Interstitial cells of Cajal: clinical relevance in pediatric gastrointestinal motility disorders.

Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca2+-activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.

Delayed primary anastomosis for repair of long-gap esophageal atresia: technique revisited.

The operative management of patients born with long-gap esophageal atresia (LGEA) remains a major challenge for most pediatric surgeons, due to the rarity and complex nature of this malformation. In LGEA, the distance between the proximal and distal esophageal end is too wide, making a primary anastomosis often impossible. Still, every effort should be made to preserve the native esophagus as no other conduit can replace its function in transporting food from the oral cavity to the stomach satisfactorily. In 1981, Puri et al. observed that in newborns with LGEA spontaneous growth and hypertrophy of the two segments occur at a rate faster than overall somatic growth in the absence of any form of mechanical stretching, traction or bouginage. They further noted that maximal natural growth arises in the first 8-12 weeks of life, stimulated by the swallowing reflex and reflux of gastric contents into the lower esophageal pouch. Since then, creation of an initial gastrostomy and continuous suction of the upper esophageal pouch followed by delayed primary anastomosis at approximately 3 months of age has been widely accepted as the preferred treatment option in most LGEA cases, generally providing good functional results. The current article offers a comprehensive update on the various aspects and challenges of this technique including initial preoperative management and subsequent gap assessment, while also discussing potential postoperative complications and long-term outcome.

C-reactive protein/albumin ratio is a prognostic indicator for predicting surgical intervention and mortality in neonates with necrotizing enterocolitis.

PURPOSE: The role of hypoalbuminemia and raised C-reactive protein (CRP) levels in predicting critical prognosis has been described extensively in adult literature. However, there are limited studies in pediatrics, particularly neonates. The CRP/albumin (CRP/ALB) ratio is often associated with higher mortality, organ failure and prolonged hospital stay. We hypothesized that the serum CRP/ALB ratio has a prognostic value in predicting surgery and mortality in neonates with necrotizing enterocolitis (NEC). METHODS: Retrospective review of all neonates with clinical and radiological evidence of non-perforated NEC that were treated in a tertiary-level referral hospital between 2009 and 2018. General patient demographics, laboratory parameters and outcomes were recorded. Receiver operating characteristics analysis was performed to evaluated optimal cut-offs and area under the curve (AUC) with 95% confidence intervals (CI). RESULTS: A total of 191 neonates were identified. Of these, 103 (53.9%) were born at ≤ 28 weeks of gestation and 101 (52.9%) had a birth weight of ≤ 1000 g. Eighty-four (44.0%) patients underwent surgical intervention for NEC. The overall survival rate was 161/191 (84.3%). A CRP/ALB ratio of ≥ 3 on day 2 of NEC diagnosis was associated with a statistically significant higher likelihood for surgery [AUC 0.71 (95% CI 0.63-0.79); p < 0.0001] and mortality [AUC 0.66 (95% CI 0.54-0.77); p = 0.0150], respectively. CONCLUSIONS: A CRP/ALB ratio of ≥ 3 on day 2 is indicative of a critical pathway in neonates with radiologically confirmed, non-perforated NEC. This could be used as an additional criterion to guide parental counselling in NEC for surgical intervention and mortality.

Transgenic animal models of congenital diaphragmatic hernia: a comprehensive overview of candidate genes and signaling pathways.

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.

Ureteral Obstruction After Endoscopic Treatment of Vesicoureteral Reflux: Does the Type of Injected Bulking Agent Matter?

PURPOSE OF REVIEW: Endoscopic injection of bulking agents for the treatment of vesicoureteral reflux (VUR) has become a therapeutic alternative to antibiotic prophylaxis and ureteral reimplantation. Although considered as a safe and efficient procedure, several studies have reported cases of ureteral obstruction (UO) after endoscopic correction of VUR. This review article evaluates the present VUR literature to estimate the incidence of UO following endoscopic injection of different substances, while also discussing the impact of injection technique and implant volume. RECENT FINDINGS: Twenty-five publications were identified that provided detailed information on 64 females and 32 males (age range, 7 months-48 years) that developed UO after endoscopic treatment of VUR using dextranomer/hyaluronic acid (Dx/HA), polyacrylate polyalcohol (PP), polydimethylsiloxane (PDMS), calcium hydroxyapatite (CaHA), polytetrafluoroethylene (PTFE), or collagen. There was some variation in the reported incidence of UO among these materials: Dx/HA (0.5-6.1%), PP (1.1-1.6%), PDMS (2.5-10.0%), CaHA (1.0%), and PTFE (0.3%). Postoperative UO was described following subureteric transurethral injection (STING), intraureteric hydrodistension implantation technique (HIT), combined HIT/STING and double HIT. The injected volume ranged widely, also depending on the type of bulking agent: Dx/HA (0.3-3.0 mL), PP (0.3-1.2 mL), PDMS (1.0-2.2 mL), CaHA (0.4-0.6 mL), and PTFE (1.5-2.0 mL). The timing of UO varied from immediately after the procedure to 63 months. Over half of patients showed asymptomatic hydroureteronephrosis on follow-up imaging, whereas the remaining presented with symptoms of acute UO or fever. UO remains a rare complication after endoscopic correction of VUR, generally reported in less than 1% of treated cases, which appears to be independent of the injected substance, volume, and technique. However, long-term follow-up is recommended as asymptomatic or delayed UO can occur, potentially leading to deterioration of renal function.

NADPH oxidase-derived H2O2 subverts pathogen signaling by oxidative phosphotyrosine conversion to PB-DOPA.

Strengthening the host immune system to fully exploit its potential as antimicrobial defense is vital in countering antibiotic resistance. Chemical compounds released during bidirectional host-pathogen cross-talk, which follows a sensing-response paradigm, can serve as protective mediators. A potent, diffusible messenger is hydrogen peroxide (H2O2), but its consequences on extracellular pathogens are unknown. Here we show that H2O2, released by the host on pathogen contact, subverts the tyrosine signaling network of a number of bacteria accustomed to low-oxygen environments. This defense mechanism uses heme-containing bacterial enzymes with peroxidase-like activity to facilitate phosphotyrosine (p-Tyr) oxidation. An intrabacterial reaction converts p-Tyr to protein-bound dopa (PB-DOPA) via a tyrosinyl radical intermediate, thereby altering antioxidant defense and inactivating enzymes involved in polysaccharide biosynthesis and metabolism. Disruption of bacterial signaling by DOPA modification reveals an infection containment strategy that weakens bacterial fitness and could be a blueprint for antivirulence approaches.

Choledochal malformations: global research, scientific advances and key controversies.

Choledochal malformations (CMs) represent a spectrum of relatively rare and complex congenital anomalies, characterized by abnormal dilatation of the biliary tract in the absence of any acute obstruction. Today, almost 20% of CMs can be detected in-utero using maternal ultrasonography. Formal scientometric analysis was used to identify where modern CM research is taking place and perhaps where our attention should be directed in the future. Thus, this article offers a comprehensive review of recent scientific advances relating to CMs including the current understanding of etiology and classification, whilst also discussing key controversies such as risk of malignant transformation and the role of newer modalities of surgical treatment. Although laparoscopic excision of CMs and biliary reconstruction is nowadays feasible and safe, care should be taken before dispensing with standard open techniques, which have minimal complication rates and proven long-term benefit.

Correction to: Choledochal malformations: global research, scientific advances and key controversies.

The last sentence in the first paragraph under subheading "Minimally invasive treatment" was incorrect in the original publication.

Expression of dispatched RND transporter family member 1 is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to be caused by a malformation of the diaphragmatic and pulmonary mesenchyme. Dispatched RND transporter family member 1 (Disp-1) encodes a transmembrane protein that regulates the release of cholesterol and palmitoyl, which is critical for normal diaphragmatic and airway development. Disp-1 is strongly expressed in mesenchymal compartments of fetal diaphragms and lungs. Recently, Disp-1 mutations have been identified in patients with CDH. We hypothesized that diaphragmatic and pulmonary Disp-1 expression is decreased in the nitrofen-induced CDH model. METHODS: Time-mated rats received nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms and lungs were microdissected on selected endpoints D13, D15 and D18; and divided into control and nitrofen-exposed specimens (n = 12 per sample, time-point and experimental group). Diaphragmatic and pulmonary Disp-1 expression was evaluated by qRT-PCR. Immunofluorescence double staining for Disp-1 was combined with diaphragmatic and pulmonary mesenchymal markers Wt-1 and Sox-9 to localize protein expression in fetal diaphragms and lungs. RESULTS: Relative mRNA levels of Disp-1 were significantly decreased in pleuroperitoneal folds/primordial lungs on D13 (0.18 ± 0.08 vs. 0.46 ± 0.41; p < 0.05/1.06 ± 0.27 vs. 1.34 ± 0.79; p < 0.05), developing diaphragms/lungs on D15 (0.18 ± 0.06 vs. 0.44 ± 0.23; p < 0.05/0.73 ± 0.36 vs. 1.16 ± 0.27; p < 0.05) and fully muscularized diaphragms/differentiated lungs on D18 (0.22 ± 0.18 vs. 0.32 ± 0.23; p < 0.05/0.56 ± 0.16 vs. 0.77 ± 0.14; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished Disp-1 immunofluorescence predominately in the diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15 and D18, associated with a clear reduction of proliferating mesenchymal cells. CONCLUSIONS: Decreased Disp-1 expression during diaphragmatic development and lung branching morphogenesis may interrupt mesenchymal cell proliferation, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

Endoscopic Injection of Dextranomer/Hyaluronic Acid as First Line Treatment in 851 Consecutive Children with High Grade Vesicoureteral Reflux: Efficacy and Long-Term Results.

PURPOSE: Endoscopic injection of dextranomer/hyaluronic acid is widely acknowledged as first line treatment of lower grade vesicoureteral reflux. We demonstrate its long-term efficacy and safety in eradicating high grade reflux. MATERIALS AND METHODS: A total of 518 girls and 333 boys with a median age of 2.3 years (range 2 months to 13.7 years) underwent endoscopic correction of high grade vesicoureteral reflux using dextranomer/hyaluronic acid. Reflux was unilateral in 415 cases and bilateral in 436, comprising 1,287 refluxing units. Reflux was grade IV in 1,153 ureters (89.6%) and grade V in 134 (10.4%). 99mTechnetium-dimercaptosuccinic acid scintigraphy identified renal scarring in 317 patients (37.3%). Followup ultrasound and voiding cystourethrogram were performed 3 months after intervention and renal ultrasound yearly thereafter. Median followup was 8.5 years (range 6 months to 16 years). RESULTS: Overall resolution rate after the first endoscopic injection was 69.5% (895 of 1,287 cases), with resolution in 70.4% of grade IV and 61.9% of grade V cases. Reflux resolved after a second injection in 259 cases (20.1%) and after a third injection in 133 (10.4%). Persistent reflux after initial treatment was significantly more common in patients younger than age 1 year and in individuals with renal scarring. No significant postoperative complications were observed and no patient required ureteral reimplantation. Following reflux resolution febrile urinary tract infection developed in 43 children (5.1%), including 24 (55.8%) during the first year, 15 (34.9%) during the second year and 4 (9.3%) during year 3 or later. Of these patients 6 had reflux recurrence and 8 had neocontralateral grade III reflux, which was successfully treated with a single endoscopic injection of dextranomer/hyaluronic acid. CONCLUSIONS: Endoscopic injection of dextranomer/hyaluronic acid is an efficient and safe long-term treatment for grade IV and V vesicoureteral reflux, and can easily be repeated in patients with treatment failure with a high subsequent resolution rate.

Congenital diaphragmatic hernia: a scientometric analysis of the global research activity and collaborative networks.

Despite a growing interest to clinicians and scientists, there is no comprehensive study that examines the global research activity on congenital diaphragmatic hernia (CDH). A search strategy for the Web of Science™ database was designed to identify scientific CDH publications. Research output of countries, institutions, individual authors, and collaborative networks was analyzed. Semi-qualitative research measures including citation rate and h-index were assessed. Choropleth mapping and network diagrams were employed to visualize results. A total of 3669 publications were found, originating from 76 countries. The largest number was published by the USA (n = 1250), the UK (n = 279), and Canada (n = 215). The USA combined the highest number of cooperation articles (n = 152), followed by Belgium (n = 115) and the Netherlands (n = 93). The most productive collaborative networks were established between UK/Belgium (n = 53), Belgium/Spain (n = 47), and UK/Spain (n = 34). Canadian publications received the highest average citation rate (22.8), whereas the USA had the highest country-specific h-index (72). Eighty-five (2.3%) articles were published by international multicenter consortiums and national research networks. The most productive institutions and authors were based in North America and Europe. Over the past decades, CDH research has increasingly become multidisciplinary and numerous innovative therapeutic strategies were introduced. CDH-related research has constantly been progressing, involving today many disciplines with main research endeavors concentrating in a few high-income countries. Recent advances in prenatal interventions and regenerative medicine therapy hold the promise of improving CDH outcome in the 21st century. International collaborations and translational research should be strengthened to allow further evolution in this field.

Gata-6 expression is decreased in diaphragmatic and pulmonary mesenchyme of fetal rats with nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia are thought to be caused by a malformation of the underlying diaphragmatic and airway mesenchyme. GATA binding protein 6 (Gata-6) is a zinc finger-containing transcription factor that plays a crucial role during diaphragm and lung development. In the primordial diaphragm, Gata-6 expression is restricted to mesenchymal compartments of the pleuroperitoneal folds (PPFs). In addition, Gata-6 is essential for airway branching morphogenesis through upregulation of mesenchymal signaling. Recently, mutations in Gata-6 have been linked to human CDH. We hypothesized that diaphragmatic and pulmonary Gata-6 expression is decreased in the nitrofen-induced CDH model. METHODS: Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on selected timepoints D13, D15 and D18, and divided into control and nitrofen-exposed specimens (n = 12 per sample, timepoint and experimental group, respectively). Diaphragmatic and pulmonary gene expression of Gata-6 was analyzed by qRT-PCR. Immunofluorescence-double staining for Gata-6 was combined with the diaphragmatic mesenchymal marker Gata-4 and the pulmonary mesenchymal marker Fgf-10 to evaluate protein expression and localization in fetal diaphragms and lungs. RESULTS: Relative mRNA expression levels of Gata-6 were significantly decreased in PPFs on D13 (0.57 ± 0.21 vs. 2.27 ± 1.30; p < 0.05), developing diaphragms (0.94 ± 0.59 vs. 2.28 ± 1.89; p < 0.05) and lungs (0.56 ± 0.16 vs. 0.71 ± 0.39; p < 0.05) on D15 and fully muscularized diaphragms (1.20 ± 1.10 vs. 2.52 ± 1.86; p < 0.05) and differentiated lungs (0.56 ± 0.05 vs. 0.77 ± 0.14; p < 0.05) on D18 of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of Gata-6 mainly in diaphragmatic and pulmonary mesenchyme, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. CONCLUSION: Decreased Gata-6 expression during diaphragmatic development and lung branching morphogenesis may disrupt mesenchymal cell proliferation, causing malformed PPFs and reduced airway branching, thus leading to diaphragmatic defects and pulmonary hypoplasia in the nitrofen-induced CDH model.

Expression of T-box transcription factors 2, 4 and 5 is decreased in the branching airway mesenchyme of nitrofen-induced hypoplastic lungs.

PURPOSE: Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major therapeutic challenge in newborns with congenital diaphragmatic hernia (CDH). T-box transcription factors (Tbx) have been identified as key components of the gene network that regulates fetal lung development. Tbx2, Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung, regulating the process of lung branching morphogenesis. Furthermore, lungs of Tbx2-, Tbx4- and Tbx5-deficient mice are hypoplastic and exhibit decreased lung branching, similar to PH in human CDH. We hypothesized that the expression of Tbx2, Tbx4 and Tbx5 is decreased in the branching airway mesenchyme of hypoplastic rat lungs with nitrofen-induced CDH. METHODS: Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetuses were killed on D15, D18 and D21, and dissected lungs were divided into control and nitrofen-exposed specimens. Pulmonary gene expression of Tbx2, Tbx4 and Tbx5 was investigated by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for Tbx2, Tbx4 and Tbx5 was combined with the mesenchymal marker Fgf10 to assess protein expression and localization in branching airway tissue. RESULTS: Relative mRNA levels of Tbx2, Tbx4 and Tbx5 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Tbx2, Tbx4 and Tbx5 in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. CONCLUSION: Decreased expression of Tbx2, Tbx4 and Tbx5 in the pulmonary mesenchyme during fetal lung development may lead to a decrease or arrest of airway branching, thus contributing to PH in the nitrofen-induced CDH model.

Antenatal retinoic acid administration increases trophoblastic retinol-binding protein dependent retinol transport in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Low pulmonary retinol levels and disrupted retinoid signaling pathway (RSP) have been implicated in the pathogenesis of congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH). It has been demonstrated that nitrofen disturbs the main retinol-binding protein (RBP)-dependent trophoblastic retinol transport. Several studies have demonstrated that prenatal treatment with retinoic acid (RA) can reverse PH in the nitrofen-induced CDH model. We hypothesized that maternal administration of RA can increase trophoblastic RBP-dependent retinol transport in a nitrofen model of CDH. METHODS: Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9) and sacrificed on D21. RA was given i.p. on D18, D19, and D20. Retinol and RA levels were measured using high-performance liquid chromatography. Immunohistochemistry was performed to evaluate trophoblastic expression of RBP. Expression levels of the primary RSP genes were determined using quantitative real-time PCR and immunohistochemistry. RESULTS: Markedly increased trophoblastic RBP immunoreactivity was observed in CDH+RA compared to CDH. Significantly increased serum and pulmonary retinol and RA levels were detected in CDH+RA compared to CDH. Pulmonary expression of RSP genes and proteins were increased in CDH+RA compared to CDH. CONCLUSION: Increased trophoblastic RBP expression and retinol transport after antenatal administration of RA suggest that retinol-triggered RSP activation may attenuate CDH-associated PH by elevating serum and pulmonary retinol levels.

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

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

Decreased Desmin expression in the developing diaphragm of the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle. DES -/- knockout mice exhibit significant reductions in stiffness and elasticity of the developing diaphragmatic muscle tissue. Furthermore, sequence changes in the DES gene have recently been identified in human cases of CDH, suggesting that alterations in DES expression may lead to diaphragmatic defects. This study was designed to investigate the hypothesis that diaphragmatic DES expression is decreased in fetal rats with nitrofen-induced CDH. METHODS: Time-mated Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18, and dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Laser-capture microdissection was used to obtain diaphragmatic tissue elements. Diaphragmatic gene expression of DES was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for DES was combined with the mesenchymal marker GATA4 to evaluate protein expression and localization in developing fetal diaphragms. RESULTS: Relative mRNA expression levels of DES were significantly decreased in pleuroperitoneal folds on D13 (1.49 ± 1.79 vs. 3.47 ± 2.32; p < 0.05), developing diaphragms on D15 (1.49 ± 1.41 vs. 3.94 ± 3.06; p < 0.05) and fully muscularized diaphragms on D18 (2.45 ± 1.47 vs. 5.12 ± 3.37; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of DES mainly in diaphragmatic MCT, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls. CONCLUSION: Decreased expression of DES in the fetal diaphragm may disturb the basic integrity of myofibrils and the cytoskeletal network during myogenesis, causing malformed MCT and leading to diaphragmatic defects in the nitrofen-induced CDH model.

Mesenchymal expression of the FRAS1/FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH. METHODS: Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue. RESULTS: Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76 ± 0.86 vs. 3.09 ± 1.15; p < 0.05 and 0.47 ± 0.26 vs. 0.82 ± 0.36; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45 ± 0.80 vs. 2.63 ± 0.84; p < 0.05 and 0.41 ± 0.16 vs. 1.02 ± 0.49; p < 0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35 ± 0.75 vs. 2.32 ± 0.92; p < 0.05 and 0.37 ± 0.24 vs. 0.70 ± 0.32; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls. CONCLUSION: Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.

Expression of hepatic lipid droplets is decreased in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Prenatal mortality in newborn infants with congenital diaphragmatic hernia (CDH) has been attributed to increased amounts of liver hernia ion through the diaphragmatic defect. Antenatal studies in human and rodent fetus with CDH further demonstrated a contribution of the developing liver in the pathogenesis of CDH. The abnormal hepatic growth in experimental animal models, therefore, indicates a disruption of normal liver development in CDH. However, the underlying structural, histological and functional changes in the liver of animals with CDH remain unclear. We design this study to test the hypothesis that the morphological and cellular liver development is altered in the nitrogen-induced CDH model. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Livers and chest were harvested on D21 and divided into two groups: control (n = 8), nitrofen with CDH (CDH, n = 8). Haematoxylin-eosin (Straub et al. Histopathology 68:617-631, 2013) staining was performed to evaluate underlying morphological changes. Apoptosis was checked by using TUNEL staining and apoptotic cell number was counted on 16-16 slides in 25 fields by two independent viewers. Hepatic lipid droplet expressions were evaluated by hepatic adipose differentiation-related protein (ARDP) expression. RESULTS: Compared to controls markedly increased hypertrophy was seen in CDH group. Significantly increased apoptotic cell numbers were detected in CDH group compared to controls (5.1 ± 1.5 vs 2.1 ± 0.6) (p < 0.05). The relative mRNA expression levels of ARDP were significantly reduced in CDH group compared to controls. Immunohistochemistry showed markedly decreased hepatic ADRP immunoreactivity in CDH fetuses compared to controls. CONCLUSIONS: Our findings provide strong evidence of hepatic hypertrophy and increased cell apoptosis in the liver of nitrofen-induced CDH. These morphological changes may affect liver lipid droplet expression function.

Decreased expression of hepatocyte growth factor in the nitrofen model of congenital diaphragmatic hernia.

PURPOSE: Pleuroperitoneal folds (PPFs) are essential for normal diaphragmatic development, representing the only source of the diaphragm's muscle connective tissue. Hepatocyte growth factor (Hgf), which is secreted in PPFs, plays a crucial role in the formation of the muscular diaphragmatic components by regulating the migration of myogenic progenitor cells into the primordial diaphragm. Hgf is also a known downstream target of Gata4 and it has been demonstrated that the expression of Hgf was significantly downregulated in PPF cells of Gata4 knockouts with congenital diaphragmatic hernia (CDH). Furthermore, mutations in PPF-derived cells have been shown to result in CDH. We hypothesized that Hgf expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH. METHODS: Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression of Hgf was analyzed by qRT-PCR. Immunofluorescence double staining for Hgf and the mesenchymal marker Gata4 or muscular progenitor marker Myogenin was performed to evaluate protein expression and localization in fetal diaphragms. RESULTS: Relative mRNA expression of Hgf was significantly downregulated in PPFs of nitrofen-exposed fetuses on D13 (3.08 ± 1.46 vs. 5.24 ± 1.93; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (2.01 ± 0.79 vs. 4.10 ± 1.50; p < 0.05) and fully muscularized diaphragms of nitrofen-exposed fetuses on D18 (1.60 ± 0.78 vs. 3.21 ± 1.89; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic immunofluorescence of Hgf in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls, which was associated with disruptions in muscle connective tissue formation and reduced myogenic progenitor cell invasion. CONCLUSION: Decreased diaphragmatic expression of Hgf may disturb the formation of muscle connective tissue in PPFs and thus prevent essential migration of muscle progenitor cells into the developing diaphragm, leading to diaphragmatic defects in the nitrofen CDH model.