducing M1 Macrophages Infiltration via COX-2 Dependent Mechanism.

OBJECTIVE AND DESIGN: Hirschsprung disease-associated enterocolitis (HAEC) is a common life-threatening complication of Hirschsprung disease (HSCR). We aimed to investigate the effectiveness, long-term safety and the underlying mechanisms of Mesenchymal stem cells (MSCs) based therapy for HAEC. MATERIAL OR SUBJECTS: Specimens from HSCR and HAEC patients were used to assess the inflammatory condition. Ednrb knock-out mice was used as HAEC model. MSCs was intraperitoneally transplanted into HAEC mice. The therapy effects, long-term outcome, safety and toxicity and the mechanism of MSCs on the treatment of HAEC were explored in vivo and in vitro. RESULTS: Intestinal M1 macrophages infiltration and severe inflammation condition were observed in HAEC. After the injection of MSCs, HAEC mice showed significant amelioration of the inflammatory injury and inhibition of M1 macrophages infiltration. The expression levels of pro-inflammatory cytokines (TNF-α and IFN-γ) were decreased and anti-inflammatory cytokines (IL-10 and TGF-ß) were increased. In addition, we found that effective MSCs homing to the inflamed colon tissue occurred without long-term toxicity response. However, COX-2 inhibitor could diminish the therapeutic effects of MSCs. Using MSCs and macrophages co-culture system, we identified that MSCs could alleviate HAEC by inhibiting M1 macrophages activation through COX-2-dependent MAPK/ERK signaling pathway. CONCLUSIONS: MSCs ameliorate HAEC by reducing M1 macrophages polarization via COX-2 mediated MAPK/ERK signaling pathway, thus providing novel insights and potentially promising strategy for the treatment or prevention of HAEC.

PHOX2B: a diagnostic cornerstone in neurocristopathies and neuroblastomas.

Paired-like homeobox 2B (PHOX2B) is a gene essential in the development of the autonomic nervous system. PHOX2B mutations are associated with neurocristopathies-Hirschsprung disease (HSCR) and congenital central hypoventilation syndrome (CCHS)-and peripheral neuroblastic tumours. PHOXB2 plays an important role in the diagnostics of these conditions.Genotyping of a PHOX2B pathogenic variant is required to establish a diagnosis of CCHS. In HSCR patients, PHOX2B immunohistochemical staining has proven to be a valuable tool in identifying this disease. Furthermore, PHOXB2 is a predisposition gene for neuroblastoma, in which PHOX2B immunohistochemical staining can be used as a highly sensitive and specific diagnostic marker. The utility of PHOX2B immunohistochemistry in pheochromocytoma and paraganglioma has also been studied but yields conflicting results.In this review, an overview is given of PHOX2B, its associated diseases and the usefulness of PHOX2B immunohistochemistry as a diagnostic tool.

Identification of the DNA methylation signature of Mowat-Wilson syndrome.

Mowat-Wilson syndrome (MOWS) is a rare congenital disease caused by haploinsufficiency of ZEB2, encoding a transcription factor required for neurodevelopment. MOWS is characterized by intellectual disability, epilepsy, typical facial phenotype and other anomalies, such as short stature, Hirschsprung disease, brain and heart defects. Despite some recognizable features, MOWS rarity and phenotypic variability may complicate its diagnosis, particularly in the neonatal period. In order to define a novel diagnostic biomarker for MOWS, we determined the genome-wide DNA methylation profile of DNA samples from 29 individuals with confirmed clinical and molecular diagnosis. Through multidimensional scaling and hierarchical clustering analysis, we identified and validated a DNA methylation signature involving 296 differentially methylated probes as part of the broader MOWS DNA methylation profile. The prevalence of hypomethylated CpG sites agrees with the main role of ZEB2 as a transcriptional repressor, while differential methylation within the ZEB2 locus supports the previously proposed autoregulation ability. Correlation studies compared the MOWS cohort with 56 previously described DNA methylation profiles of other neurodevelopmental disorders, further validating the specificity of this biomarker. In conclusion, MOWS DNA methylation signature is highly sensitive and reproducible, providing a useful tool to facilitate diagnosis.

RMRP-related short stature: A report of six additional Japanese individuals with cartilage hair hypoplasia and literature review.

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal-onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < -2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed "RMRP-related short stature".

The Effect of the Ganglionic Segment Inflammatory Response to Postoperative Enterocolitis in Hirschsprung Disease.

INTRODUCTION: We examined the relationship between proinflammatory cytokines that occur in the inflammatory reaction in the intestine in Hirschsprung disease (HD) and Hirschsprung-associated enterocolitis (HAEC). METHODS: Thirty cases (M:27, F:3) operated on due to HD. The cases were divided into three groups: group 1 with pre and post operative EC, group 2 with post-operative, and group 3 with pre-operative EC. The intestinal segments were evaluated by immunohistochemistry for interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). RESULTS: IL-1ß staining was significantly higher in the ganglionic zone of groups with enterocolitis compared to the control group (p = 0.012). TNF-α staining in the transitional zone of Group 3 and IL-1ß staining in the ganglionic zone of Group 1 was significantly higher than the control group (p = 0.030, p = 0.020). CONCLUSION: In our study, older age at diagnosis and more than 20% IL-1ß staining in the ganglionic segment were found to be risk factors for HAEC. It is noteworthy that the increase in IL-1ß can be associated with HAEC.

Histological transitional zone pull-through in Hirschsprung disease. Postoperative functional results and current recommendations.

BACKGROUND: Surgeons create a neorectum to repair patients with Hirschsprung's disease (HD), which should be formed from a normoganglionic bowel. However, the neorectum is occasionally created with a transition zone (TZ) bowel. A neorectum created with a TZ has been postulated as a cause of postoperative enterocolitis or constipation. This study compares the incidence of enterocolitis and constipation in patients with TZ neorectum and normoganglionic bowel. METHODS: We conducted a retrospective review of patients with rectosigmoid HD who underwent primary pull-through. Patients were divided into normoganglionic neorectum (NNR) and TZ neorectum. The diagnosis was based on the final histopathologic report of the proximal margin. The incidence of enterocolitis and constipation was compared between these two groups. RESULTS: A total of 98 HD patients were analyzed. Seventy-one patients fulfilled the inclusion criteria. 65 (92%) had a NNR, and six patients (8%) had a TZ neorectum. From these patients, 42 (59%) presented with enterocolitis or constipation. However, there was no significant difference between both groups. CONCLUSION: The present study showed no difference in the incidence of enterocolitis or postoperative constipation in HD patients with normoganglionic or TZ neorectum. These results suggest that TZ neorectum does not cause postoperative obstructive symptoms.

INTRODUCCIÓN: Los cirujanos crean un neo-recto para tratar a los pacientes con enfermedad de Hirschsprung (EH), que debe formarse con intestino normogangliónico; sin embargo, en ocasiones el neo-recto se forma con intestino de la zona de transición. Se ha postulado que un neo-recto en zona de transición causa enterocolitis o estreñimiento postoperatorio. El objetivo de este estudio fue comparar la frecuencia de enterocolitis y estreñimiento en pacientes con neo-recto en zona de transición y con neo-recto normogangliónico. MÉTODOS: Se llevó a cabo una revisión retrospectiva de pacientes con EH recto sigmoideo que se sometieron a descenso primario. Los pacientes se dividieron en el grupo neo-recto normogangliónico y el grupo con neo-recto en zona de transición. El diagnóstico del neo-recto se estableció con el informe histopatológico definitivo del margen proximal. Se comparó la frecuencia de enterocolitis y estreñimiento entre estos dos grupos. RESULTADOS: Se analizó un total de 98 pacientes con EH, de los cuales 71 pacientes cumplieron los criterios de inclusión; 65 (92%) con neo-recto normogangliónico y seis (8%) con neo-recto en zona de transición. Posteriormente, 42 (59%) pacientes presentaron enterocolitis asociada a Hirschsprung (HAEC) o estreñimiento; sin embargo, no hubo diferencia significativa entre ambos grupos. CONCLUSIONES: El presente estudio no demostró una diferencia en la frecuencia de HAEC o estreñimiento postoperatorio en pacientes con EH con neo-recto normogangliónico o en zona de transición. Estos resultados sugieren que un neo-recto en zona de transición no causa síntomas obstructivos postoperatorios.

Suction rectal biopsy (SRB) in Hirschsprung's Disease: Is a single macroscopically adequate sample sufficient?

Hirschsprung's Disease (HD) is a congenital disorder causing severe constipation in infants and children. Suction rectal biopsy (SRB) is the preferred technique for obtaining tissue samples for histopathological evaluation. In low-resource settings like Malaysia, cost-effective diagnostic approaches are necessary, making single sample SRB valuable. This study evaluates the diagnostic accuracy and sufficiency of a single macroscopically adequate sample in suction rectal biopsies for the histopathological confirmation of HD. We conducted a retrospective study of children who underwent suction rectal biopsies for the diagnosis of HD at Hospital Raja Perempuan Zainab II (HRPZII), Kota Bharu, Kelantan. A total of 68 patients were included in the study. The inadequacy rate for bedside SRB was 14%, comparable to current literature. Our study found no statistically significant association between sample inadequacy and gestational age, gender, birth weight, or weight at biopsy. Complication rates were 0%, consistent with literature reports. Calretinin staining, an additional technique, was performed in 23 biopsy episodes, with a 4.3% inadequacy rate, compared to 20% in specimens not subjected to calretinin staining. The cost of SRB almost doubled with each additional sample taken, significant in low-resource environments. In conclusion, single sample SRBs can be adequately diagnostic and cost-effective in low-resource settings, providing valuable insights for healthcare facilities in Malaysia and other developing countries. The use of adjunctive techniques such as calretinin staining may improve diagnostic accuracy while maintaining cost-effectiveness. Further prospective studies with larger sample sizes are needed to validate these findings.

A novel mouse model of intestinal neuronal dysplasia: visualization of the enteric nervous system.

PURPOSE: Intestinal neuronal dysplasia (IND) is a congenital anomaly affecting gastrointestinal neural innervation, but the pathogenesis remains unclear. The homozygous Ncx/Hox11L.1 knockout (Ncx-/-) mice exhibit megacolon and enteric ganglia anomalies, resembling IND phenotypes. Sox10-Venus transgenic mouse were used to visualize enteric neural crest cells in real time. This study aims to establish a novel mouse model of Sox10-Venus+/Ncx-/- mouse to study the pathogenesis of IND. METHODS: Sox10-Venus+/Ncx-/- (Ncx-/-) (n = 8) mice and Sox10-Venus+/Ncx+/+ controls (control) (n = 8) were euthanized at 4-5 weeks old, and excised intestines were examined with fluorescence microscopy. Immunohistochemistry was performed on tissue sections with neural marker Tuj1. RESULTS: Ncx-/- mice exhibited dilated cecum and small intestine. Body weight of Ncx-/- mice was lower with higher ratio of small intestine length relative to body weight. The neural network (Sox10-Venus) was observed along the intestine wall in Ncx-/- and control mice without staining. Ectopic and increased expression of Tuj1 was observed in both small intestine and proximal colon of Ncx-/- mice. CONCLUSION: This study has established a reliable animal model that exhibits characteristics similar to patients with IND. This novel mouse model can allow the easy visualization of ENS in a time- and cost-effective way to study the pathogenesis of IND.

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.

Hirschsprung disease.

Hirschsprung disease (HSCR) is a rare congenital intestinal disease that occurs in 1 in 5,000 live births. HSCR is characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the intestine. Most patients present during the neonatal period with the first meconium passage delayed beyond 24 h, abdominal distension and vomiting. Syndromes associated with HSCR include trisomy 21, Mowat-Wilson syndrome, congenital central hypoventilation syndrome, Shah-Waardenburg syndrome and cartilage-hair hypoplasia. Multiple putative genes are involved in familial and isolated HSCR, of which the most common are the RET proto-oncogene and EDNRB. Diagnosis consists of visualization of a transition zone on contrast enema and confirmation via rectal biopsy. HSCR is typically managed by surgical removal of the aganglionic bowel and reconstruction of the intestinal tract by connecting the normally innervated bowel down to the anus while preserving normal sphincter function. Several procedures, namely Swenson, Soave and Duhamel procedures, can be undertaken and may include a laparoscopically assisted approach. Short-term and long-term comorbidities include persistent obstructive symptoms, enterocolitis and soiling. Continued research and innovation to better understand disease mechanisms holds promise for developing novel techniques for diagnosis and therapy, and improving outcomes in patients.

Suppression of PGE2/EP2 signaling alleviates Hirschsprung disease by upregulating p38 mitogen-activated protein kinase activity.

Hirschsprung disease (HSCR) is a congenital disorder caused by the failure of enteric neural crest cells (ENCCs) to colonize the distal bowel, resulting in absence of enteric nervous system. While a range of molecules and signaling pathways have been found to contribute to HSCR development, the risk factors and pathogenesis of this disease in many patients remain unknown. We previously demonstrated that increased activity of the prostaglandin E2 (PGE2)/PGE2 receptor subtype EP2 pathway can be a risk factor for HSCR. In this study, an Ednrb-deficient mouse model of HSCR was generated and used to investigate if PGE2/EP2 pathway could be a potential therapeutic target for HSCR. We found that downregulation of PGE2/EP2 signaling by siRNA-mediated ablation of a PGE2 synthase or pharmacologic blockage of EP2 enhanced ENCC colonization in the distal bowel of Ednrb-/- mice and alleviated their HSCR-like symptoms. Furthermore, blockage of EP2 was shown to promote ENCC migration through upregulating p38 mitogen-activated protein kinase activity, which was downregulated in the colon of Ednrb-/- mice and in the distal aganglionic bowel of HSCR patients. These data provide evidence that maternal exposure during embryonic development to an environment with dysregulated activation of the PGE2/EP2 pathway may predispose genetically susceptible offspring to HSCR, and avoidance or early disruption of maternal events (e.g. inflammation) that possibly enhance PGE2/EP2 signaling during pregnancy would reduce the occurrence and severity of this disease. KEY MESSAGES : Knockdown of PTGES alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling promotes ENCC migration via enhancing p38 activity.

Systematic orientation of fresh rectal suction biopsies improves histopathological diagnostics in hirschsprung's disease - a method description and preliminary report.

BACKGROUND: Optimizing rectal suction biopsy (RSB) diagnostics in Hirschsprung's disease (HD) may shorten diagnostic time and prevent need for repeated biopsies. AIM: To explore whether systematic orientation of fresh RSB specimens increased biopsy quality, diagnostic times, diagnostic efficacy, and histopathologic workload, and to explore these outcome measures for aganglionic specimens. MATERIALS/METHODS: This was an observational case-control study conducted at a national referral center for HD on data collected from the local HD-diagnostic register. From 2019 each fresh RSB was oriented by the collector in a notch in a foam cushion, placed in a separate cassette, and sent in formalin for pathological analysis. Outcome measures of oriented RSB samples collected 2019-2021 were compared to those of non-oriented RSB samples collected 2015-2018. Staining/immunohistochemistry consisted of hematoxylin eosin, S-100 and calretinin. RESULTS: 78 children with 81 RSBs and 242 biopsy analyzes were included. The frequency of high-quality RSB specimens was higher in oriented: 40% (42/106) versus non-oriented 25% (34/136) (p = 0.018), the diagnostic turnaround time was shorter: 2 days (1-5) versus 3 days (2-8) (p = 0.015), and the number of additional sectioning/leveling/re-orientation per biopsy was lower: 7 (3-26) versus 16 (7-72) (p = 0.011). Specifically for aganglionic specimens, the frequency of high-quality biopsies was generally higher in oriented than in non-oriented RSB specimens: 47% (28/59) versus 14% (7/50) (p < 0.001); the diagnostic efficacy was higher 95% (19/20) versus 60% (9/15) (p = 0.027) and the diagnostic turnaround time shorter: 2 days (2-3) versus 3 days (2-8) (p = 0.036). CONCLUSIONS: Systematic orientation of fresh RSB specimens improves HD diagnostics. Improvement was consistent in aganglionic specimens.

Development of the aganglionic colon following surgical rescue in a cell therapy model of Hirschsprung disease in rat.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

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

BACKGROUND: Hirschsprung'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. OBJECTIVES: To develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus. METHODS: Using 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. RESULTS: The 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. CONCLUSION: The 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.

Tissue Shrinkage of Resected Specimens in Hirschsprung's Disease: Why Pediatric Surgeons Think the Bowel Specimen was Longer Than Indicated in the Pathology Report.

BACKGROUND: Hirschsprung disease (HD) is an aganglionosis of variable length starting at the rectosigmoid colon with surgery as sole therapeutic option. The length of the resected bowel segment is a crucial information for the treating surgeons and influences the prognosis of the patient. It is often artificially altered due to post operative tissue shrinkage. The objective of this study is to quantify the extent tissue shrinkage of HD specimens. MATERIAL AND METHODS: Colorectal HD specimens were measured at the time of surgery and at the time of cut-up, either fresh or after formalin fixation and statistically analyzed. RESULTS: Sixteen colorectal specimens were included. Following formalin fixation the specimen length decreased by 22.7% (P < .001). Without formalin fixation the specimens shrank by an average of 24.9% (P = .05). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (P = .76). CONCLUSION: This study showed that there is significant tissue shrinkage in HD specimens. The 2 different cohorts revealed that tissue shrinkage is mostly caused by tissue retraction/alteration after organ removal but also to a lesser extent by fixation with formalin. Surgeons and (neuro-)pathologists should be aware of the sizeable shrinking artifact to avoid unnecessary confusion.