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Purpose: The enteric nervous system (ENS), comprising neurons and glial cells, organized as interconnected ganglia within the gut wall, controls peristalsis and the production of secretions. The RET receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development. Humans with mutations in the RET locus have Hirschsprung's disease (HSCR), and mice lacking RET exhibit total intestinal aganglionosis. Although a number of mutations with the potential for causing HSCR have been reported, their precise correlation with phenotype and symptom severity in HSCR is not clearly understood. Our study investigates the correlation between mutations in the RET locus and symptom severity in HSCR.Methods: We performed a comprehensive nucleotide analysis of the RET coding region in 18 HSCR patients and 87 controls, performed cellular biological analysis by Western blotting using the expression vector, and analyzed cell proliferation with anti-Ki67 antibody under immunofluorescence confocal microscopy (ICM).Results: We identified three novel mutations, D489N, L769L, and V778D in the RET coding region in our HSCR patients. In the allelic distribution of D489N and L769L, the difference between HSCR patients and controls reached statistical significance (p = 0.0373 and p = 0.0004, respectively), whereas no statistical difference was observed in the allelic distribution of V778D (p = 0.1073). One HSCR patient who died from total colonic aganglionosis had a combination of homozygous mutation of D489N, L769L, and heterozygous mutation of V778D. Western blotting of full mutant RET from this patient showed significantly increased 150kD-band, which corresponds to the immature form compared with wild-type and single mutant RET. ICM showed that overexpression of full mutant RET significantly reduced cellular proliferation in comparison with wild-type and single mutant RET.Conclusion: A combination of mutations in the RET locus may correlate with symptom severity in HSCR as a consequence of reduced cellular proliferation secondary to altered maturation of RET. [ABSTRACT FROM AUTHOR]

Purpose: Postoperative anastomotic stricture (PAS) is a well-known complication after correcting choledochal cyst (CC). Although the exact cause of PAS is unknown, various risk factors, such as Todani classification type IV-A, hepaticoduodenostomy, and narrow anastomosis have been reported to be associated with PAS. As far as we know, there is no report with a cumulative analysis of such risk factors of PAS. This systematic review and meta-analysis aimed to investigate the risk factors of PAS following surgical correction of CC in children., Methods: A systematic literature search for relevant articles was performed in four databases using the combinations of the following terms "Congenital biliary dilatation", "Congenital choledochal cyst", "Choledochal cyst", "Stenosis", "Stricture", and "Complication" for studies published between 1973 and 2022. The relevant cohorts of PAS were systematically searched for clinical presentation and outcomes., Results: The search strategy identified 795 reports. Seventy studies met the defined inclusion criteria, reporting a total of 206 patients with PAS. There is no prospective study in this search. The incidence of PAS was 2.1%. The proportion of Todani classification of the patient with PAS was higher in type IV-A with significant difference (2.0% in type I and 10.1% in type IV-A (p = 0.001)). Fourteen studies reported a comparison between hepaticojejunostomy and hepaticoduodenostomy. There was no significant difference between the two groups (p = 0.36). Four studies reported the diameter of the anastomosis at the primary surgery. The mean diameter was 12.5 mm. Nine studies reported a comparison between laparoscopic surgery and open surgery. Pooled odds ratio of PAS did not show a statistical difference (p = 0.29)., Conclusions: This study suggests that close careful follow-up is important in the patients with type IV-A of CC who underwent excision surgery, considering the possibility of PAS., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Laparoscopic repairs for children with inguinal hernia have been established with various technical modifications. Laparoscopic percutaneous extraperitoneal closure (LPEC) is one of the most recognized techniques. Although the rate of complications of LPEC is similar to conventional repairs, the major cause of recurrence is still incompletely understood. The purpose of this study is to evaluate detail profile of the cause of recurrence in children treated with LPEC., Methods: A systematic literature search was performed using the combinations of the following terms "pediatric inguinal hernia", "LPEC", "complication", and "recurrence" for studies published between 2002 and 2020. The relevant cohorts of recurrence of LPEC in children were systematically searched for clinical outcomes., Results: 35 studies met defined inclusion criteria, reporting a total of 121 patients who had recurrence after LPEC. The mean age at primary operations was 46.7 ± 52.0 months. The gender proportions were 63.9% (male) and 36.1% (female). The rate of treatment side was 44.5% (right), 37.0% (left) and 18.5% (bilateral). The hernia sac was closed with absorbable suture materials (5.3%) and non-absorbable (94.7%). The recurrence occurred in 6.9 ± 8.5 months postoperatively. Operative findings of reoperation were loosening of the knot of internal rings (61.1%), low ligation of the inguinal canal (16.7%), and skipping (22.2%)., Conclusions: This study suggests that male infants have a higher risk of recurrence after LPEC, and the majority of recurrences can occur in a year postoperatively. It is important to confirm carefully that the suture is tight and high without skipping., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by incomplete formation of the diaphragm. Because CDH herniation occurs at the same time as preacinar airway branching, normal lung development becomes severely disrupted, resulting almost invariably in pulmonary hypoplasia. Despite various research efforts over the past decades, the pathogenesis of CDH and associated lung hypoplasia remains poorly understood. With the advent of molecular techniques, transgenic animal models of CDH have generated a large number of candidate genes, thus providing a novel basis for future research and treatment. This review article offers a comprehensive overview of genes and signaling pathways implicated in CDH etiology, whilst also discussing strengths and limitations of transgenic animal models in relation to the human condition.

Anorectal malformations (ARM) represent a broad spectrum of patients with different level of the rectum and type of a fistula. Standardized approaches are usually successful, but patients occasionally present with an unusual course of fistula which requires a modified surgical strategy. We present here three male ARM patients with an atypical fistula which did not have connection with the urinary tract, but ran near the fistula. Case 1 has a low-type ARM with a rectoscrotal fistula running deep and partly involved in the corpus spongiosum. Anorectoplasty was performed through an anterior sagittal incision and the anterior wall of the fistula was laid open leaving the posterior wall undetached. Case 2 was diagnosed with an intermediate-type ARM with a long rectoscrotal fistula running near and parallel the urethra. Posterior sagittal anorectoplasty (PSARP) was performed leaving the fistula untouched. Case 3 presented with an intermediate-type ARM with a rectoperineal fistula adherent to the urethra. The patient was treated by PSARP leaving the fistula and part of the muscle coat of the rectum in situ. All the cases were smoothly discharged and no urological complication nor problem associated with the residual fistula was observed at the latest follow-up (17 months-2 years). Preoperative distal colostography with the aid of diverting colostomy was importantly useful for deciding surgical procedure.

Purpose: The cadmium (Cd) chick model has been described as a reliable model of omphalocele. Skeletal anomalies, including lumber lordosis, can be seen in the Cd chick model, as well as in the human omphalocele. Bone deformations, such as lordosis, are associated with high bone mineral density (BMD). Recently, three-dimensional microcomputed tomography (3DMCT) has been used to investigate skeletal development in small animal embryos. We used 3DMCT to test the hypothesis that the BMD is increased in the Cd-induced omphalocele chick model., Methods: After a 60-h incubation, chicks were exposed to either chick saline or Cd in ovo. Chick embryos were harvested at embryonic day 16.5 (E16.5) and were divided into control (n = 8) and Cd (n = 9). Chicks were then scanned by 3DMCT. The body volume, bone volume, bone/body volume ratio, bone mineral quantity and BMD were analysed statistically (significance was accepted at p < 0.05)., Results: Bone mineral density (mg/cm 3 ) was significantly increased in the Cd group compared to control group (235.3 ± 11.7 vs 223.4 ± 4.6, p < 0.05), whereas there was no significant difference in the bone/body volume ratio between the Cd group and the control group (0.7 ± 0.1 vs 0.6 ± 0.0). The body volume (cm 3 ) (0.3 ± 0.2 vs 0.3 ± 0.1), bone volume (cm 3 ) (0.2 ± 0.2 vs 0.2 ± 0.1), and bone mineral quantity (mg) (51.3 ± 41.6 vs 41.5 ± 16.5) were not significantly different between the two groups., Conclusions: Increased BMD may be associated with lordosis of the vertebral column in the Cd-induced omphalocele chick model, stimulating osteogenesis by activating the canonical Wnt signalling pathway.

Aim: Detailed implications of age at laparoscopic transanal pull-through (LTAPT) on postoperative bowel function (POBF) in Hirschsprung's disease (HD) are somewhat obscure because of a spectrum of factors., Methods: Age at surgery was used to categorize 106 consecutive postoperative HD cases treated by our modified LTAPT (JLTPAT) between 1997 and 2015; group A: < 3 months old (n = 31); group B: 3-11 months old (n = 44); group C: 1-3 years old (n = 19); and group D: ≥ 4 years old (n = 12). POBF was assessed by reviewing outpatient records 1, 3, 5, 7, and 10 years after JLTAPT prospectively and scoring each of 5 criteria on a scale of 0-2; best score = 10., Results: Only operative time was statistically longer in group D versus groups A, B, and C. Differences in gender ratios, blood loss, duration of follow-up, and POBF scores were not statistically significant. Mean POBF scores over time were: group A: 6.8, 7.6, 8.4, 8.6, and 8.4; group B: 7.1, 7.8, 8.3, 8.5, and 9.0; group C: 6.9, 7.9, 8.1, 8.3, and 8.6; group D: 7.0, 7.4, 8.2, 8.1, and 8.5, respectively., Conclusion: Age at JLTAPT was not correlated with POBF in HD.

Background: We assessed the quality of life (QOL) of postoperative esophageal atresia (EA) with tracheoesophageal fistula (TEF) cases, comparing open with thoracoscopic repair., Methods: A retrospective review of consecutive EA/TEF repairs (2001-2014) was performed, excluding cases with birth weight less than 2000 g and severe cardiac/chromosomal anomalies. Of 37 cases, 13 had thoracoscopic repair (TR) and 24 had open repair (OR) according to the operating surgeon's preference. QOL was determined regularly by scoring responses to a standard questionnaire about oral intake, vomiting, bougienage, coughing, growth retardation, learning ability, and thoracic deformity. Lower scores reflected poorer outcome. QOL after TR and OR was compared 1 year postoperatively (POQ) and after starting school (ScQ)., Results: Subject demographics were similar. Apart from two anastomotic leaks that resolved spontaneously after TR, there were no intraoperative complications or recurrence of TEF. Laparoscopic fundoplication was required for gastroesophageal reflux in four cases (OR 1; TR 3) (p = ns). QOL scores went from 6.5 → 11.5 in OR and 4.6 → 11.3 in TR, respectively. Final ScQ scores were similar, but POQ was significantly higher after OR (p < 0.05)., Conclusion: Initial QOL scores were significantly lower after TR, but by school age QOL scores were similar.

Purpose: To evaluate the optimal ventilation mode during thoracoscopic repair (TR) of neonatal congenital diaphragmatic hernia (CDH), we compared high-frequency oscillatory ventilation (HFOV) with conventional mechanical ventilation (CMV)., Methods: Twenty-three neonatal CDH cases who underwent TR without intraoperative inhalation of nitric oxide at our institution between 2007 and 2016 were reviewed. Patients were initially ventilated with HFOV, which was converted to CMV if the HFOV settings were decreased to FiO 2 <0.4, stroke volume <4 mL/kg and mean airway pressure <12 cmH 2 O. Arterial blood gases in the perioperative period were compared between HFOV and CMV., Results: Seventeen patients were ventilated with HFOV (group I), and six patients were ventilated with CMV (group II). Preoperative PaCO 2 was significantly higher and pH was significantly lower in group I compared with group II. In both groups I and II, intraoperative PaCO 2 increased significantly and pH decreased significantly compared with preoperation. Although intraoperative PaCO 2 and pH were not different between the groups, group II showed greater worsening of intraoperative PaCO 2 and pH as compared to their respective preoperative values., Conclusions: HFOV seems to prevent deterioration of hypercapnia and acidosis to a greater extent than CMV during TR in neonatal cases of CDH, although patients can also be ventilated with CMV.

Purpose: We reviewed all rectal mucosal/submucosal biopsies (RMSBx) performed between 1986 and 2015 focusing on specimen quality, incidence of complications during and after biopsy, and parental satisfaction., Methods: From 1986 to 2004, RMSBx were performed without general anesthesia (GA) (N-GA; n = 98) and from 1995 to 2015 were performed under GA (GA; n = 525). In GA cases, some sections were stained intraoperatively and examined by a pathologist and pediatric surgeon immediately to identify ganglion cells., Results: Mean ages at RMSBx were similar (2.7 vs. 2.5 years; p = NS). There were significantly more inadequate specimens in N-GA [18/98 (18.4 %) vs. 0/525 (0 %); p < 0.0001]. Incidence of rectal bleeding requiring transfusion was significantly lower in GA [0/525 (0 %) versus 2/98 (2.0 %); p = 0.024]. Parents of GA subjects willingly consented to RMSBx when told GA would facilitate diagnosis. Incidentally, RMSBx was more expensive in GA (US$1320 versus US$294; using ¥120 = US$1)., Conclusion: RMSBx performed under GA are safe and all specimens obtained included submucosa appropriate for optimum diagnosis in all the cases.

Purpose: To evaluate factors related to conversion to open repair (OR) during thoracoscopic repair (TR) in congenital diaphragmatic hernia (CDH)., Methods: CDH subjects who were diagnosed prenatally or within 6 h of birth and underwent TR at our institution from 2007 to 2015 were reviewed. Two groups were defined: Group A, subjects who tolerated TR; and Group B, subjects who required conversion to OR., Results: Twenty-nine subjects (Group A, 20; Group B, 9) underwent TR. Patients' demographics were not significantly different between the two groups. In Group A, pre-operative arterial blood gas analysis showed mean PaCO2 37.4 mmHg, mean pH 7.45, and mean PaO2 201.6 mmHg, which were not significantly different from those of Group B (PaCO2 40.6 mmHg, pH 7.43, and PaO2 251.1 mmHg). Two Group B cases required conversion due to cardiopulmonary instability. The remaining seven cases were converted for technical reasons (large defect in two, anterior location of the defect in two, bleeding from short gastric vessels or spleen in two, and insufficient operative view in one)., Conclusions: TR can be performed safely in selected cases of CDH. CDH with a large defect and/or an unusual location of the defect remains a challenge for pediatric surgeons.

Purpose: To report our experience of correcting penile ventral curvature associated with minor or no hypospadias., Methods: We reviewed 43 penile ventral curvature patients treated by a single surgeon from 1997 to 2015. Of these, 23 had minor hypospadias. Curvature was corrected using degloving, chordectomy, dorsal plication (DP), tunica albuginea incision (TAI), or a combination of these. Outcome was confirmed by induced artificial erection and post-operative appearance., Results: Mean age at curvature correction was 3.2 ± 2.6 years. 17/43 had degloving and chordectomy (DC), 16/43 had DP after DC, and 10/43 had TAI after DC, because of ventral shortening and severe curvature caused by a short hypoplastic urethra. Other procedures required were primary meatoplasty (n = 4) and urethroplasty (UP; n = 1) at the time of curvature correction, and UP after correction of curvature (n = 11). Complications included recurrence of curvature after DP (n = 3/16; 18.8 %) and urethral stenosis after UP with tubed peritoneum (n = 1/10; 10 %). There were no recurrences of curvature in TAI cases. Parents reported penile cosmesis as good (n = 38; 88.4 %), acceptable (n = 4; 9.3 %), or poor (n = 1; 2.3 %)., Conclusion: We recommend TAI followed by UP for correcting penile ventral curvature with short hypoplastic urethra. Tubed peritoneum is not recommended for UP.

Purpose: Laparoscopically assisted anorectoplasty (LAARP) was compared to posterior sagittal anorectoplasty (PSARP) in the treatment of male imperforate anus associated with either recto-prostatic fistula (RPF) or recto-bulbar fistula (RBF)., Method: 19 RPF patients (12 treated by LAARP and 7 by PSARP) and 26 RBF patients (14 treated by LAARP and 12 by PSARP) between 1995 and 2014 were retrospectively assessed using a fecal continence evaluation questionnaire (FCE) (with a maximum score of 10), an FCE score coefficient variation, as well patients' MRI scores, anorectal angle values (AA), and incidence of postoperative complications. Statistical significance was determined at p < 0.05., Results: Both groups were similar in mean age and mean weight at repair, as well as sacral status. Postoperatively, mean MRI scores, mean AA, and biochemistry were also similar (p = NS). All cases treated with LAARP showed consistently higher and less variable FCES values, fewer wound infection incidence, but greater rectal mucosal prolapse unrelated with sacrum status. Significantly lower doses of postoperative analgesia were needed in all LAARP cases (p < 0.05)., Conclusion: Technical outcomes appear to be similar based on imaging studies, but FCES-assessed functional outcomes appear to favor LAARP for treatment of both RPF and RBF.

Purpose: We report the efficacy of staged segmental urethroplasty (SSUP) versus non-staged urethroplasty (NSUP) for treating scrotal/perineal hypospadias (SPH)., Methods: Between 1997 and 2015, 29 SPH patients underwent UP (SSUP: n = 15; NSUP: n = 14). Incidences of urethrocutaneous fistula (UF), stenosis of the neourethra (SNU), diverticula formation, and residual chordee (RC) were compared. Differences were statistically significant if p < 0.05., Results: The difference in mean age at NSUP (3.2 ± 1.3 years) and at the final stage of SSUP (5.5 ± 2.4 years) was significant (p < 0.05). Mean operative times for NSUP and SSUP (total for all stages) were not significantly different (231.5 ± 117.5 versus 272.5 ± 99.4 min); however, the incidence of postoperative complications was significantly less in SSUP (n = 1; UF) compared with NSUP (n = 6; 2 cases of UF, 3 cases of SNU, and 1 case of RC; (p < 0.05). Mean follow-up was significantly shorter in SSUP; 1.4 ± 1.2 years versus 7.0 ± 4.5 years in NSUP (p < 0.05)., Conclusion: SSUP would appear to be effective for treating SPH because of a significantly lower incidence of UF, SNU and RC during the first postoperative year, the period when complications have been reported to arise most frequently.

Aim: Thoracoscopic lobectomy (TL) and open lobectomy (OL) were compared for treating congenital pulmonary airway malformation (CPAM) with preoperative complications, specifically pneumonia/abscess formation (PA)., Methods: The medical records of 46 CPAM patients treated by lobectomy at our institution from 1990 to 2014 were reviewed retrospectively. Four groups, TL for patients without PA (n = 17; TL-), TL for patients with PA (n = 8; TL+), OL for patients without PA (n = 16; OL-), and OL for patients with PA (n = 5; OL+) were compared for operative time, intra/postoperative complications, blood loss, duration of chest tube insertion, postoperative analgesia, pre: postoperative white blood cell (WBC) ratio, and duration of hospitalization., Results: Operative time for TL+ was longest, but not statistically significant. Incidences of intra/postoperative complications were similar in all groups. Blood loss was significantly less for TL+ versus OL+ (p < .05). WBC ratio was significantly lower in TL+ versus OL+ (p < .05), similar for TL+ and TL-, and significantly higher in OL+ versus OL- (p < .01). Chest tube insertion was significantly longer in OL- versus TL- (p < .01)., Conclusion: PA would not appear to be a contraindication to perform TL in CPAM. TL is associated with less surgical stress than OL despite longer operative time.

Purpose: Total bilirubin (T-bil) is used universally for monitoring post-portoenterostomy (PE) biliary atresia (BA) patients although other biochemical markers [BM; AST/ALT and platelet count (PC)] are also prognostic. We compared open PE (OPE) with laparoscopic PE (LPE) using T-bil, AST/ALT, and PC (3BM) as more comprehensive indicators of postoperative clinical status., Methods: Subjects were 31 PE cases (LPE: n = 17; OPE: n = 14). BA classification was type III (n = 16), type II (n = 1) in LPE and type III (n = 12), type I (n = 1), type II (n = 1) in OPE., Results: Mean ages and weights at PE were similar: 65.5 days, 4.4 kg (LPE) versus 69.3 days, 4.1 kg (OPE); and mean follow-up was 2.5 years for both LPE and OPE. Jaundice clearance (T-bil ≤1.2 mg/dL) was achieved in 16/17 (94.1 %) after LPE versus 10/14 (71.4 %) after OPE (p = NS), but 3BM were closer to normal after OPE. At the time of review, 13/17 LPE cases (76.5 %) were alive with native livers and 4/17 had received LTx (23.5 %) and 10/14 OPE cases (71.4 %) were alive with native livers and 4/14 had received LTx (28.6 %)., Conclusions: Although JC was better after LPE, 3BM were better after OPE. Further follow-up will prove the comprehensive prognostic value of 3BM.

Purpose: We monitored arterial blood gases during thoracoscopic (TR) and open repair (OR) of congenital diaphragmatic hernia (CDH) to assess the safety of intraoperative hypercapnia and acidosis., Methods: We reviewed 30 neonatal CDH cases (OR = 10, TR = 20) diagnosed prenatally or within 6 h of birth at out institution from 2002 to 2014 not requiring inhaled nitric oxide (NO) intraoperatively. OR, routine until 2006 was replaced by TR in 2007. All subjects were managed identically., Results: Five TR cases requiring conversion to OR were excluded. Prenatal diagnosis, gestational age at birth, gender, birth weight, and side of CDH were similar. Preoperative PaCO2 and pH were not significantly different. However, while intraoperative increase in mean PaCO2 (38.8-62.8 mmHg; p < 0.01) and decrease in mean pH (7.44-7.25; p < 0.01) were significant in TR, intraoperative PaCO2 was <70 mmHg in 12/15 cases and intraoperative pH was >7.20 in 11/15 cases. Both PaCO2 and pH reverted to normal on completion of surgery; pre- and postoperative results were comparable. There were no postoperative complications., Conclusions: It would appear that neonatal cases of CDH not requiring NO can tolerate TR, despite transient reversible deterioration in acid/base balance, indicating that TR is safe for the treatment of selected cases of CDH.

During surgery for choledochal cyst (CC), any intrapancreatic CC (IPCC) must also be excised to prevent postoperative pancreatitis and stone formation. We report our technique for laparoscopic total IPCC excision (n = 16; mean age 6.0 years). We insert a fine ureteroscope with a light source into the opened CC through an extra 3.9-mm trocar placed in the epigastrium through a minute incision to identify the pancreatic duct orifice. By pulling the end of the ureteroscope emerging from the trocar gently to withdraw the tip from the pancreatic duct to where distal dissection was ceased under laparoscopic view, the IPCC can be measured. If longer than 5 mm, the distal CC is dissected further caudally until it is less than 5 mm. For accuracy, the distal CC is elevated with a suture that is exteriorized and clamped to provide constant traction. The IPCC was able to be measured in 11/16 (68 %). Initial lengths measured were 3-10 mm (5.2 ± 2.7 mm). Final IPCC were all 5 mm or less. Surgery was uncomplicated without any pancreatic duct injury and postoperative recovery was unremarkable. Follow-up MRI at 32 months showed no IPCC in any case. Measuring the IPCC enables total CC excision, thus reducing the potential for postoperative complications.

Purpose: We present EDCAT (epidural catheter-assisted Deflux(®) treatment) for treating vesicoureteral reflux (VUR) and confirming ureteric patency after Deflux(®) treatment., Methods: We treated 147 ureters in 101 children (M:F 62:39; VUR ≤ grade III: n = 72; VUR ≥ grade IV: n = 75) using EDCAT between 2011 and 2014. EDCAT involves injecting 1-3 mL of diluted indigo carmine solution through an epidural anesthesia catheter inserted into the Deflux(®)-treated ureter and observing for up to 15 min to confirm patency., Results: For EDCAT, mean age was 4.9 years and mean operative time was 30.1 ± 12.1 min; overall cure of VUR after initial treatment was 87.7 % for VUR grades I-V, 88.9 % for VUR ≤ grade III, and 86.6 % for VUR ≥ grade IV; VUR was cured in 129/147 after 1 treatment, 7/18 after 2 treatments, 7/11 after 3 treatments, and persistent in 4/4. We experienced 1 case of obstruction after an EDCAT catheter was removed before confirming patency. EDCAT catheters were left overnight in 2 ureters in 2 cases when patency could not be confirmed after 15 min to prevent obstruction. Both did not develop obstruction. Four ureters with recurrence of VUR (all grade ≤ II) are being observed., Conclusion: EDCAT allows ureteric patency to be confirmed immediately and prevents obstruction.

Bronchogenic cysts are congenital cystic lesions of foregut origin, usually intrapulmonary or mediastinal, while esophageal cysts generally originate within the esophagus. To the best of our knowledge, this is the first report of a case of a completely intramural bronchogenic cyst of the cervical esophagus in a neonate.

Purpose: Soft tissue interposition (STI) during hypospadias repair (HR) purportedly prevents postoperative urethrocutaneous fistula (PUF) by supporting the neourethra. We report our experience., Methods: Data from 243 hypospadias patients treated by a single surgeon from 1997 to 2014 by urethroplasty (UP) with STI (n = 229; UP + STI) and UP without STI (n = 14; UP-STI) were collated prospectively and compared for incidence of PUF. Re-operative UP were excluded., Results: Hypospadias was distal (n = 55), mid-shaft (n = 59), proximal/penoscrotal (n = 109), scrotal (n = 15), and perineal (n = 5). UP was single-staged in 86, multi-staged in 157; mean age at UP was 3.1 ± 2.4 years. Soft tissue used for STI was prepucial inner dartos fascia (inner dartos: n = 88), ventral dartos fascia (ventral dartos: n = 15), pedicled external spermatic fascia (ESF: n = 84), adipose tissue surrounding the spermatic cord (pericordal: n = 9), scrotal adipose tissue (n = 8), or a combination of tissues (combined: n = 25). Mean follow-up was 6.4 ± 4.6 (range 0.6-16.8) years. Overall incidence of PUF was 10/243 (4.1 %); 7/229 (3.1 %) for UP + STI and 3/14 (21.4 %) in UP-STI (p < 0.05); incidence versus type of hypospadias was 1/55 for distal (1.8 %), 3/59 for mid-shaft (5.1 %), 5/109 for proximal/penoscrotal (4.6 %), 0/15 for scrotal (0 %), and 1/5 for perineal (20 %); incidence versus type of STI was 7/88 for inner dartos, 0/15 for ventral dartos, 0/84 for ESF, 0/9 for pericordal adipose tissue, 0/8 for scrotal adipose tissue, and 0/25 for combined. All PUF were repaired successfully. Satisfaction with penile cosmesis was acceptable (10.3 %) or good (89.7 %) without any testicular complications or scrotal deformity., Conclusion: STI, especially ESF, would appear to effectively prevent PUF in HR.

Introduction: Urethrocutaneous fistula (UCF) complicating hypospadias surgery is associated with compromised tissue and perfusion at the UCF site, especially if recurrent. We report our technique for UCF repair., Methods: Between 1997 and 2014, we treated 35 UCF in 26 postoperative hypospadias patients; 12 UCF were recurrent (mean 2.5; range 1-5). Mean age at UCF repair was 9.3 years (range 2-22). Our repair involves making a superficial incision 3-5 mm around the fistula orifice, placing multiple stay sutures in the outer edge of this incision and dissecting only the epidermis under traction for 7-10 mm. This technique does not compromise underlying connective tissue or tissue perfusion. The skin layer of the inner edge of the circumferential incision is trimmed completely and the fistula closed using 7/0 absorbable interrupted sutures. A pedicled external spermatic fascia, or tunica vaginalis flap is then mobilized to cover the repair site through a subcutaneous tunnel and the skin closed. A urethral catheter is placed and removed the next day. Duration of follow-up was calculated as the period from discharge home until the last outpatient clinic attendance., Results: Repair was successful in all cases. Penile cosmesis was acceptable to good without any testicular complications or scrotal deformity. At mean follow-up of 7.4 years (range 0.4-17.3) there have been no recurrences., Conclusions: Our technique allows UCF to be repaired effectively and is also indicated for recurrences.

Purpose: There are few reports about postoperative outcome of penoplasty (PP). We present the results of mid- to long-term follow-up of PP performed for congenital megaprepuce (CMP)., Methods: Data from 65 CMP cases treated by PP performed by a single surgeon from 2000 to 2014 were collected prospectively. All cases were treated using the technique reported by Cuckow (Pediatric surgery. Springer, Berlin, pp 543-554, 2006)., Results: Mean age at PP was 5.9 years (range 0.4-13.9). All cases presented as infants and some 12 cases (18.5 %) had PP when 10 or more years old. There were no intra- and postoperative complications. Mean duration of follow-up was 3.6 years (range 0.1-17.5). Duration of follow-up was 4 years or less in 48 (73.8 %), 5-9 years in 13 (20.0 %), and 10 or more years in 4 (6.2 %). While postoperative penile cosmesis was good in 63/65 (96.9 %) cases without scrotal deformity, 2/65 (3.1 %) had redundant penile skin excised upon the recommendation of the treating surgeon even though the patients and parents were unconcerned., Conclusion: Mid- to long-term follow-up of our PP cases shows that outcome is cosmetically acceptable and stable.

Purpose: We assessed the effect of high partial pressure of arterial carbon dioxide (PaCO2) due to pneumoperitoneum (PP) on growth (height/weight) and development (gross/fine motor function, receptive/expressive communication, and social interaction), by comparing outcome after portoenterostomy (PE) for biliary atresia (BA) using laparoscopic PE (LPE: n = 13) and open PE (OPE: n = 13) cases performed between 2005 and 2014., Methods: Our PE is based on Kasai's original PE. All data were collated prospectively., Results: Differences in duration of follow-up (LPE: 38.8 months; OPE: 38.1 months), jaundice clearance (LPE: 12/13 = 92.3 %; OPE: 9/13 = 69.2 %), survival with the native liver (LPE: 10/13 = 76.9 %; OPE: 9/13 = 69.2 %), incidence of cholangitis, hypersplenism, and incidence of esophageal varices were not significant. Mean intraoperative PaCO2 was significantly higher in LPE (LPE: 50.1 mmHg; OPE: 40.7 mmHg, p < 0.05). Liver function impairment was not statistically different, although LPE results were slightly worse. There was no overall delay in growth observed, although height/weight gain was more consistent in LPE. The pattern of developmental delay observed was similar for LPE and OPE suggesting that developmental delay is not PE-related; in other words, PP is not implicated in developmental delay., Conclusions: PP during LPE would appear to have no adverse effects on overall growth/development and liver function in BA patients.

Aim: We assessed the outcome of 72 cases of type-C esophageal atresia (EAc) treated by open (OR) or thoracoscopic (TR) repair from 2000 to 2013. During OR, direct lung retraction (DLR) is required but during TR, CO2 insufflation causes lung collapse so DLR is unnecessary., Methods: Patient demographics, operative time, respiratory tract impact (RTI: incidence of atelectasis, timing of extubation, need for reintubation, and duration of chest tube insertion), narcotic usage, commencement of oral feeding, and length of hospitalization (LOH) were compared., Results: Seven long-gap cases were excluded, leaving 65 EAc subjects (TR = 25, OR = 40). Patient demographics, operative time, and duration of chest tube insertion were similar. No TR case required DLR. Significant differences were found in TR for narcotic usage (1.6 vs. 3.1 days), commencement of feeding (7.8 vs. 10.5 days), incidence of atelectasis (8 vs. 30 %), initial extubation (2.8 vs. 5.6 days), and LOH (33 vs. 46 days), (all p < 0.05, respectively). Postoperative tracheal tube blockage caused by excessive secretions (4 vs. 10 %) and reintubation (4 vs. 17.5 %) were less in TR but not significant. There were three anastomotic leakages in TR, one in OR., Conclusion: TR is less traumatic than OR because DLR is unnecessary, resulting in lower RTI and smooth recovery.

Aim: During retroperitoneoscopic renal surgery (RPRS), the open technique (OT) for trocar insertion is always associated with CO2 leakage around the first trocar, while the closed technique (CT) involves advancing an optical trocar into the retroperitoneal space (RS) under direct vision. We are the first to compare OT with CT., Methods: Forty-one cases of RPRS performed for pyeloplasty (31), hemi-nephrectomy (4), nephrectomy (4), stone removal (1), and cyst excision (1) were studied. The initial 5 mm trocar was inserted using OT (n = 11) or CT (n = 30). The tip of a telescope was used for blunt dissection of the retroperitoneum to create the RS., Results: Ages and weights at RPRS were similar (OT versus CT: 7.3 versus 7.1 years; 25.4 versus 25.0 kg; respectively). In CT, RS access was significantly faster (38.2 versus 5.6 min), RS was created significantly quicker (77.7 versus 31.9 min), and narcotic usage was significantly less (4.0 versus 1.5 days). In OT, CO2 leaked in 11/11 and the retroperitoneum was torn in 3/11. In CT there was no leakage and no tearing., Conclusions: CT is quicker and safer than OT during RPRS.

Accurate division and sealing of lung parenchyma particularly in cases of total or near total incomplete fissure are crucial for preventing air leakage following thoracoscopic pulmonary lobectomy (TPL). However, conventional endoscopic stapling devices cannot be used during TPL in small children because of limited space. Consequently, Ligasure (LS) and Enseal (ES) devices are being used instead. We are the first to compare LS and ES for efficacy and efficiency during TPL. Of 26 TPL (6 upper, 3 middle, and 17 lower) performed for congenital adenomatoid malformation (n = 16) and sequestration (n = 10), incomplete fissure was found in 14. TPL (LS = 11; ES = 15) was performed conventionally in the lateral decubitus position with single lung ventilation using four 5 mm trocars. All cases had a chest tube inserted intraoperatively that was left in situ. Patient demographics, location of pathology, incidence of incomplete fissure, mean age/weight at TPL, mean blood loss, and mean operative time were all similar. However, duration of chest tube insertion was significantly shorter in ES because there was less postoperative air leakage (1.3 vs. 3.9 days; p < 0.05). ES would appear to seal lung parenchyma more effectively during TPL based on the shorter duration of chest tube insertion.

Background/purpose: Despite technical advances in the surgical/medical care of anorectal malformation (ARM), persistent unsatisfactory postoperative bowel habit has been attributed to histopathologic abnormalities of the distal rectum/pouch (DRP) and hypoplasia of anal sphincter muscles (ASM). We used Sox10-Venus mice with ARM induced by all-trans retinoic acid (ATRA) to investigate neural crest cell (NCC) innervation in the DRP and ASM., Method: Pregnant Sox10-Venus mice were administered single doses of 50, 70, or 100 mg/kg of ATRA on embryonic day 8.5 (E8.5) then sacrificed on either E16.5 or E19.5. Bowel specimens comprising the anorectum were examined using fluorescence microscopy without immunohistochemical staining (FMIS). Anti-PGP9.5 was used to delineate ganglion cells and anti-SMA for smooth muscles., Results: The appropriate dose of ATRA for inducing ARM was 50 mg/kg. Under FMIS, all ARM embryos (n = 5; all high type; 3 male:2 female) had less NCC innervation with thick Venus-positive nerve fibers in the DRP compared with normal embryos (n = 8); there was abnormal NCC innervation in the DRP and absent ASM in ARM mice., Conclusion: We are the first to delineate abnormal enteric nervous system innervation in the DRP of ARM mice without using immunohistochemical staining techniques thus allowing specimens to be examined without any distortion.

Introduction: We report four cases of long-gap esophageal atresia (LGEA) treated with thoracoscopic esophagoesophagostomy (TEE) after sequential extrathoracic esophageal elongation (SEEE: Kimura's technique)., Methods: All initially had gastrostomy, then SEEE. The proximal end of the esophagus was introduced into the apex of the thorax under direct vision during TEE., Results: Mean birth weight was 1.9 kg. Mean gestational age was 35.0 weeks (range 30-39); mean age at initial esophagostomy was 43.2 days (range 15-110); SEEE was performed for a mean of 3 times (range 2-4) at mean intervals of 5.3 months (range 2-10), with the upper esophageal segment lengthened by a mean of 2.1 cm each time (range 1.1-3.5). Mean age and weight at TEE were 22.3 months and 9.8 kg, respectively. Gap was initially 4.5 vertebrae (range 4-5). Mean operating time was 9.6 h. TEE was successful in 3; 1 required thoracotomy, then re-anastomosis after 11 months of anastomosis leakage; 2 cases with post-TEE anastomosis leakage were treated conservatively. Postoperatively, all cases required fundoplication and esophageal dilatation (2, 3, 5, 8 times, respectively). At mean follow-up of 3.6 years, 2 eat normally and 2 eat minced food., Conclusions: TEE after SEEE appears to be a feasible option for treating LGEA.

Background/purpose: In Hirschsprung's disease (HD), thick extrinsic nerve fibers can be associated with the aganglionic segment in the anorectum. We surgically disrupted the migration of vagal neural crest cell-derived cells (vagal NCC) in embryos from transgenic mice we created previously (SOX10-VENUS Tg) which have the SOX10 gene labeled with Venus (V), a green fluorescent protein, to observe sacral NCC activity in the anorectum., Method: Proximal colon harvested from SOX10-VENUS Tg embryos on day 10.5 (n = 10) was transected at the ascending colon. V-positive sacral NCC in the anorectum were observed during organ culture under fluorescence stereoscopic microscopy, and compared with non-transected control specimens (n = 10)., Results: In transected specimens, no V-positive sacral NCC were identified initially in the anorectum. By day 2, there were thick beaded sacral NCC in the anorectum in 6/10 (60 %) that migrated steadily to the transected end over 3-4 days. In controls, thinner and shorter V-positive sacral NCC began migrating cranially on day 2, and were met by distally migrating vagal NCC., Conclusion: Disruption of vagal NCC migration appears to induce sacral NCC activity in the anorectum, suggesting that thick extrinsic nerve fibers seen in HD may be a secondary phenomenon.

Purpose: Nitrofen model of congenital diaphragmatic hernia (CDH) has been widely used to investigate the pathogenesis of pulmonary hypoplasia (PH). Fibroblast growth factor (FGF) signaling pathway plays a fundamental role in fetal lung development. FGF7 and FGF10, which are critical for lung morphogenesis, have been reported to be downregulated in nitrofen-induced PH. FGF signaling is mediated by a family of four single transmembrane receptors, FGFR1-4. FGFR2 and FGFR3 have been shown to be expressed predominantly in the late stages of developing lungs. In addition, the upregulation of FGFR2 gene expression has been associated with severe defects in lung development and resulted in arrested alveologenesis similar to PH seen in the nitrofen model. Furthermore, FGFR3(-/-)FGFR4(-/-) double mutants showed thinner mesenchyme and larger air spaces. We designed this study to test the hypothesis that FGFR gene expression is upregulated in the late stages of lung development in the nitrofen CDH model., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Cesarean section was performed and fetuses were harvested on D18 and D21. Fetal lungs were divided into three groups: control, nitrofen without CDH [CDH(-)], and nitrofen with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary gene expression levels of FGFR1-4 were analyzed by real-time RT-PCR. Immunohistochemistry was also performed to evaluate protein expression/distribution at each time-point., Results: The relative messenger RNA expression levels of pulmonary FGFR2 and FGFR3 on D21 were significantly increased in CDH(-) (6.38 ± 1.93 and 7.84 ± 2.86, respectively) and CDH(+) (7.09 ± 2.50 and 7.25 ± 3.43, respectively) compared to controls (P < 0.05 and P < 0.01, respectively), whereas no significant alteration was observed on D18. There were no differences in FGFR1 and FGFR4 expression at both time-points. Increased immunoreactivity of FGFR2 and FGFR3, mainly in the distal epithelium and mesenchyme, was observed in the nitrofen-induced hypoplastic lungs on D21 compared to controls., Conclusion: Upregulation of FGFR2 and FGFR3 pulmonary gene expression in the late stages of fetal lung development may disrupt FGFR-mediated alveologenesis resulting in PH in the CDH model.

Background: Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression., Results: Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 μg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 μg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group., Conclusions: Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.

Purpose: Although the precise pathogenesis of ventral body wall (VBW) defects is not clearly understood, it has recently postulated that disruption of somite development during early embryogenesis may cause failure of proper VBW formation. The administration of cadmium (Cd) after 60 h of incubation induces omphalocele spectrum in the chick embryo. Previous studies have shown that one of the earliest histological changes seen in this model is abnormal cell death in the somite, occurring at 4 h post treatment (4H). However, the molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Presenilins are expressed in somites and play an important role in vertebrate development, including somitogenesis and thus VBW formation. We designed this study to test the hypothesis that gene expression levels of presenilin 1 (PSEN1) and presenilin 2 (PSEN2) are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele in the chick model., Methods: After 60 h of incubation, chick embryos were exposed to either saline or 50 μM cadmium and divided into two groups: control and Cd (n = 8 at each time point for each group). Real-time RT-PCR was performed to evaluate the relative mRNA expression levels of PSEN1 and PSEN2 in the Cd-induced omphalocele chick model. Differences between two groups at each time point were analysed statistically and the significance was accepted at p < 0.05. Immunofluorescence confocal microscopy was performed to evaluate the protein expression/distribution of presenilins in the somite of chick embryo., Results: The relative mRNA expression levels of PSEN1 and PSEN2 were significantly downregulated in the Cd group at 4H compared with controls (p < 0.005) (Table). However, there were no significant differences at the other time points. At 4H, immunofluorescence of presenilins (green) was markedly diminished in the Cd-treated embryos, whereas strong immunofluorescence of them was seen in the somite (dermomyotome) in controls (Fig. 1). 1 Immunofluorescence Confocal Microscopy for PSEN1 and PSEN2 in the dermomyotome of the somite in the trunk level of chick embryo 4H post treatment. Intensity of PSEN1 immunofluorescence (green) was markedly diminished in Cd-treated embryos, whereas strong PSEN1 expression was seen in the dermomyotome in controls. PSEN2 immunofluorescence was also decreased in the Cd-treated embryos, whereas strong PSEN2 immunofluorescence (green) was observed predominantly in the dermomyotome in controls. Immunofluorescence in orange is DNA counter staining by DAPI CONCLUSION: We provide evidence, for the first time, that gene expression of presenilins is downregulated during the narrow window of very early embryogenesis in the Cd chick model. Decreased expression of presenilins may contribute to omphalocele phenotype in Cd chick model, by disrupting somite development.

Purpose: The exact pathogenesis of pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Smad1, one of the bone morphogenesis protein (BMP) receptor downstream signaling proteins, plays a key role in organogenesis including lung development and maturation. Smad1 knockout mice display reduced sacculation, an important feature of pulmonary hypoplasia. Wnt inhibitor factor 1 (Wif1) is a target gene of Smad1 in the developing lung epithelial cells (LECs). Smad1 directly regulates Wif1 gene expression and blockade of Smad1 function in fetal LECs is reported to downregulate Wif1 gene expression. We designed this study to test the hypothesis that pulmonary Smad1 and Wif1 gene expression is downregulated during saccular stage of lung development in the nitrofen CDH model., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18, and D21. Fetal lungs were dissected and divided into 2 groups: control and nitrofen (n = 9 at each time point, respectively). Pulmonary gene expression of Smad1 and Wif1 were analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution of Smad1 and Wif1., Results: The relative mRNA expression levels of Smad1 and Wif1 were significantly downregulated in the nitrofen group compared to controls on D18 and D21 (*p < 0.01, **p < 0.05). Immunoreactivity of Smad1 and Wif1 was also markedly decreased in nitrofen lungs compared to controls on D18 and D21., Conclusion: We provide evidence, for the first time, that the pulmonary gene expression of Smad1 and Wif1 is downregulated on D18 and D21 (saccular stage of lung development) in the nitrofen-induced hypoplastic lung. These findings suggest that the downregulation of Smad1/Wif1 gene expression may contribute to pulmonary hypoplasia in the nitrofen CDH model by retardation of lung development during saccular stage.

Skip segment Hirschsprung's disease (SSHD) involves a skip area of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. We report a case of SSHD in infant with total colonic aganglionosis (TCA). Although SSHD is a rare and controversial condition, it should be considered a definite entity in the evaluation of Hirschsprung's disease.

Purpose: The nitrofen model of congenital diaphragmatic hernia (CDH) is widely used to investigate the pathogenesis of CDH. However, the exact pathomechanism of the diaphragmatic defect is still unclear. Diaphragmatic muscularization represents the last stage of diaphragmatic development. Myogenic differentiation 1 (MyoD) and myogenic factor 5 (Myf5) play a crucial role in muscularization. MyoD(-/-) : Myf5(+/-) mutant mice show reduced diaphragmatic size, whereas MyoD(+/-) : Myf5(-/-) mutants have normal diaphragms. We designed this study to investigate diaphragmatic gene expression of MyoD and Myf5 in the nitrofen CDH model., Methods: Pregnant rats received nitrofen or vehicle on day 9 of gestation (D9), followed by cesarean section on D18 and D21. Fetal diaphragms (n = 40) were micro-dissected and divided into CDH group and controls. MyoD and Myf5 mRNA-expression were determined using Real-time PCR. Immunohistochemistry was performed to evaluate protein expression of MyoD and Myf5., Results: Relative diaphragmatic mRNA expression levels and immunoreactivity of MyoD were decreased in the CDH group on D18 and D21. Myf 5 mRNA and protein expression were not altered in the CDH group., Conclusion: This is the first study showing that MyoD expression is selectively decreased in the diaphragm muscle in the nitrofen model of CDH.

Purpose: In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. The earliest histological changes in this model are observed commencing at 4-h post treatment (4H). The molecular mechanisms by which Cd acts during the critical period of embryogenesis remain unclear. Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) regulate many fundamental biological processes and are involved in various embryonic morphogenesis, including ventral body wall (VBW) formation. Homozygous BMP1 mutant mice cause VBW defects. It has been reported that BMPR1A conditional knockouts also exhibit omphalocele phenotype. We designed this study to test the hypothesis that gene expression levels of BMP1 and BMPR1A are downregulated during the critical period of embryogenesis in the Cd chick model., Methods: After 60-h incubation, chick embryos were exposed to either Cd or saline and then harvested 1H, 4H and 8H. Chicks were divided into control (n = 24) and Cd (n = 24). RT-PCR was performed and differences between the two groups were tested statistically (significance was accepted at p < 0.05). Immunohistochemical study was also performed to evaluate those proteins expression/distribution., Results: The gene expression levels of BMP1 and BMPR1A at 4H were significantly downregulated in Cd group compared to controls. Immunoreactivity of BMP1 and BMPR1A was also markedly decreased in Cd-treated embryos compared to controls., Conclusion: Disruption of BMPR1A-mediated BMP1 signalling during the narrow window of early embryogenesis may interfere with normal VBW formation, causing omphalocele phenotype in the Cd chick model.

Purpose: Recent studies have suggested that retinoids may be involved in the molecular mechanisms of pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). Connective tissue growth factor (CTGF) plays a key role in foetal lung development and remodelling during later gestation. CTGF knockout mice exhibit PH with similar characteristics to the human and nitrofen-induced PH. Prenatal administration of retinoic acid (RA) has been shown to stimulate alveologenesis in nitrofen-induced PH. In vitro studies have revealed that RA can induce CTGF gene expression. We hypothesized that pulmonary gene expression of CTGF is downregulated during the later stages of lung development, and that prenatal administration of RA upregulates CTGF in the nitrofen CDH model., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on D18, D19 and D20. Foetuses were harvested on D21 and divided into control, CDH, control + RA and CDH + RA group. Pulmonary CTGF gene and protein expression levels were determined using RT-PCR and immunohistochemistry., Results: On D21, CTGF relative mRNA expression levels were significantly downregulated in CDH group compared to controls. After RA treatment, expression levels of CTGF were significantly upregulated in CDH + RA and control + RA compared to the CDH group. Immunohistochemical studies confirmed these results., Conclusion: Downregulation of pulmonary CTGF gene and protein expression during later stages of lung development may interfere with normal alveologenesis in the nitrofen CDH model. Upregulation of CTGF pulmonary gene expression after prenatal RA treatment may promote lung growth by promoting alveologenesis in the nitrofen-induced CDH model.

Purpose: Although, recent studies have suggested that disruption of somitogenesis may be involved in ventral body wall (VBW) defects; the molecular mechanisms of VBW defects remain unclear. In the chick embryo, the administration of cadmium (Cd) induces VBW defects similar to the human omphalocele. In this model, the earliest histological change in the somite occurs commencing at 4 h post-Cd treatment (4 h). PITX2 is expressed in somites, and PITX2 mutants have been shown to display VBW defects. PITX2 interacts with lymphoid enhancer factor-1 (LEF1) to regulate somite myogenesis. We designed this study to investigate the hypothesis that PITX2 and LEF1 genes are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model., Materials and Methods: Chick embryos were exposed to Cd or saline after 60 h incubation and harvested at 1, 4, and 8 h posttreatment. Chicks were then divided into two groups: control (n = 24), and Cd (n = 24). RT-PCR was performed and analyzed statistically (significant difference was accepted at p < 0.05). Immunohistochemistry was also performed to evaluate expression/distribution of those proteins., Results: The mRNA expression levels of PITX2 and LEF1 at 4 h were significantly decreased in the Cd group compared with controls, whereas there were no differences at the other time points. Immunoreactivity of those proteins at 4 h was also markedly decreased in somites in the Cd-treated embryos compared with controls., Conclusions: Downregulation of PITX2 and LEF1 genes may interfere with ventral body wall formation in Cd chick model causing omphalocele by disrupting somite myogenesis.

Purpose: Nitrofen-induced congenital diaphragmatic hernia (CDH) model has been widely used to investigate the pathogenesis of pulmonary hypoplasia (PH) in CDH. Recent studies have suggested that retinoids may be involved in the molecular mechanisms of PH in CDH. Prenatal treatment with retinoic acid (RA) has been reported to improve the growth of hypoplastic lung in the nitrofen CDH model. Midkine (MK), a RA-responsive growth factor, plays key roles in various organogenesis including lung development. In fetal lung, MK mRNA expression has its peak at E13.5-E16.5 and is markedly decreased during mid-to-late gestation, indicating its important role in early lung morphogenesis. We designed this study to investigate the hypothesis that the pulmonary MK gene expression is downregulated in the early lung morphogenesis in the nitrofen-induced PH, and to evaluate the effect of prenatal RA treatment on pulmonary MK gene expression in the nitrofen-induced CDH model., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into control, nitrofen with or without CDH [CDH(+) or CDH(-)]. In addition, RA was given on days D18, D19, and D20 and fetal lungs were harvested on D21, and then divided into control + RA and nitrofen + RA. The pulmonary gene expression levels of MK were evaluated by real-time RT-PCR and statistically analyzed. Immunohistochemistry was also performed to examine protein expression/distribution of MK in fetal lung., Results: The relative mRNA expression levels of MK were significantly downregulated in nitrofen group compared to controls at D15 ((§)p < 0.01), whereas there were no significant differences at D18 and D21. MK gene expression levels were significantly upregulated in nitrofen + RA (0.71 ± 0.17) compared to the control (0.35 ± 0.16), CDH(-) (0.24 ± 0.15), CDH(+) (0.39 ± 0.19) and control + RA (0.47 ± 0.13) (*p < 0.05). Immunoreactivity of MK was also markedly decreased in nitrofen lungs compared to controls on D15, and increased in nitrofen + RA lungs compared to the other lungs on D21., Conclusion: Downregulation of MK gene on D15 may contribute to primary PH in the nitrofen CDH model by disrupting early lung morphogenesis. Upregulation of MK gene after RA treatment in the nitrofen-induced hypoplastic lung suggests that RA may have a therapeutic potential to rescue PH in CDH through RA-responsive growth factor signaling.

Purpose: Administration of heavy metal cadmium (Cd) after 60-h incubation induces omphalocele spectrum in the chick embryo. Although previous studies have shown that the earliest detectable histological changes in the chick Cd model occurs commencing at 4-h post-treatment (4H). However, the molecular mechanism by which Cd acts in the critical period of early embryogenesis still remains unclear. Zic3, a Gli superfamily transcription factor, is expressed in somites and plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Gli3 is also expressed in somites and interacts with Zic3 physically and functionally. It has been reported that Gli3 homozygous double mutants display omphalocele. Zic3 mutant mice have also been known to result in omphalocele phenotype. We designed this study to test the hypothesis that Gli3 and Zic3 gene expression is downregulated during the critical period of very early embryogenesis in the Cd-induced omphalocele in the chick model., Methods: After 60-h incubation, chick embryos were exposed to either saline or 50 μM cadmium and divided into two groups: control and Cd (n = 24 for each group). Real-time reverse transcription polymerase chain reaction was performed to evaluate the relative mRNA expression levels of Gli3 and Zic3 in the Cd-induced omphalocele chick model. Differences between the two groups at each time point were analyzed statistically and the significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate the expression/distribution of those proteins in chick embryo., Results: The relative mRNA expression level of Gli3 and Zic3 was significantly decreased in the Cd group at 4H when compared with controls (p < 0.05). However, there were no significant differences at the other time points. At 4H, immunoreactivity of GLI3 and ZIC3 was also markedly decreased in Cd-treated embryos, whereas strong expression of them was seen in the somite in controls., Conclusion: We provide evidence, for the first time, that Gli3 and Zic3 gene expression is downregulated during the narrow window of very early embryogenesis in Cd chick model. Disruption of Gli3-Zic3 interaction in the critical period for ventral body wall formation may contribute to omphalocele phenotype in Cd chick model.

Purpose: In the chick embryo, administration of the heavy metal Cadmium (Cd) induces omphalocele phenotype. Cd is a potent inhibitor of antioxidant enzymes and causes accumulation of reactive oxygen species (ROSs) such as hydrogen peroxide. Previous work with the Cd chick model has demonstrated that increased levels of MDA, as a marker for oxidative stress, 24 h post Cd treatment (24H) are identical in chick embryos exposed to Cd. Furthermore, of the several antioxidants assessed, only N-acetylcysteine (NAC) has been shown to reduce MDA levels to control values in the Cd-treated chick embryo. However, the molecular mechanisms by which NAC acts to maintain oxidative stress in the Cd-induced ventral body wall defect chick model remains to be unclear. We designed this study to investigate the hypothesis that gene expression levels of antioxidant enzymes are downregulated in malformed embryos exposed to Cd compared to controls and to determine the effect of pre-treatment with NAC on the expression levels of genes encoding antioxidant enzymes., Methods: After 60 h incubation, chick embryos were pre-treated with NAC and exposed to either chick saline or Cd. Chicks were then harvested at 24H and divided into five groups: control, Cd group without malformation [Cd(-)], Cd group with malformation [Cd(+)], NAC + Cd(-) and NAC + Cd(+). Real-time PCR was performed to evaluate the relative mRNA expression levels of antioxidant enzymes, including superoxide dismutase (SOD)-1, SOD2, catalase (CAT) and glutathione peroxidase (GPX)-4. Differences between five groups were tested by Tukey-Kramer post-hoc test following one-way ANOVA. Statistical significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate protein expression., Results: The mRNA expression levels of SOD2 and CAT were significantly decreased in Cd(+) as compared to controls, whereas there was no significant difference between controls and Cd(-) (p < 0.05 vs. controls). In addition, gene expression levels of SOD2 and CAT were significantly increased in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+) (p < 0.05 vs. controls). However, there were no significant differences in the expression levels of SOD1 and GPX4 among any groups. Increased immunoreactivity of SOD2 and CAT was also observed in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+)., Conclusion: Our results suggest that SOD2 and CAT may play an important role in preventing Cd-induced teratogenesis. Prenatal treatment with drugs which can upregulate SOD2 and CAT transcripts may have a therapeutic potential in preventing omphalocele phenotype.

Purpose: The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm's tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1(-/-) mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model., Methods: Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05., Results: Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21., Conclusion: Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.

Purpose: Pulmonary hypoplasia (PH), the leading cause of mortality in congenital diaphragmatic hernia (CDH), is associated with arrested alveolarization. Late gestation lung protein 1 (LGL1) plays a crucial role in the regulation of alveolarization. Inhibition of LGL1 impairs alveolar maturation in fetal rat lungs. LGL1 heterozygotus knockout mice display delayed lung maturation. It is well known that prenatal administration of retinoic acid (RA) stimulates alveologenesis in nitrofen-induced PH. In vitro studies have reported that RA is a key modulator of LGL1 during alveologenesis. We hypothesized, that pulmonary gene expression of LGL1 is downregulated in the late stage of lung development, and that prenatal administration of RA upregulates pulmonary LGL1 expression in the nitrofen CDH model., Methods: Pregnant rats were exposed to nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on D18, D19 and D20. Fetal lungs were dissected on D21 and divided into control, control + RA, CDH and CDH + RA group. Expression levels of LGL1 were determined using RT-PCR and immunohistochemistry., Results: On D21, LGL1 relative mRNA expression levels were significantly downregulated in CDH group compared to controls. After RA treatment, gene expression levels of LGL1 were significantly upregulated in CDH + RA and control + RA compared to CDH group. Immunohistochemical studies confirmed these results., Conclusion: Downregulation of pulmonary LGL1 gene expression in the late stage of lung development may interfere with normal alveologenesis. Upregulation of LGL1 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in the nitrofen CDH model.

Purpose: Prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs (HL) in the nitrofen model of congenital diaphragmatic hernia (CDH). Parathyroid hormone-related protein (PTHrP) promotes alveolar maturation by stimulating surfactant production, regulated by PTHrP receptor (PTHrP-R). PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia. We have recently reported that nitrofen inhibits PTHrP signaling in the nitrofen-induced HL. Because both PTHrP and PTHrP-R genes have RA-inducible element, we hypothesized that prenatal administration of RA upregulates pulmonary gene expression of PTHrP and PTHrP-R in the nitrofen-induced HL., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given on days D18, D19 and D20. Fetal lungs were obtained on D21 and divided into four groups: control, control + RA, nitrofen, nitrofen + RA. RT-PCR and Immunohistochemistry were performed to investigate the pulmonary PTHrP and PTHrP-R gene and protein expression in each group, respectively., Results: The pulmonary gene expression levels of PTHrP and PTHrP-R were significantly increased in nitrofen + RA group compared to nitrofen group (p < 0.05). Immunoreactivity of PTHrP and PTHrP-R was also remarkably increased in nitrofen + RA group compared to nitrofen group., Conclusions: Upregulation of PTHrP and PTHrP-R genes after prenatal treatment with RA in the nitrofen-induced HL suggests that RA may have a therapeutic potential in reverting lung hypoplasia in CDH, by stimulating surfactant production and alveolar maturation.

Purpose: In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. HoxB2 and HoxB4, expressed in cell types that contribute to ventral body wall (VBW) formation, act together to mediate proper closure of the VBW, involving a key downstream transcription factor, Alx4. HoxB2 and HoxB4 knockout mice display VBW defects with specific downregulation of Alx4 gene expression, while homozygous Alx4 knockouts show omphalocele phenotype. Although the earliest histological changes in the Cd chick model occur commencing at 4H post treatment, the exact timing and molecular mechanism by which Cd acts is still unclear. We hypothesized that HoxB2, HoxB4 and Alx4 genes are downregulated during the critical timing of very early embryogenesis in the Cd-induced omphalocele chick model., Methods: After 60H incubation, chick embryos were harvested at 1H, 4H and 8H after treatment with saline or Cd and divided into controls and Cd group (n = 24 for each group). RT-PCR was performed to investigate the gene expression of HoxB2, HoxB4 and Alx4 and statistically analyzed (significance was accepted at p < 0.05). Immunohistochemical staining was also performed to evaluate the protein expression/distribution of HoxB2, HoxB4 and Alx4 in the chick embryo., Results: The expression levels of HoxB2, HoxB4 and Alx4 gene at 4H were significantly downregulated in the Cd group as compared to controls, whereas there were no significant differences at the other time points. Immunoreactivity of HoxB2, HoxB4 and Alx4 at 4H is also markedly decreased in the ectoderm and the dermomyotome in the Cd chick model as compared to controls., Conclusion: Downregulation of HoxB2, HoxB4 and Alx4 expression during the narrow window of early embryogenesis may cause omphalocele in the Cd chick model by interfering with molecular signaling required for proper VBW formation. Furthermore, these results support the concept that HoxB2, HoxB4 and Alx4 genes work together to mediate proper VBW formation.

Background: The precise mechanism of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) still remains unclear. Recently, prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs in the nitrofen model of CDH. The serine/threonine protein kinase B (AKT) plays a key role in lung morphogenesis through epithelial-mesenchymal interaction in phosphatidylinositide 3-kinase (PI3K)-dependent manner. It has been reported that the lung morphogenesis in explants in mice is interfered by inhibitors of PI3K-AKT signaling pathway. Furthermore, we have recently shown that nitrofen inhibits PI3K-AKT signaling during mid-to-late lung morphogenesis in the nitrofen-induced hypoplastic lung. We hypothesized that prenatal administration of RA upregulates pulmonary gene expression of PI3K and AKT in the nitrofen-induced hypoplastic lung., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). 5 mg/kg of RA was given on D18, D19 and D20. The fetuses were harvested on D21, and fetal lungs were obtained and divided into four groups: control, control + RA, nitrofen, nitrofen + RA. The mRNA expression levels of PI3K and AKT were analyzed in each lung by real-time RT-PCR and statistically analyzed. Immunohistochemistry was also performed to evaluate protein expression of PI3K and AKT in the fetal lungs at D21., Results: The pulmonary gene expression levels of PI3K and AKT were significantly upregulated in nitrofen + RA group compared to nitrofen group and control + RA group (p < 0.05), whereas there were no significant differences between controls and control + RA group. Immunoreactivity of PI3K and AKT was markedly increased in nitrofen + RA lungs compared to nitrofen-induced hypoplastic lungs., Conclusions: Upregulation of PI3K and AKT genes after prenatal treatment with RA in the nitrofen-induced hypoplastic lung suggests that RA may have a therapeutic potential in modulating lung alveologenesis by stimulating epithelial-mesenchymal interaction via PI3K-AKT signaling.

Purpose: Pulmonary hypoplasia and persistent pulmonary hypertension (PPH) aggravate clinical courses in congenital diaphragmatic hernia (CDH). Endothelin 1 enhances PPH by vasoconstriction and proliferation of vessel walls. Up-regulation of pulmonary Endothelin Receptors A and B (EDNRA, EDNRB) has been reported in human CDH and animal models, but the onset of those alterations during lung development remains unclear. We hypothesized that pulmonary expression of EDNRA and EDNRB is up-regulated at early gestational stages in the nitrofen model., Methods: Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Embryos were sacrificed on D15, D18 and D21 and divided into nitrofen- and control group. Pulmonary RNA was extracted and mRNA levels of EDNRA and EDNRB were determined by real-time PCR. Immunohistochemistry for protein expression of both receptors was performed., Results: mRNA levels of EDNRA and EDNRB were significantly increased in the nitrofen group on D15, D18 and D21. Immunohistochemistry revealed increased pulmonary vascular expression of EDNRA and EDNRB compared to controls., Conclusion: Altered expression of EDNRA and EDNRB is an early event in lung morphogenesis in the nitrofen model. We speculate that pulmonary arteries in CDH become excessively muscularised in early fetal life, becoming unable to adapt normally at birth.

Purpose: Administration of cadmium (Cd) causes omphalocele in the chick embryo. The earliest histological changes in the chick Cd model are the breakdown of adherens junctions (AJs). Calreticulin (CRT) plays a key role in Ca(2+) signaling and cell adhesion. Ca(2+) signaling in the Cd chick model is known to be altered. The calcium-dependent adhesion molecule, E-cadherin, and its associate, beta-catenin, are key components of AJs regulated by CRT. CRT knockouts display omphalocele. We hypothesized that CRT, E-cadherin and beta-catenin are downregulated during early embryogenesis in the Cd chick model., Methods: After 60 h (H) incubation, chicks were harvested 1H, 4H, and 8H post treatment with saline or Cd and divided into controls and Cd. RT-PCR was performed to evaluate mRNA levels of CRT, E-cadherin and beta-catenin in the Cd chick model., Results: The mRNA levels of CRT were significantly decreased in the Cd group at 1H compared to controls (p < 0.05). The mRNA levels of E-cadherin and beta-catenin were significantly decreased at 4H in the Cd group compared to controls (p < 0.05). There were no significant differences at 8H., Conclusion: Downregulation of CRT, E-cadherin and beta-catenin genes may cause omphalocele in the Cd chick model by disrupting CRT-mediated Ca(2+) signaling and AJs.

Purpose: Despite remarkable progress in resuscitation and intensive care, the morbidity and mortality rates in congenital diaphragmatic hernia (CDH) remain high due to severe pulmonary hypoplasia. The pathogenesis of pulmonary hypoplasia associated with CDH is still not clearly understood. Pulmonary parathyroid hormone-related protein (PTHrP) is expressed in the type II epithelial cells and stimulates surfactant production by a paracrine feedback loop regulated by PTHrP receptor (PTHrP-R), which is expressed in the mesenchyme, during terminal airway differentiation. It has been reported that PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia, disrupting alveolar maturation before birth. We designed this study to test the hypothesis that gene expression of PTHrP and PTHrP-R is downregulated in the late stages of lung morphogenesis in the nitrofen-induced hypoplastic lung., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, 18, and 21 and divided into three groups: control, nitrofen without CDH [CDH(-)], and nitrofen with CDH [CDH(+)] (n = 8 at each time point for each group, respectively). Total mRNA was extracted from fetal lungs and mRNA expression of PTHrP and PTHrP-R was analyzed by real-time RT-PCR and the significant differences between the groups were accepted at P < 0.05 by statistical analysis. Immunohistochemical studies were also performed to evaluate PTHrP and PTHrP-R protein expression at each time point., Results: Pulmonary mRNA expression of PTHrP-R was significantly decreased in both nitrofen groups [CDH(-) and CDH(+)] compared to controls at D18 and 21. The mRNA level of PTHrP was significantly decreased at D21 in both nitrofen groups compared to controls. Immunoreactivity of PTHrP and PTHrP-R at D18 and 21 was diminished in the distal epithelium and in the mesenchyme, respectively, in the nitrofen-induced hypoplastic lung compared to control lungs. There were no significant differences in both gene/protein expression of PTHrP and PTHrP-R on D15., Conclusion: Downregulation of PTHrP and PTHrP-R gene expression during late lung morphogenesis may cause pulmonary hypoplasia in the nitrofen CDH model, disrupting alveolar maturation and surfactant production by interfering with mesenchymal-epithelial interactions.