Prospective evaluation of common hepatic duct histopathology at the time of choledochal cyst excision ranging from children to adults.

PURPOSE: To evaluate common hepatic duct just distal to the HE anastomosis (d-CHD) prospectively for mucosal damage, inflammation, fibrosis, dysplasia, carcinoma in situ, malignant transformation, effects of serum amylase, and symptoms at presentation in CC cases ranging from children to adults. METHODS: Cross-sections of d-CHD obtained at cyst excision 2018-2023 from 65 CC patients; 40 children (< 15 years old), 25 adults (≥ 15) were examined with hematoxylin and eosin, Ki-67, S100P, IMP3, p53, and Masson's trichrome to determine an inflammation score (IS), fibrosis score (FS), and damaged mucosa rate (DMR; damaged mucosa expressed as a percentage of the internal circumference). RESULTS: Mean age at cyst excision ("age") was 18.2 years (range: 3 months-74 years). Significant inverse correlations were found for age and DMR (p = 0.002), age and IS (p = 0.011), and age and Ki-67 (p = 0.01). FS did not correlate with age (p = 0.32) despite significantly increased IS in children. Dysplasia was identified in a 4-month-old girl with cystic CC. Serum amylase was elevated in high DMR subjects. CONCLUSIONS: High DMR, high IS, and evidence of dysplasia in pediatric CC suggest children are at risk for serious sequelae best managed by precise histopathology, protocolized follow-up, and awareness that premalignant histopathology can arise in infancy.

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.

The impact of the recipient intestinal site on the differentiation of transplanted enteric neural crest cells.

PURPOSE: It has long been established that the failure of enteric neural crest cells (ENCCs) to colonize the entire gut results in aganglionosis at the distal colon in Hirschsprung disease (HD). However, it is still unclear how the intestinal microenvironment of the distal aganglionic gut differs from that of the proximal ganglionic gut in HD versus normal gut. We have recently succeeded in transplanting ENCC into aganglionic gut in endothelin receptor B (Ednrb) knockout (KO) mice. to advance the development of cell therapy for HD, it is essential to determine if the transplanted ENCCs differentiate normally in aganglionic gut. Therefore, we designed this study to investigate the impact of the environment of the recipient intestinal tract, at various sites of aganglionic gut, on the differentiation of transplanted ENCCs. METHODS: ENCCs were isolated from Sox10 Venus transgenic (Tg) mouse gut on embryonic day 18.5 (E18.5) and neurospheres (NS) were generated. Then, NS were transplanted into aganglionic KO and wildtype (WT) gut that had been transected just distal to the ENCC wavefront (KO-wf: n = 6, WT: n = 7), and into distal KO gut transected at a site equivalent to that of the WT (KO-d: n = 6) on E12.5. ENCC differentiation was evaluated using whole-mount immunohistochemistry with Tuj-1 (neuronal marker) and GFAP (glial marker) antibodies. RESULTS: The transplanted ENCCs migrated to form the myenteric and submucosal plexus in all groups. The ratio of the area of Tuj-1-positive cells/GFAP-positive cells in migrated cells in the recipient gut was found to be significantly lower in KO-d compared to KO-wf and WT, while there was no significant difference between KO-wf and WT groups. This suggests that neuronal/glial differentiation was decreased in KO-d compared to that in KO-wf and WT groups. CONCLUSION: Our study highlights the differences in ENCC differentiation depending on the site of transplantation. To further develop cell therapy for HD, it is important to consider the impact of the recipient intestinal environment on transplanted ENCCs.

Early Clinical Outcomes of the First Commercialized Human Autologous Ex Vivo Cultivated Oral Mucosal Epithelial Cell Transplantation for Limbal Stem Cell Deficiency: Two Case Reports and Literature Review.

The first product in the world for ex vivo cultivated oral mucosal epithelial cell transplantation (COMET) to treat limbal stem cell deficiency (LSCD), named Ocural®, was launched in June 2021 in Japan. COMET was performed on two patients, including the first case in the post-marketing phase of Ocural®. Pathological and immunohistochemical examinations were also carried out using specimens obtained before and after COMET and the spare cell sheet. In case 1, the ocular surface remained free from epithelial defects for approximately six months. In case 2, although defect of the cornea-like epithelia was observed after COMET for one month, it was resolved after the insertion of lacrimal punctal plugs. In case 1, adjuvant treatment was interrupted due to an accident during the second month after COMET, resulting in conjunctival ingrowth and corneal opacity. Eventually, a lamellar keratoplasty was required at six months after COMET. Immunohistochemistry revealed the presence of markers for stem cells (p63, p75), proliferation (Ki-67), and differentiation (Keratin-3, -4, and -13) in both the cornea-like tissue after COMET and a cultivated oral mucosal epithelial cell sheet. In conclusion, Ocural® can be accomplished without major complications, and the stem cells derived from oral mucosa might be successfully engrafted.

Increased enteric neural crest cell differentiation after transplantation into aganglionic mouse gut.

PURPOSE: In recent years, many studies have made considerable progress in the development of stem cell-based therapies for Hirschsprung's disease (HD). However, the question of whether enteric neural crest-derived cells (ENCCs) that are transplanted into the aganglionic gut can migrate, proliferate, and differentiate in a normal manner remains unanswered. Thus, we designed this study to compare the behavior of ENCCs transplanted into the aganglionic gut of endothelin receptor B knockout (Ednrb-KO) mice versus wild-type (WT) mice. METHODS: ENCCs were isolated from the fetal guts of Sox10 transgenic mice, in which ENCCs were labeled with an enhanced green fluorescent protein, Venus, on an embryonic day 18.5 (E18.5). Neurospheres were generated and transplanted into the aganglionic region of either Ednrb-KO mice gut, or WT mice gut that had not yet been colonized, on E12.5. Time-lapse imaging of the transplanted ENCCs was performed after 24, 48, and 72 h of culture. Neuronal differentiation was evaluated using whole-mount immunohistochemistry. RESULTS: Sox10-positive ENCCs were seen to successfully migrate into the myenteric region of the aganglionic gut following transplantation in both the Ednrb-KO and WT mice. The ratio of Tuj1-positive/Sox10-positive cells was significantly increased after 72 h of culture compared to 24 h in the Ednrb-KO mice, which suggests that the transplanted ENCCs differentiated over time. In addition, at the 72 h timepoint, neuronal differentiation of transplanted ENCC in the aganglionic gut of Ednrb-KO mice was significantly increased compared to that of WT mice. CONCLUSIONS: The results of our study demonstrated that transplanted ENCCs migrated into the myenteric region of the aganglionic recipient gut in mice. The increased neuronal differentiation of transplanted ENCC in Endrb-KO mice gut suggests that the microenvironment of this region affects ENCC behavior following transplantation. Further research to explore the characteristics of this microenvironment will improve the potential of developing cell therapy to treat HD patients.

Differentiation of enteric neural crest cells transplanted from SOX10-Venus mouse embryonic stem cells into the gut of the endothelin receptor B null mouse model.

PURPOSE: Failure of enteric neural crest-derived cells (ENCCs) to correctly colonize the embryonic gut results in Hirschsprung's disease (HD). Embryonic stem cells (ESCs) have the potential to differentiate into all tissue-specific cells and lineages, including ENCCs. We investigated the cellular differentiation of ESCs from Sox10-Venus + mice into both control and endothelin receptor-B knockout (Ednrb KO) mouse gut to assess each region. METHODS: We established ESCs from Sox10-Venus + mice. These cells were cultured for 2 days, then selected and co-cultured with either a dissociated control or Sox10-Venus - Ednrb KO mouse gut (both small intestine and colon) on embryonic day (E) 13.5. Four days later, cells were immunolabeled for Tuj1 and visualized using confocal microscopy. RESULTS: Confocal microscopy revealed that transplanted Sox10-Venu + cells from ESCs migrated extensively within the host gut. Moreover, Tuj1-positive neurites were detected in the transplanted ESCs. Tuj1 expression was significantly decreased in aganglionic HD colon compared to controls (p < 0.05) and the HD small intestine (p < 0.05). CONCLUSIONS: This study demonstrated that an appropriate host environment is crucial for normal and complete colonization of the gut. Further investigations are required to confirm whether modifying this environment can improve the results of this model.

High intestinal vascular permeability in a murine model for Hirschsprung's disease: implications for postoperative Hirschsprung-associated enterocolitis.

PURPOSE: Intestinal vascular permeability (VP) in a murine model for Hirschsprung's disease (HD) and postoperative Hirschsprung-associated enterocolitis (HAEC) were investigated. METHODS: Intestinal VP was determined using a Miles assay using 1% Evans blue injected into a superficial temporal vein of newborn endothelin receptor-B KO HD model (KO) and syngeneic wild-type (WT) mice (n = 5, respectively). Extravasated Evans blue in normoganglionic ileum (Ng-I), normoganglionic proximal colon (Ng-PC) and aganglionic distal colon (Ag-DC) was quantified by absorbance at 620 nm. Quantitative polymerase chain reaction (qPCR) for Vascular Endothelial Growth Factor A (VEGF-A), VEGF-B, CDH5, SELE and CD31, and immunofluorescence for CD31 were performed. RESULTS: VP was significantly higher in Ng-I, Ng-PC, and Ag-DC from KO than WT (p < 0.01, p < 0.05, and p < 0.05, respectively). qPCR demonstrated upregulated VEGF-A in Ng-I and Ag-DC, VEGF-B in Ng-I, and SELE in Ng-I and Ng-PC (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.05, respectively), and downregulated CDH5 in Ng-I and Ng-PC from KO (p < 0.05, respectively). Expression of CD31 mRNA in Ng-I and Ag-DC from KO was significantly higher on qPCR (p < 0.05) but differences on immunofluorescence were not significant. CONCLUSIONS: VP may be etiologic for postoperative HAEC throughout the intestinal tract even after excision of aganglionic bowel.

In vitro investigation of the differentiation of enteric neural crest-derived cells following transplantation of aganglionic gut in a mouse model.

PURPOSE: Cell therapy is a promising approach to treat enteric neuropathies such as Hirschsprung disease (HD). Recent studies have reported that enteric neurons derived from stem cells (ENCCs) can be grafted into the HD colon. Thus, we investigated the migration and generation of enteric neurospheres from SOX10-VENUS+ mice after transplantation into control or Ednrb KO mice, which are a model of HD. METHODS: Single-cell suspensions were isolated from the fetal guts of SOX10-VENUS+ mice E13.5 and dissociated. These cells were cultured for 7 days under non-adherent conditions to generate neurospheres, which were co-cultured with dissociated control or SOX10-VENUS- Ednrb KO mouse gut on E13.5. 4 days later, these cells were fixed and the expression of the neuronal marker, Tuj1, was evaluated. RESULTS: Transplanted neurospheres had undergone abundant neuronal migration and differentiation of ENCCs in the control gut compared with the HD gut. The average length and intersections were significantly decreased in HD colon compared with controls (p < 0.05), and a similar pattern was observed in the HD small intestine (p < 0.05). CONCLUSIONS: We demonstrated that transplanted ENCCs did not differentiate properly in HD gut. These results highlight the importance of the neuronal environment in the recipient gut for enteric nervous system development.

Influence of aging on the peripheral nerve repair process using an artificial nerve conduit.

The influence of aging on the induction of nerve regeneration in artificial nerve conduits has yet to be clarified. In the present study, artificial nerve conduit transplantation and histological analysis using the sciatic nerve of young and elderly mice were performed. Using 20 male C57BL/6 mice, an artificial nerve conduit was transplanted to the sciatic nerve at 8 weeks (Young group) or 70 weeks of age (Aged group), and the sciatic nerve was evaluated histologically at 1, 4 and 12 weeks after surgery. Using hematoxylin and eosin staining, the state of induction of nerve regeneration in the artificial nerve conduit was evaluated. Additionally, immunohistochemical staining was used to investigate an angiogenic marker [vascular endothelial growth factor A (VEGFA)], Schwann cell markers [sex determining region Y-box 10 (SOX10) and S100 calcium-binding protein ß (S100ß)] and a nerve damage marker [nerve growth factor (NGF)]. The results revealed that the induction of nerve regeneration was significantly higher in the Young group than in the Aged group. In addition, VEGFA and SOX10 expression at 1 week, SOX10 expression at 4 weeks and SOX10, S100ß and NGF expression at 12 weeks in the proximal stump were significantly higher in the Young group than in the Aged group. At the center of the artificial nerve conduit, S100ß and NGF expression at 4 weeks, and VEGFA, SOX10, S100ß and NGF expression at 12 weeks were significantly higher in the Young group than in the Aged group. In the distal stump, no significant difference was noted in immunostaining at any week between the two groups. The present study suggested that the nerve regeneration-inducing functions decrease due to aging.

Decreased expression of ß1 integrin in enteric neural crest cells of the endothelin receptor B null mouse model.

BACKGROUND: Interactions between enteric neural crest-derived cells (ENCC) and the surrounding intestinal microenvironment, such as the extracellular matrix (ECM), are critical for regulating enteric nervous system (ENS) development. Integrins are the major receptors for ECM molecules, such as laminin, which have been reported to be involved in the pathogenesis of Hirschsprung's disease. In this study, we examined the expression of ß1 integrin in the endothelin receptor B (Ednrb) knock out (KO) mouse gut, which presents with an aganglionic colon. METHODS: A Sox10-Venus-positive Ednrb KO mouse, where ENCC is labeled with fluorescent protein, 'Venus', was created. Sox10-Venus-positive Ednrb wild type (WT) were used as controls. Small intestine, proximal colon and distal colon were dissected on E13.5 and E15.5 and ß1 integrin expression of the gut tissue was examined by immunohistochemistry and real time RT-PCR. The cells of the gut dissected on E11.5 were isolated and cultured for 2 days. Venus-positive ENCC were immunostained with ß1 integrin and Tuj-1, which is a marker for neurons. RESULTS: The expression of ß1 integrin was not significantly different between KO and WT in all parts of the gut examined. However, the ß1 integrin expression in the isolated ENCC was significantly decreased in KO compared to WT. The average threshold area was 42.98 ± 17.47% in KO and 73.53 ± 13.77 in WT (p < 0.001). CONCLUSIONS: We demonstrated that ß1 integrin expression was specifically decreased in ENCC in Ednrb KO mice. Our results suggest that impaired interaction between integrin and its ligands may disturb normal ENS development, resulting in an aganglionic colon.

DPP4 inhibitor reinforces cell junction proteins in mouse model of short bowel syndrome.

PURPOSE: Bacterial overgrowth commonly occurs and favors bacterial translocation in short bowel syndrome (SBS). Glucagon-like peptide-2 (GLP-2) is effective for treating SBS, but is rapidly inactivated by dipeptidyl peptidase IV (DPP4). DPP4 inhibitor (DPP4I) is known to be effective for treating SBS. Here, we investigated cell junction protein function following DPP4I administration in a mouse model of SBS. METHODS: Mice were divided into four groups: naïve (n = 5), naïve + DPP4I (n = 6), control (n = 6), and DPP4I (n = 5). All control and DPP4I mice had 50% of their proximal small bowel resected. DPP4I or normal saline was administered orally twice daily from days 1-7 postoperatively. The functions of cell junction proteins were assessed by RT-PCR and immunohistochemistry. Body weights and blood glucose levels were recorded. RESULTS: E-Cadherin was significantly higher in the DPP4I group than in the control group. E-Cadherin, occludin, and claudin-4 were significantly higher in the naïve group than in the control group. Positive staining for E-cadherin and occludin varied widely between the control and DPP4I groups. CONCLUSION: Up-regulation of E-cadherin and occludin by DPP4I may be correlated with the anti-inflammatory action of DPP4I. Therefore, DPP4I may reduce bacterial translocation in SBS.

Endothelin receptor B affects the perfusion of newborn intestine: possible mechanism of necrotizing enterocolitis development.

BACKGROUND: Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. Hypoxia is known as one of the major risk factors for the development of NEC. Endothelin, known to regulate vasoconstriction, has two receptors (A and B). However, the role of endothelin receptor B (EDNRB) in neonatal intestinal injury remains unclear. We aimed to investigate whether EDNRB is involved in NEC pathophysiology. METHODS: Following ethical approval (#44032), EDNRB hetero knockout mice pups (EDNRB±) and their wild-type (WT) littermates were studied. NEC was induced from postnatal day 5-9 (P5-P9) by hypoxia, gavage feeding of formula and administration of lipopolysaccharide. On P9, the ileum was harvested. RESULTS: NEC induction in WT mice was associated with mucosal injury. However, EDNRB± NEC mice had reduced mucosal injury. Similarly, EDNRB± mice had significantly lower expression of IL-6 mRNA compared to WT NEC mice. Pimonidazole immunostaining was also significantly lower in EDNRB± compared to WT NEC, suggesting reduced tissue hypoxia. CONCLUSIONS: Partial knockout of EDNRB results in reduced NEC severity and reduced tissue hypoxia. Intestinal perfusion and hypoxia are important elements of NEC pathogenesis. These findings are relevant to the understanding of NEC pathophysiology and to the development of novel preventive strategies for NEC.

Role of FBXW7 in the quiescence of gefitinib-resistant lung cancer stem cells in EGFR-mutant non-small cell lung cancer.

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC.

Compromised vitality of spermatozoa after contact with colonic mucosa in mice: implications for fertility in colon vaginoplasty patients.

AIM OF THE STUDY: Colon vaginoplasty (CV) is often performed for cloacal malformation (CM). We used mice to study the vitality of spermatozoa after contact with colonic mucosa as a factor contributing to infertility. METHODS: Spermatozoa isolated from the epididymides of C57BL/6J male mice (n = 23) were syringed directly into the vaginas (Vag-group) or colons (Colo-group) of female mice (n = 45). Vitality was determined by assessing motility using computer-assisted sperm analysis, viability by staining with SYBR-14 and propidium iodide, and fertility by in vitro fertilization, prior to deposition, and at 5, 10, 30, and 60 min after deposition. MAIN RESULTS: Motility was significantly decreased in Colo only at 10 and 60 min. Viability of Colo spermatozoa was significant at all assessment times, except at 10 min. Normal fertilization was observed with all Vag spermatozoa, but with Colo, there was arrest of embryo development with spermatozoa collected at 5 and 10 min, and no fertilization with spermatozoa collected at 30 and 60 min. CONCLUSIONS: The vitality of spermatozoa is compromised by contact with colonic mucosa which could contribute to infertility in CM after CV, because their ovaries and fallopian tubes are considered to be normal.

Innervation of the entire internal anal sphincter in a mouse model of Hirschsprung's disease: a first report.

BACKGROUND: Impaired function of the internal anal sphincter (IAS) may be implicated in postoperative obstructed defecation (POD) that may complicate Hirschsprung's disease (HD) patients. While innervation of part of the IAS in HD has been reported, accurate details based on anatomic landmarks that can explain the clinical morbidity seen in POD are lacking, and there appear to be no studies that specifically document the innervation of the "entire" IAS in HD. We used endothelin receptor-B knockout mice to represent HD (HD-mice) and C57B6 wild mice as controls (C-mice) to investigate the innervation of the entire IAS to assess the pathophysiology of POD experimentally. METHODS: The end-point of the longitudinal muscle layer was used to define the border between the IAS and the circular muscle layer (CML). Specimens of anorectum from HD- and C-mice were immunostained with PGP 9.5 and S100 as general nerve markers, nNOS and VIP as parasympathetic nerve markers, TH as a sympathetic nerve marker, and calretinin as a reliable diagnostic marker for HD. Immunostained cells/fibers were quantified using ImageJ. RESULTS: On fluorescence microscopy, PGP 9.5, nNOS, and calretinin were significantly lower in the IAS of HD-mice than in C-mice (p < 0.05, respectively), while there were no significant differences between HD-mice and C-mice for S100, VIP, or TH. CONCLUSION: We are the first to confirm that the expression of histochemical markers of innervation is abnormal throughout the "entire" IAS in HD-mice. Application of this finding may be beneficial for preventing POD and requires further research.

Altered expression of laminin alpha1 in aganglionic colon of endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Laminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut. METHODS: We harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb -/- mice (n = 8) were compared with Sox10-VENUS+/Ednrb +/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS: The relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5. CONCLUSIONS: Altered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration.

Increased expression of Semaphorin 3A in the endothelin receptor-B null mouse model of Hirschsprung disease.

PURPOSE: Semaphorins are guidance cues for developing neurons, implicated in the determination of the migratory pathway of neural crest-derived neural precursors during enteric nervous system development. Recently, it has been reported that Semaphorin 3A (SEMA3A) expression is up-regulated in the aganglionic colon in Hirschsprung disease (HD) patients, suggesting that increased SEMA3A expression may be a risk factor for HD. Thus, the aim of our study was to determine the expression of SEMA3A using Sox10-Venus mice gut. METHODS: We harvested the gut on postnatal day 2 (P2). SOX10-Venus+/EDNRB-/- mice were compared with SOX10-Venus+/EDNRB+/+ mice as controls. QRT-PCR was performed to determine gene expression of SEMA3A (n=8). Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS: On P2, gene expression levels of SEMA3A were significantly increased in the HD group compared to controls in the proximal and distal colon (p<0.05). Laser scanning microscopy revealed SEMA3A expression was localized within the submucosa and muscle layer of the gut in both HD and controls. In HD, SEMA3A was highly expressed in the proximal and distal colon. CONCLUSIONS: In the present study, we demonstrated that SEMA3A expression is increased in the EDNRB-/- HD model on P2, suggesting that SEMA3A may interfere with ENCC migration, resulting in an absence of enteric neurons.

Sensory innervation of the anal canal and anorectal line in Hirschsprung's disease: histological evidence from mouse models.

AIM: We used non-Hirschsprung's disease (HD) Sox10-Venus Transgenic mice (non-HDSV-mice), an endothelin receptor-B knockout mouse model of HD (HD-mice), and C57B6C3 wild controls (C-mice) to identify the correlation between the anorectal line (ARL) and successful transanal pull-through (TAPT). METHODS: In non-HDSV-mice, intestinal neural crest-derived cells can be visualized with Venus,-a green fluorescent protein-without histochemical staining. We exposed the anal canal in each non-HDSV-mouse and marked the ARL directly with red ink. Specimens of anus and rectum from HD- and C-mice were immunostained with sensory nerve markers substance P and calcitonin gene related peptide (CGRP) and Hematoxylin and Eosin. RESULTS: Stereoscopic microscopy confirmed a squamous-columnar epithelial junction corresponding to the red ink in non-HDSV-mice. Fluorescence microscopy showed intense Venus expression proximal to the ARL and little enteric nerve expression distally. Substance P and CGRP expression were strong in the basal layer of the anal transitional zone (ATZ) in both HD- and C-mice; i.e., distal sensory innervation was normal in HD-mice. CONCLUSIONS: The ARL delineated a distinct demarcation in sensory innervation that is normal even in HD-mice. Thus, the initial incision during TAPT should be based on the ARL because it is readily identifiable and intimately involved with bowel function.

Semaphorin 3A expression following intestinal ischemia/reperfusion injury in Sox10-Venus mice.

PURPOSE: Semaphorin 3A (Sema3A) is a protein secreted during development of the nervous system that plays an important role in neuronal pathophysiology. However, there is no known correlation between Sema3A and intestinal ischemia/reperfusion (I/R) injury. We assessed Sema3A expression and distribution in relation to enteric nervous system (ENS) damage seen after intestinal I/R injury in Sox10-Venus mice. METHODS: Intestinal I/R injury was induced by vascular occlusion for 3 h. Ileal specimens were harvested 0, 3, 12, 24, 48, and 96 h after reperfusion. Stereoscopic microscopy and fluorescence microscopy were used to assess sox10-Venus+ cells and PGP9.5+ cells. RESULTS: By 3 h after reperfusion, Sema3A expression had increased to a maximum and Sox10-Venus+ cells had faded to a minimum in harvested ileal segments. Both differences were statistically significant. By 96 h after reperfusion, both Sema3A and Sox10-Venus+ cell fluorescence had reverted to original levels. Hematoxylin and eosin staining identified histologic damage mimicking Sema3A expression, while PGP9.5+ cell response was minimal. CONCLUSION: We are the first to demonstrate a correlation between Sema3A expression and ENS damage following intestinal I/R in Sox10-Venus mice.

Altered differentiation of enteric neural crest-derived cells from endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HD) is caused by a failure of enteric neural crest-derived cells (ENCC) to colonize the bowel, resulting in an absence of the enteric nervous system (ENS). Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize ENCC with the green fluorescent protein, Venus. The aim of this study was to isolate Sox10-Venus+ cells, which are differentiated neurons and glial cells in the ENS, and analyze these cells using Sox10-Venus mice gut. METHODS: The mid-and hindgut of Sox10-Venus+/Ednrb +/+ and Sox10-Venus+/Ednrb -/- at E13.5 and E15.5 were dissected and cells were dissociated. Sox10-Venus+ cells were then isolated. Expression of PGP9.5 and GFAP were evaluated neurospheres using laser scanning microscopy. RESULTS: 7 days after incubation, Sox10-Venus+ cells colonized the neurosphere. There were no significant differences in PGP9.5 expressions on E13.5 and E15.5. GFAP was significantly increased in HD compared to controls on E15.5 (P < 0.05). CONCLUSIONS: Our results suggest increased glial differentiation causes an imbalance in ENCC lineages, leading to a disruption of normal ENS development in this HD model. Isolation of ENCC provides an opportunity to investigate the ENS with purity and might be a useful tool for modeling cell therapy approaches to HD.