Ccr2 Dependent Monocytes Exacerbate Intestinal Inflammation and Modulate Gut Serotonergic Signaling Following Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) leads to acute gastrointestinal dysfunction and mucosal damage, resulting in feeding intolerance. Ccr2+ monocytes are crucial immune cells that regulate the gut's inflammatory response via the brain-gut axis. Using CCR2KO mice, we investigated the intricate interplay between these cells to better elucidate the role of systemic inflammation after TBI. METHODS: A murine-controlled cortical impact model was utilized, and results were analyzed on post-injury days (PID) 1 and 3. The experimental groups included (1) Sham C57Bl/6 wild-type (WT), (2) TBI WT, (3) Sham CCR2KO and (4) TBI CCR2KO. Mice were euthanized on PID 1 and 3 to harvest the ileum and study intestinal dysfunction and serotonergic signaling using a combination of quantitative real-time PCR (qRT-PCR), immunohistochemistry, FITC-dextran motility assays, and flow cytometry. Student's t-test and one-way ANOVA were used for statistical analysis, with significance achieved when p < 0.05. RESULTS: TBI resulted in severe dysfunction and dysmotility of the small intestine in WT mice as established by significant upregulation of inflammatory cytokines iNOS, Lcn2, TNFα, and IL1ß and the innate immunity receptor toll-like receptor 4 (Tlr4). This was accompanied by disruption of genes related to serotonin synthesis and degradation. Notably, CCR2KO mice subjected to TBI showed substantial improvements in intestinal pathology. TBI CCR2KO groups demonstrated reduced expression of inflammatory mediators (iNOS, Lcn2, IL1ß, and Tlr4) and improvement in serotonin synthesis genes, including tryptophan hydroxylase 1 (Tph1) and dopa decarboxylase (Ddc). CONCLUSION: Our study reveals a critical role for Ccr2+ monocytes in modulating intestinal homeostasis after TBI. Ccr2+ monocytes aggravate intestinal inflammation and alter gut-derived serotonergic signaling. Therefore, targeting Ccr2+ monocyte-dependent responses could provide a better understanding of TBI-induced gut inflammation. Further studies are required to elucidate the impact of these changes on brain neuroinflammation and cognitive outcomes. STUDY TYPE: Original Article (Basic Science, level of evidence N/A).

Complete Colonic Duplication and Perineal Fistula: Transanal Mucosectomy of the Ectopic Rectum.

Background Colonic duplication may present in different anatomic variants. The surgical approach towards these anomalies can be challenging and has implications for subsequent future continence. Case Description We report on a 1-year-old girl with congenital heart defect and pacemaker who was referred to us with an anorectal malformation. The patient was stooling from both an anus and a perineal fistula. Examination under anesthesia revealed an orthotopic and age-appropriate sized anus with surrounding sphincter and a second rectal lumen ending as a perineal fistula. A computed tomography and contrast enema indicated colonic duplication. Exploratory laparotomy showed a duplicated terminal ileum leading to two ceca and appendices, which joined to a duplicated colon with a septum and common mesentery. At the rectosigmoid junction, one part of the duplication ended as a perineal fistula, the second one led to the (orthotope) anus. The common colonic wall was divided using a stapler. The rectal duplication leading to the perineal fistula was not completely resected but treated by mucosectomy only (Soave plane) leaving its muscular cuff in place. Finally, an ileostomy was created. The postoperative course was uneventful. A contrast enema prior to ostomy takedown demonstrated a well-configurated colon and rectum without stenosis or impaction. The girl is currently continent with a complete resolution of her constipation. Conclusion In cases of complete colonic duplication division of the common wall is simple and safe. Mucosectomy of the ectopic rectum limits pelvic dissection and preserves the entire muscular wall of the duplicated orthotope rectum.

Global Development of Research on Anorectal Malformations over the Last Five Decades: A Bibliometric Analysis.

PURPOSE: Anorectal malformations (ARM) are one of the most challenging congenital malformations in pediatric surgery. We aimed to assess the research activity on ARM over the last five decades. METHODS: Data on original research publications were retrieved from the Web of Science Core Collection (1970-2020), and analyzed for countries, authors, scientific journals, and top-ten papers. Scientific quantity was assessed by the number of publications. Research quality was estimated from the number of citations, average citation rate per item (ACI), and h-index. RESULTS: A total number of 1595 articles with 19,419 citations (ACI = 12.2; h-index = 54) were identified. The annual number of publications and citations significantly increased over time (p < 0.0001). The USA (n = 386; 24.2%), Japan (n = 153; 9.6%), and China (n = 137; 8.6%) were the most productive countries; and the USA (n = 7850; ACI = 20.3; h-index = 44), Japan (n = 1937; ACI = 12.6; h-index = 21), and the Netherlands (n = 1318; ACI = 17.3; h-index = 22) were the top cited countries. Articles were preferentially published in JPS (n = 391; 24.5%), PSI (n = 181; 11.3%), and EJPS (n = 56; 3.5%). Top-ten cited papers focused on classification (n = 1), surgical technique (n = 3), associated syndromes (n = 2), postoperative outcome (n = 3), and basic research (n = 1). CONCLUSION: This bibliometric study provides valuable insights into the global development of ARM research, and shows that clinical studies and international collaborations dominate in this field.

Accidental bladder injury during elective inguinal hernia repair: a preventable complication with high morbidity.

INTRODUCTION: Bladder injury (BI) represents a rare complication of inguinal hernia surgery. Protrusions of the urinary bladder through the deep inguinal ring ("bladder ears") have been reported with an incidence of 9% in infants younger than 6 months of age and may be misinterpreted as the hernia sac. This literature review was designed to determine incidence and outcomes of bladder injuries during pediatric inguinal hernia repair. METHODS: A literature review of the literature (1967-2017) was performed using the keywords "bladder ears", "inguinal hernia", "iatrogenic bladder injury" and "bladder hernia". Publications were reviewed for epidemiology, presentation and extent of injury, treatment and outcome. RESULTS: Thirteen articles reporting on 30 cases of BI during inguinal hernia repair from 1967 to 2017 were included (19 boys, 2 girls, 9 unknown). Median age at herniotomy was 10.5 months (1 month-6 years). Out of 30 children, 14 (47%) experienced mild complications. Sixteen patients (53%) had severe complications after initial surgery and needed revisional surgery. Complications were noticed up to 4 years after the initial surgery. In 9 (56%) of the 16 severe cases, major damage to the bladder wall and impairment of bladder capacity occurred. In seven patients (44%), secondary closure was successful. In ten patients (63%), the bladder was partially resected, and in one child (6%), the entire bladder was removed. CONCLUSIONS: The degree of accidental BI during inguinal hernia repair was severe in in the majority of reported cases in the literature. Surgeons should be aware of the high prevalence of "bladder ears" in infants to prevent injury to the urinary tract.

Upregulation of S1P1 and Rac1 receptors in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Sphingolipids play a crucial role in pulmonary development. The sphingosine kinase 1 (SphK1) modulates the synthesis of sphingolipid sphingosine-1-phosphate (S1P). S1P regulates cell proliferation and angiogenesis via different receptors, S1P1, S1P2 and S1P3, which all influence the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1). We designed this study to test the hypothesis that the S1P/Rac1 pathway is altered in the nitrofen-induced CDH model. METHODS: Pregnant rats received nitrofen or vehicle on D9. On D21, fetuses were killed and divided into nitrofen and control group (n = 12). QRT-PCR, western blotting and confocal-immunofluorescence microscopy were performed to reveal pulmonary gene and protein expression levels of SphK1, S1P1, S1P2, S1P3 and Rac1. RESULTS: Pulmonary gene expression of S1P1 and Rac1 was significantly increased in the CDH group compared to controls, whereas S1P2 and S1P3 expression was decreased. These results were confirmed by western blotting and confocal microscopy. SphK1 expression was not found to be altered. CONCLUSION: The increased expression of S1P1 and Rac1 in the pulmonary vasculature of nitrofen-induced CDH lungs suggests that S1P1 and Rac1 are important mediators of PH in this model.

Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632).

AIM: Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute to failure of VBW closure. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute to the development of VBWD. METHODS: At 60 h incubation, chick embryos were explanted into shell-less culture and treated with 50 µL of vehicle for controls (n = 33) or 50 µL of 500 µM of Y-27632 for the experimental group (Y-27, n = 56). At 8 h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E-cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A further cohort of embryos was treated in ovo by dropping 50 µL of vehicle or 50 µL of different concentrations of Y-27632 onto the embryo and allowing development to 12 and 14 days for further assessment. RESULTS: Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly decreased in treated embryos compared with controls (p < 0.05). Thickened actin filament bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were dissociated with reduced actin distribution in affected embryos. Clumping of vinculin expression was found in the neural tube and somites, whereas reduced expression of microtubules was observed in Y-27 embryos compared with controls. At 12 and 14 days of development, affected embryos presented with an enlarged umbilical ring and herniation of abdominal contents through the defect. CONCLUSION: ROCK inhibition alters cytoskeletal arrangement during early chick embryogenesis, which may contribute to failure of anterior body wall closure causing VBWD at later stages of development.

Syndromic Hirschsprung's disease and associated congenital heart disease: a systematic review.

PURPOSE: Hirschsprung's disease (HD) occurs as an isolated phenotype in 70% of infants and is associated with additional congenital anomalies or syndromes in approximately 30% of patients. The cardiac development depends on neural crest cell proliferation and is closely related to the formation of the enteric nervous system. HD associated with congenital heart disease (CHD) has been reported in 5-8% of cases, with septation defects being the most frequently recorded abnormalities. However, the prevalence of HD associated with CHD in infants with syndromic disorders is not well documented. This systematic review was designed to determine the prevalence of CHD in syndromic HD. METHODS: A systematic review of the literature using the keywords "Hirschsprung's disease", "aganglionosis", "congenital megacolon", "congenital heart disease" and "congenital heart defect" was performed. Resulting publications were reviewed for epidemiology and morbidity. Reference lists were screened for additional relevant studies. RESULTS: A total of fifty-two publications from 1963 to 2014 reported data on infants with HD associated with CHD. The overall reported prevalence of HD associated with CHD in infants without chromosomal disorders was 3%. In infants with syndromic disorders, the overall prevalence of HD associated with CHD ranged from 20 to 80 % (overall prevalence 51%). Septation defects were recorded in 57% (atrial septal defects in 29%, ventricular septal defects in 32%), a patent ductus arteriosus in 39%, vascular abnormalities in 16%, valvular heart defects in 4% and Tetralogy of Fallot in 7%. CONCLUSION: The prevalence of HD associated with CHD is much higher in infants with chromosomal disorders compared to infants without associated syndromes. A routine echocardiogram should be performed in all infants with syndromic HD to exclude cardiac abnormalities.

Outcome of right-sided diaphragmatic hernia repair: a multicentre study.

AIM: Right-sided congenital diaphragmatic hernia (R-CDH) occurs in 14 to 25% of all CDH cases. The current literature comparing the outcome of R-CDH vs left CDH (L-CDH) is inconsistent, with some studies reporting higher and others lower mortality in R-CDH compared to L-CDH. The aim of our multicentre study was to analyse characteristics and outcome of R-CDH. METHODS: We retrospectively reviewed the medical records of 178 consecutive infants with CDH who underwent surgical repair of CDH at three European tertiary pediatric surgical centres from three different countries between 2000 and 2009. The analysis focused on demographic data, morbidity and mortality in R-CDH compared with L-CDH. RESULTS: Out of a total of 178 children, 32 (18.0%) right-sided and 146 (82.0 %) left-sided cases of CDH were identified. Prenatal diagnosis was made in 8 R-CDH vs 67 L-CDH (25.0 vs 45.9%, p = 0.030). Median gestational age in R-CDH was 39 weeks (range 29-42 weeks) and 39 weeks in L-CDH (range 28-43 weeks, p = 0.943). Median birth weight in R-CDH was 3233 g (range 905-4480 g) and in L-CDH was 3060 g (range 1065-5240 g, p = 0.184). Major associated anomalies were present in 19 R-CDH vs 46 L-CDH (59.4 vs 31.5%, p = 0.003). Extracorporeal membrane oxygenation (ECMO) was required in 3 R-CDH vs 19 L-CDH (9.4 vs 13.0%, p = 0.571). A diaphragmatic patch was used in 13 R-CDH and 59 L-CDH (40.6 vs 40.4%, p = 0.982). Fundoplication for GERD was required in 1 R-CDH and 19 L-CDH (3.1 vs 13.0 %, p = 0.109). No significant differences were observed in recurrence rate (9.4 vs 8.9%, p = 0.933). Postoperative mortality rate was significantly higher in R-CDH compared to L-CDH (21.9 vs 8.2%, p = 0.023). In R-CDH, prenatal diagnosis and patch repair correlated with mortality by univariate regression (p = 0.005 and p = 0.019). CONCLUSION: This multicentre study shows that prenatal diagnosis and patch repair were associated with an increased mortality rate in R-CDH. However, the morbidity following repair of R-CDH was not significantly different from that in L-CDH in survivors.

Increased pulmonary vascular expression of Krüppel-like factor 5 and activated survivin in experimental congenital diaphragmatic hernia.

AIM OF THE STUDY: The high morbidity and mortality in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by increased pulmonary artery smooth muscle cell (SMC) proliferation, suppressed apoptosis as well as endothelial dysfunction. Krüppel-like factor 5 (KLF5) belongs to a family of transcription factors that has diverse functions during cell differentiation and embryonic development. KLF5 is preferentially expressed in proliferating SMCs but reduced in differentiated cells. KLF5 induces the expression of Survivin, a 16.5 kDa protein overexpressed in almost all malignancies but hardly detected in normal differentiated tissues. Survivin has been shown to inhibit apoptosis, promote cell proliferation, and enhance angiogenesis. Recent studies have implicated activation of KLF5 and Survivin in the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that KLF5 and Survivin expression are increased in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen (n = 16) and control group (n = 16). Quantitative real-time PCR, western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of KLF5, Survivin, and phosphorylated Survivin (p-Survivin). MAIN RESULTS: Confocal microscopy revealed markedly increased pulmonary vascular KLF5 and p-Survivin expression in lungs of nitrofen-exposed fetuses compared to controls. These results were confirmed by western blotting, showing increased pulmonary expression of KLF5 and p-Survivin. Furthermore, the relative pulmonary gene expressions of KLF5 and Survivin were significantly increased in the CDH group compared to controls (p < 0.005 rsp. p < 0.01). CONCLUSION: This study provides striking evidence of increased gene and protein expression of KLF5 and activated Survivin in the pulmonary vasculature of nitrofen-induced CDH, suggesting that increased expression of KLF5 may activate p-Survivin expression and play an important role in the pathogenesis of PH in nitrofen-induced CDH.

Prevalence of Hirschsprung's disease in premature infants: a systematic review.

PURPOSE: Intestinal dysmotility in preterm infants has often been attributed to immature enteric nervous system. It is frequently reported that Hirschsprung's disease (HD) is rare in premature infants. The exact prevalence of HD in premature infants is not well documented. The classical signs of HD may often not be identified due to the complexity of symptoms of prematurity itself. This systematic review was designed to determine the prevalence and presentation of HD in premature infants. METHODS: A systematic review of the literature using the keywords "Hirschsprung's disease", "aganglionosis", "congenital megacolon", "premature" and "preterm" was performed. Resulting publications were reviewed for epidemiology and morbidity. Only infants born <37 weeks of gestation or described as preterm birth by the authors were included. Reference lists were screened for additional relevant studies. RESULTS: Twenty-six publications from 1964 to 2013 reported data on premature infants with HD. Out of a total number of 4,147 infants, prematurity was recorded in 257 cases, giving a prevalence rate of 6 % of preterm infants diagnosed with HD. During 1964-1999, reported prevalence of HD in premature infants ranged from 1.7 to 9.2 % (overall prevalence 5 %) and during 2000-2013 prevalence ranged from 4 to 19.4 % (overall prevalence 14 %). The prevalence of total colonic aganglionosis in premature infants was 13 % (15 out of 118 infants). Mean gestational age of preterm infants was 34.5 (± 0.7) weeks and mean age at diagnosis ranged from 18.3 days to 3.9 months. Abdominal distension was observed in 80 % of preterm infants, delayed passage of meconium in 57 and 37 % of premature infants presented with bile-stained vomiting. CONCLUSION: In recent years, higher prevalence of HD has been reported in premature infants compared to previous years. Hirschsprung's disease should be considered in preterm infants presenting with features of intestinal obstruction.

Congenital anomalies of the kidney and urinary tract (CAKUT) associated with Hirschsprung's disease: a systematic review.

PURPOSE: Congenital anomalies of the kidney and urinary tract (CAKUT), a term introduced in the late 1990 s accounts for 30-50 % of cases of end-stage renal disease in children. The association of urogenital anomalies and Hirschsprung's disease (HSCR) based on the common genetic background of enteric nervous system and human urinary tract development has been well described in the literature. However, the reported prevalence of HSCR associated with CAKUT seems to be underestimated. The aim of this systematic review was to determine the prevalence of this association and show its relationship to other syndromes. METHODS: A systematic literature search was conducted for relevant articles published between 1955 and 2014. Two online databases were searched for the terms "Hirschsprung's disease", "congenital anomalies of the kidney and urinary tract", "urogenital anomalies" and "urological anomalies". All published studies containing adequate clinical data were included. Resulting publications were reviewed for epidemiology, genetic testing, operative treatment and morbidity. Reference lists were screened for additional cases. RESULTS: A total of 32 articles reported 222 cases of HSCR associated with either CAKUT, "urological" or "urogenital" anomalies from 1955 to 2014. Gender was reported in a total of 68 cases, with 54 (79 %) males and 14 (21 %) females. Extent of aganglionosis was reported in 67 cases and included classical rectosigmoid disease in 38, long-segment aganglionosis in 12, total colonic aganglionosis in 12 and total intestinal aganglionosis in 5 patients. 18 articles reported 204 cases of either CAKUT, "urological" or "urogenital" anomalies in a case series of 5.693 HSCR patients, resulting in an overall prevalence of 3.6 % of this association. Within this collective of 18 studies only seven were, regardless of the date of publication compatible with CAKUT criteria introduced and published in the late 1990 s. These seven studies reported a total of 72 patients with associated CAKUT among 757 HSCR patients resulting in a prevalence of 9.5 %. After introduction of the CAKUT acronym, only three studies specifically investigated the association of HSCR and CAKUT stating a prevalence of 14.3 % resulting in an almost fivefold increase compared to the reported prevalence of HSCR and associated urological and urogenital anomalies. The remaining 14 publications reported 18 single cases of HSCR patients with associated CAKUT phenotypes. Of these 18 cases, 11 (61 %) cases were associated with other syndromes or syndromatic features or reported chromosomal anomalies. CONCLUSION: This review confirms that the recognition of CAKUT in HSCR patients has been underestimated in the past. The results suggest that when confronted with HSCR in a patient, a thorough urological investigation may be indicated. The high prevalence of associated syndromes in HSCR with CAKUT may further suggest a syndromic association.

Upregulation of serotonin-receptor-2a and serotonin transporter expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and pulmonary hypertension (PH). PH is characterized by structural changes resulting in vascular remodeling. Serotonin, a potent vasoconstrictor, plays a central role in the development of PH. It exerts its constricting effects on the vessels via Serotonin receptor 2A (5-HT2A) and induces pulmonary smooth muscle cell proliferation via the serotonin transporter (5-HTT). This study was designed to investigate expressions of 5-HT2A and 5-HTT in the pulmonary vasculature of rats with nitrofen-induced CDH. METHODS: Rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n=32). Pulmonary RNA was extracted and mRNA level of 5HT2A was determined by qRT-PCR. Protein expression of 5HT2A and 5-HTT was investigated by western blotting. Confocal immunofluorescence double-staining for 5-HT2A, 5-HTT, and alpha smooth muscle actin were performed. RESULTS: Pulmonary 5-HT2A gene expression levels were significantly increased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy confirmed increased pulmonary protein expression in CDH lungs compared to controls. CONCLUSION: Increased gene and protein expression of 5HT2A and 5-HTT in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that 5HT2A and 5-HTT are important mediators of PH in nitrofen-induced CDH.

Prenatal administration of retinoic acid increases the trophoblastic insulin-like growth factor 2 protein expression in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: The high mortality rate in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH). Insulin-like growth factor 2 (IGF2) is an important regulator of fetal growth. The highest levels of IGF2 expression are found in the placenta, which are negatively regulated by decidual retinoid acid receptor alpha (RARα). It has been demonstrated that prenatal administration of retinoic acid (RA) suppresses decidual RARα expression. Previous studies have further shown that prenatal administration of RA can reverse PH in nitrofen-induced CDH model. In IGF2 knockout animals, low levels of IGF2 are associated with decreased placental growth and PH. We therefore hypothesized that nitrofen decreases trophoblastic IGF2 expression and prenatal administration of RA increases it through decidual RARα in the nitrofen-induced CDH model. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given intraperitoneally on D18, D19 and D20. Fetuses were harvested on D21 and divided into three groups: control, CDH and nitrofen+RA. Immunohistochemistry was performed to evaluate decidual RARα and trophoblastic IGF2 expression. Protein levels of IGF2 in serum, intra-amniotic fluid and left lungs were measured by enzyme-linked immunosorbent assay. RESULTS: Significant growth retardation of placenta and left lungs was observed in the CDH group compared to control and nitrofen+RA group. Markedly increased decidual RARα and decreased IGF2 immunoreactivity were found in the CDH group compared to control and nitrofen+RA group. Significantly decreased IGF2 protein levels were detected in serum, intra-amniotic fluid and left lungs in the CDH group compared to control and nitrofen+RA group. CONCLUSION: Our findings suggest that nitrofen may disturb trophoblastic IGF2 expression through decidual RARα resulting in retarded placental growth and PH in the nitrofen-induced CDH. Prenatal administration of RA may promote lung and placental growth by increasing trophoblastic IGF2 expression.

ROCK inhibitor (Y-27632) disrupts somitogenesis in chick embryos.

AIM: In chick embryos, administration of cadmium (Cd) induces ventral body wall defects (VBWD) similar to human omphalocele. It has been shown that failure of proper VBW formation may be due to disruption of somite development during early embryogenesis. In the VBWD chick model, Cd causes abnormal cell death in the somitic region resulting in improperly developed somites and tortuosity of the neural tube. However, the exact molecular mechanisms leading to VBWD still remain unclear. Wnt signaling is crucial during embryogenesis and plays a key role in normal somite formation. The Rho-associated coiled-coil containing protein kinase (ROCK) is involved in the non-canonical Wnt pathway which controls actin cytoskeleton assembly and cell contractility, and contributes to several developmental processes including somitogenesis. ROCK gene expression levels have recently been reported to be significantly decreased in the Cd-induced VBWD chick model. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) in the absence of Cd disrupts somitogenesis and could contribute to the development of VBWD during early embryogenesis. METHODS: After 60 h of incubation chick embryos were transferred from eggs to culture dishes containing 20 µM of Y-27632 for experimental group (Y-27, n = 22) or chick saline for controls (n = 14). Following 24 h in the incubator they were assessed for stage development and gross abnormalities in morphology using the dissecting microscope. Western blot was performed to confirm Y-27632 inhibition of ROCK downstream signaling using an antibody against phosphorylated cofilin-2. RESULTS: 20 (90.9 %) embryos from Y-27 group and all controls were alive at examination. Morphological abnormalities were detected in 14 (70 %) Y-27 embryos. Somites appeared improperly developed, flattened in the cranio-caudal direction, and elongated in transverse direction in relation to controls. Chick embryos in Y-27 also presented with tortuosity of the neural tube in the lumbosacral region. Western blot analysis showed inhibition of cofilin-2 phosphorylation in affected embryos in comparison to controls. CONCLUSION: Our study provides evidence that ROCK inhibitor results in the disruption of normal somitogenesis in chick embryos which may contribute to the failure of fusion of the anterior abdominal wall causing VBWD.

Increased trophoblastic apoptosis mediated by neutrophil gelatinase-associated lipocalin (NGAL) activation in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Retinoids play a key role in fetal lung development. It has been suggested that the maternal-fetal retinol transport is disrupted by trophoblastic apoptosis. The mechanism underlying nitrofen-induced apoptosis in placenta is not fully understood. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in the fetal part of the maternal-fetal interface. NGAL is part of the immune barrier and serves primarily as a transport protein transferring biologically hazardous molecules in a safe and controlled way. It has been shown that over-activation of NGAL induces apoptosis. We hypothesized that increased placental NGAL expression induces trophoblastic apoptosis in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Placenta harvested on D21 and divided into two groups: control and nitrofen with CDH. Immunohistochemistry was performed to evaluate trophoblasts (by cytokeratin expression), NGAL expression, and apoptotic trophoblastic cells (using TUNEL assay). Total RNA was extracted from each placenta and the relative mRNA expression levels of NGAL were analyzed using RT-PCR. RESULTS: Immunohistochemistry showed NGAL immunoreactivity both in control and CDH in the fetal part of the fetal-maternal interface of placenta. Markedly increased NGAL expression was detected in CDH group compared to controls. Relative mRNA expression levels of NGAL gene were significantly increased in the CDH group compared to control in the placenta (5.924 ± 0.93 vs. 1.895 ± 0.54, p < 0.001). Markedly increased numbers of apoptotic trophoblastic cells were seen in the maternal-fetal interface in the CDH group compared to controls. CONCLUSIONS: NGAL activation may lead to increased trophoblastic apoptosis in the maternal-fetal interface in the nitrofen model of CDH. These changes may therefore cause disturbance in maternal-fetal retinol transport affecting fetal lung morphogenesis.