Laparoscopic versus Open Inguinal Hernia Repair Is Feasible in Infants with Caudal Anesthesia and Spontaneous Respiration.

INTRODUCTION: Minimally invasive surgery (i.e., laparoscopy) and minimally invasive anesthesia (i.e., caudal anesthesia with spontaneous respiration) have separately shown benefits for inguinal hernia repair in infants, yet to what degree these techniques can be combined remains unknown. This study investigated whether laparoscopy impacts the feasibility of performing caudal anesthesia with spontaneous respiration in infants. METHODS: Prospectively collected data of all infants less than 12 months old and over 3 kg weight who underwent laparoscopic indirect hernia repair (LAP) at our department from 2019 to 2021 were compared with a historical control-matched group of infants who underwent open repair (OPEN) from 2017 to 2021. We assessed the patients' characteristics, anesthesia, and surgical data as well as intra- and postoperative complications. RESULTS: A total of 87 infants were included (LAP n = 29, OPEN n = 58). Caudal anesthesia with spontaneous respiration was feasible in 62.1% of cases (LAP n = 55.2%, OPEN n = 65.5%; nonsignificant). Neither group registered anesthetic intra- or postoperative complications. Sedatives were utilized in 97% of LAP patients versus 56.9% of OPEN patients (p < 0.00001). The airway was secured with a laryngeal mask in 89.7% of patients during LAP versus 41.4% during OPEN (p < 0.00001). No significant differences were found regarding the use frequency of opioids (48.3% LAP vs. 34.5% OPEN; nonsignificant) or neuromuscular blockers (6.9% LAP vs. 5.2% OPEN; nonsignificant). CONCLUSION: This is the first comparative study on caudal anesthesia and spontaneous respiration in infants undergoing laparoscopic versus open inguinal hernia surgery. Laparoscopy increased the need for ventilatory support and sedatives but did not significantly impair the feasibility of caudal anesthesia and spontaneous respiration.

Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes-Brocks syndrome 2.

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1-/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

Vena Cava Thrombosis after Congenital Diaphragmatic Hernia Repair: Multivariate Analysis of Potential Risk Factors.

INTRODUCTION: The treatment of newborns with congenital diaphragmatic hernia (CDH) is associated with a significant complication rate. Information on major thrombotic complications and their incidence in newborns with CDH is lacking. The aims of our analysis were to evaluate the frequency of vena cava thrombosis and to determine its predictors within a consecutive series of patients with CDH. MATERIALS AND METHODS: We retrospectively analyzed charts of all neonates of our department that underwent CDH repair from 2007 to 2021, focusing on vena cava thrombosis. Vena cava thrombosis was diagnosed sonographically and classified as complete or partial venous occlusion. Complete occlusion was confirmed by cavography. Variables evaluated were CDH side, liver position, central vein line, surgical approach, and extracorporeal membrane oxygenation (ECMO). Univariate and multivariate tests were utilized. RESULTS: Among 57 neonates who underwent CDH repair, vena cava thrombosis was diagnosed in 14 (24.6%), seven of whom had complete occlusion of the vena cava. Factors associated with vena cava thrombosis were femoral or saphenous venous catheter (p = 0.044), right sided CDH (p = 0.027) and chylothorax (p < 0.0001). ECMO was not associated with vena cava thrombosis. Seven patients (50%) with vena cava thrombosis were treated interventionally with angioplasty and seven (50%) conservatively with anticoagulation only. Mortality was not higher in patients with compared with patients without vena cava thrombosis. CONCLUSION: The incidence of vena cava thrombosis in newborns with CDH in our series is high. Routine postoperative abdominal sonography focusing on vena cava thrombosis is mandatory in all patients with CDH. Patients who developed vena cava thrombosis were more likely to develop chylothorax after CDH repair. Considering the good outcome of medical therapy of partial vena cava thrombosis, it may be discussed whether low dose anticoagulation may be provided to all newborns with CDH.

Appendiceal Carcinoids in Children-Prevalence, Treatment and Outcome in a Large Nationwide Pediatric Cohort.

Background and Objectives: Appendiceal carcinoids are rare neuroendocrine tumors and mainly found incidentally during histopathological examination following appendectomy. This observational cohort study was performed to determine the prevalence, treatment modalities and outcomes in children diagnosed with an appendiceal carcinoid tumor. Materials and Methods: Data from the largest German statutory health insurance "Techniker Krankenkasse" were analyzed within an 8-year period: January 2010 to December 2012 and January 2016 to December 2020. Patient characteristics, surgical technique, type of surgical department, diagnostic management, and postoperative morbidity were analyzed. Results: Out of 40.499 patients following appendectomy, appendiceal carcinoids were found in 44 children, resulting in a prevalence of 0.11%. Mean age at appendectomy was 14.7 (±2.6) years. Laparoscopic approach was performed in 40 (91%) cases. Right-sided hemicolectomy was performed in 8 (18%) patients. Additional diagnostic work-up (CT and MRI) was recorded in 5 (11%) children. Conclusions: This large nationwide pediatric study shows that 1 in 1000 patients was found to have a neuroendocrine tumor of the appendix (prevalence 0.11%), emphasizing its low prevalence in the pediatric age group. The majority of patients were treated with appendectomy only. However, treatment modalities are still variable. Longer follow-up analyses are needed to evaluate published guidelines and recommendations to aim for a limited surgical approach.

Vacuum-assisted closure (VAC) prevents wound dehiscence following posterior sagittal anorectoplasty (PSARP): An exploratory case-control study.

BACKGROUND: Wound dehiscence (WD) of the anocutaneous anastomosis or perineal body after posterior sagittal anorectoplasty (PSARP) is common. We aimed to evaluate the efficacy of a perineal vacuum-assisted closure (VAC) for prevention of WD following repair of anorectal malformations (ARM) with rectoperineal and rectovestibular fistula. METHODS: A retrospective dual-center case-control study of children undergoing PSARP without colostomy between 2011 and 2019 was performed. The VAC group received preoperative bowel preparation (PBP), postoperative application of a VAC, loperamide (only Location A), intravenous antibiotics (IA), and total parenteral nutrition (TPN). The non-VAC group underwent PBP, loperamide (Location A), IA, and TPN without VAC. Primary outcome was WD at the anocutaneous anastomosis or reconstructed perineal body within the first 14 days after surgery. RESULTS: The study population included 18 patients (VAC group) and 20 children (non-VAG group) with rectoperineal and rectovestibular fistula. The incidence of WD in the VAC group was 0% compared to 25% in the non-VAC group (0/18 vs. 5/20, p = 0.04). No VAC related complications occurred. CONCLUSION: Postoperative application of a VAC embedded in a perioperative treatment protocol has the potential to prevent wound dehiscence of the neoanus and reconstructed perineal body following PSARP. TYPE OF STUDY: Case-control study. LEVEL OF EVIDENCE: Level III.

Severity grading of unexpected events in paediatric surgery: evaluation of five classification systems and the Comprehensive Complication Index (CCI®).

BACKGROUND: Postoperative adverse events may be associated with substantial morbidity and mortality. Numerous severity grading systems for these events have been introduced and validated but have not yet been systematically applied in paediatric surgery. This study aimed to analyse the advantages and disadvantages of these classifications in a paediatric cohort. METHODS: Unexpected events associated with interventional or organizational problems in the department of paediatric surgery during 2017-2020 were prospectively documented daily for all children. Events were classified according to the Clavien-Dindo grading system during monthly morbidity and mortality conferences. All events were also classified according to five additional grading systems: T92, contracted Accordion, expanded Accordion, Memorial Sloan Kettering Cancer Center, and Comprehensive Complication Index (CCI)®. RESULTS: Of 6296 patients, 673 (10.7 per cent) developed adverse events and 240 (35.7 per cent) had multiple events. Overall, 1253 adverse events were identified; of these, 574 (45.2 per cent) were associated with surgical or medical interventions and 679 (54.8 per cent) included organizational problems. The grading systems demonstrated high overall correlation (rpears = 0.9), with minor differences in sentinel events. The Clavien-Dindo classification offered the most detailed assessment. However, these details had only limited additional value. The CCI® scores were correlated with other grading systems (rpears = 0.9) and were useful in analysing multiple events within individual patients. CONCLUSION: Grading systems demonstrated similar scoring patterns for minor and sentinel events, with none being superior for unexpected events in children. However, the CCI® can be a major improvement in assessing morbidity in patients with multiple events. Its use is therefore recommended in prospective studies on paediatric surgery.

Ultrasound Monitoring after Pelvis-Sparing Dismembered Pyeloplasty: High Sensitivity and Low Specificity for the Success of Operation.

INTRODUCTION: A decrease in the anteroposterior diameter (APD) of the renal pelvis on ultrasound has been postulated to be indicative of sufficient pelvic drainage after pyeloplasty. Traditionally, pyeloplasty is combined with a reduction of the renal pelvis. We have recently demonstrated that resection of the pelvis during pyeloplasty is not necessary. We aimed to evaluate the efficacy of ultrasound APD measurements during follow-up to identify sufficient pelvic drainage in these patients. MATERIALS AND METHODS: Data from children (0-16 years) who underwent pelvis-sparing pyeloplasty in our institution from 2007 to 2018 were analyzed retrospectively. We included only those patients for whom pre- and postoperative ultrasound and renal scan data were available. Patients with a decrease versus patients with an increase in APD were analyzed with regard to urinary drainage and reoperation. RESULTS: Seventy-three patients who underwent follow-up at a mean of 3 months after operation were included; 61 showed a decrease in APD. Renal scan showed sufficient urinary drainage in 58 of them, with none requiring reoperation. Twelve patients had an increase in APD. Six of these showed free urinary drainage on renal scan; another six showed insufficient drainage, of whom five required reoperation. The positive predictive value of a decrease in APD was 1, and the negative predictive value of increase in APD was 0.42. CONCLUSION: To our knowledge, this is the first study evaluating the efficacy of ultrasound measurements to identify patients with decompensated urinary drainage during early follow-up after pyeloplasty with pelvis sparing. Post- versus preoperative decrease in renal pelvis diameter appears to be sufficient to rule out recurrence of obstruction. Renal scan seems to be indicated only in cases with post- versus preoperative increase in the APD of the renal pelvis on ultrasound.

Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia.

INTRODUCTION: Extensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone. METHODS: In a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC. In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC. RESULTS: Increased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control). CONCLUSION: Prenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.

Downregulated Elastin Microfibril Interfacer 1 Expression in the Pulmonary Vasculature of Experimental Congenital Diaphragmatic Hernia.

AIM: Pulmonary hypertension (PH) is a severe complication of congenital diaphragmatic hernia (CDH). Transforming growth factor-ß (TGFß) signaling is suggested to be involved in PH development by regulating embryonic angiogenesis, cell proliferation, and cell differentiation. Altered TGFß signaling has been demonstrated in experimental CDH lungs. Elastin microfibril interfacer 1 (Emilin-1) is an extracellular matrix glycoprotein expressed in endothelial and vascular smooth muscle cells and known to regulate TGFß processing and arterial diameter. We designed this study to investigate the pulmonary vascular expression of Emilin-1 in nitrofen-induced CDH rats. MATERIALS AND METHODS: Following ethical approval (REC913b, REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH group and control group. Quantitative real-time polymerase chain reaction (n = 11 each group), Western blot analysis, and confocal microscopy were used to determine the gene and protein expression of Emilin-1. MAIN RESULTS: Relative Emilin-1 messenger RNA (ribonucleic acid) levels were significantly downregulated in CDH lung tissue compared with controls (CDH: 0.043 ± 0.003; control: 0.067 ± 0.004; p < 0.001). Western blotting confirmed the decreased pulmonary Emilin-1 protein expression in CDH lungs. Confocal microscopy demonstrated a markedly diminished expression of Emilin-1 in the CDH pulmonary vasculature compared with controls. CONCLUSION: To our knowledge, this study demonstrates for the first time a decreased Emilin-1 gene and protein expression in the pulmonary vasculature of nitrofen-induced CDH. Emilin-1 deficiency through its interaction with TGFß may result in abnormal vascular remodeling resulting in PH in this model.

Non-invasive test using palmitate in patients with suspected fatty acid oxidation defects: disease-specific acylcarnitine patterns can help to establish the diagnosis.

BACKGROUND: The aim of the present study was to establish a non-invasive, fast and robust enzymatic assay to confirm fatty acid oxidation defects (FAOD) in humans following informative newborn-screening or for selective screening of patients suspected to suffer from FAOD. MATERIAL/METHODS: The reliability of this method was tested in whole blood from FAOD patients with specific enzymatic defects. Whole blood samples were assayed in 30 medium chain- (MCADD, age 0 to 17 years), 6 very long chain- (VLCADD, age 0 to 4 years), 6 long chain hydroxy- (LCHAD, age 1 to 6 years), 3 short chain- (SCADD, age 10 to 13 years) acyl-CoA-dehydrogenase- and 2 primary carnitine transporter deficiencies (CTD, age 3 to 5 years). Additionally, 26 healthy children (age 0 to 17 years) served as controls. Whole blood samples were incubated with stable end-labeled palmitate; labeled acylcarnitines were analyzed by tandem mass spectrometry and compared with controls and between patient groups (Mann-Whitney Rank Sum Test). Concentrations of specific labeled acylcarnitine metabolites were compared between particular underlying MCADD- (ANOVA), VLCADD- and LCHADD- genetic variants (descriptive data analysis). RESULTS: 11 different acylcarnitines were analyzed. MCADD- (C8-, C10-carnitine, C8/C10- and C8/C4-carnitine), VLCADD- (C12-, C14:1-, C14:2-carnitine, C14:1/C12- and C14:2/C12-carnitine), LCHADD (C16-OH-carnitine) as well as CTD- deficiency (sum of all acylcarnitines) samples could be clearly identified and separated from control values as well as other FAOD, whereas the sum of all acylcarnitines was not conclusive between FAOD samples. Furthermore, C4- (SCADD), C14- (VLCADD) and C14-OH-carnitines (LCHADD) were discriminating between the FAOD groups. Metabolic parameters did not differ significantly between underlying MCADD variants; similar results could be observed for VLCADD- and LCHADD- variants. CONCLUSION: This functional method in whole blood samples is relatively simple, non-invasive and little time consuming. It allows to identify MCADD-, VLCADD-, LCHADD- and carnitine transporter deficiencies. The genetic phenotypes of one enzyme defect did not result in differing acylcarnitine patterns in MCADD, VLCADD or LCHADD in vitro.

Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypertension (PH) is a common complication of congenital diaphragmatic hernia (CDH). Voltage-gated potassium channels KCNQ1, KCNQ4, and KCNQ5 are expressed by rodent pulmonary artery smooth muscle cells, contributing to their vascular tone. We hypothesized that KCNQ1, KCNQ4, and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH rats. METHODS: After ethical approval (REC913b), time-pregnant rats received nitrofen or vehicle on gestational day (D)9. D21 fetuses were divided into CDH and control group (n=22). QRT-PCR and western blotting were performed to determine gene and protein expression of KCNQ1, KCNQ4, and KCNQ5. Confocal microscopy was used to detect these proteins in the pulmonary vasculature. RESULTS: Relative mRNA level of KCNQ5 (p=0.025) was significantly downregulated in CDH lungs compared to controls. KCNQ1 (p=0.052) and KCNQ4 (p=0.574) expression was not altered. Western blotting confirmed the decreased pulmonary KCNQ5 protein expression in CDH lungs. Confocal-microscopy detected a markedly diminished KCNQ5 expression in pulmonary vasculature of CDH fetuses. CONCLUSIONS: Downregulated pulmonary expression of KCNQ5 in CDH lungs suggests that this potassium channel may play an important role in the development of PH in this model. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. LEVEL OF EVIDENCE: 2b (Centre for Evidence-Based Medicine, Oxford).

Imbalance of NFATc2 and KV1.5 Expression in Rat Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia.

Aim of the Study Nuclear factor of activated T-cell (NFATc2), a Ca2+/calcineurin-dependent transcription factor, is reported to be activated in human and animal pulmonary hypertension (PH). KV1.5, a voltage-gated K+ (KV) channel, is expressed in pulmonary artery smooth muscle cells (PASMC) and downregulated in PASMC in patients and animals with PH. Furthermore, activation of NFATc2 downregulates expression of KV1.5 channels, leading to excessive PASMC proliferation. The aim of this study was to investigate the pulmonary vascular expression of NFATc2 and KV1.5 in rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Materials and Methods After ethical approval, time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day 9 (D9). When sacrificed on D21, the fetuses (n = 22) were divided into CDH and control groups. Using quantitative real-time polymerase chain reaction and western blotting, we determined the gene and protein expression of NFATc2 and KV1.5. Confocal microscopy was used to detect both proteins in the pulmonary vasculature. Results Relative mRNA levels of NFATc2 were significantly upregulated and KV1.5 levels were significantly downregulated in CDH lungs compared with controls (p < 0.05). Western blotting confirmed the imbalanced pulmonary protein expression of both proteins. An increased pulmonary vascular expression of NFATc2 and a diminished expression of KV1.5 in CDH lungs compared with controls were seen in confocal microscopy. Conclusions This study demonstrates for the first time an altered gene and protein expression of NFATc2 and KV1.5 in the pulmonary vasculature of nitrofen-induced CDH. Upregulation of NFATc2 with concomitant downregulation of KV1.5 channels may contribute to abnormal vascular remodeling resulting in PH in this model.

Decreased Endoglin expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia rat model.

AIM OF THE STUDY: Pulmonary hypertension (PH) remains a therapeutical challenge in neonates born with congenital diaphragmatic hernia (CDH). Endoglin (Eng), an auxiliary receptor component of the transforming growth factor ß (TGFß) signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodelling. Genetic studies have linked TGFß and Eng mutations to human arterial PH and other cardiovascular syndromes. Eng interacts with the TGFß receptors 1 and 2 (Tgfßr1, Tgfßr2). We designed this study to investigate the hypothesis that Eng is altered in the pulmonary vasculature of rats with nitrofen-induced CDH subjected to its interdependency with Tgfßr1 and Tgfßr2. METHODS: After ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received either nitrofen or olive oil on gestational day (D9). The foetuses (n = 22) were sacrificed and divided into CDH and control group on D21. Gene and protein expressions of Eng, Tgfßr1 and Tgfßr2 were assessed via qRT-PCR and western blotting. Immunofluorescence staining for Eng was combined with CD34 to evaluate Eng expression in the pulmonary vasculature. MAIN RESULTS: Relative mRNA levels of Eng, Tgfßr1 and Tgfßr2 were significantly downregulated in CDH lungs compared to controls (Eng CDH 0.341 ± 0.022, Eng Ctrl 0.471 ± 0.031, p = 0.0015; Tgfßr1 CDH 0.161 ± 0.008, Tgfßr1 Ctrl 0.194 ± 0.01, p = 0.0114; Tgfßr2 CDH 0.896 ± 0.099, Tgfßr2 Ctrl 1.379 ± 0.081, p = 0.0006) Western blotting confirmed the reduced pulmonary protein expression of these three proteins in the CDH lungs. A markedly diminished endothelial expression of Eng in the pulmonary vasculature of nitrofen-exposed foetuses compared to controls was seen in laser scanning confocal-microscopy. CONCLUSION: This study demonstrates for the first time a reduced expression of Endoglin in the pulmonary vasculature of nitrofen-induced CDH. Abnormal Eng/Tgfßr1/Tgfßr2 signalling may contribute to impaired vascular remodelling and development of PH in this CDH animal model.

The Role of Activin Receptor-Like Kinase 1 Signaling in the Pulmonary Vasculature of Experimental Diaphragmatic Hernia.

AIM: The high morbidity and mortality in newborn infants diagnosed with congenital diaphragmatic hernia (CDH) is widely recognized to be due to pulmonary hypoplasia and persistent pulmonary hypertension (PH). The underlying structural and molecular pathomechanisms causing PH are not fully understood. Recently, activin receptor-like kinase 1 (ALK-1), an endothelial cell (EC) receptor, has been implicated in the pathogenesis of PH. ALK-1 transmits signals via a Smad pathway stimulating EC proliferation and migration leading to structural lung remodeling consecutively resulting in PH. Increased pulmonary expression of ALK-1 has been reported in patients with severe PH as well as in experimental models of PH. We designed this study to investigate the hypothesis that pulmonary ALK-1 expression is 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 polymerase chain reaction, Western blotting, and confocal-immunofluorescence microscopy were performed to determine pulmonary gene and protein expression as well as vascular localization of expressed ALK-1. RESULTS: Pulmonary gene expression levels of ALK-1 were significantly upregulated in nitrofen-treated lung tissue compared with controls. Western blotting showed increased pulmonary protein expression for ALK-1 in the CDH group when compared with control lung tissue. Confocal microscopy demonstrated markedly increased medial and adventitial thickness of pulmonary arteries in the CDH group and revealed increased ALK-1 protein expression of the pulmonary vasculature of CDH pups compared with controls. CONCLUSION: Upregulated gene and increased protein expression of ALK-1 in the pulmonary vasculature of nitrofen-induced CDH suggest that increased expression of ALK-1 may play a crucial role in the molecular pathogenesis of vascular remodeling induced PH in experimental CDH.

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.

Kif7 expression is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Developmental mutations that inhibit diaphragmatic and pulmonary mesenchyme formation have been shown to cause congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia (PH). Kinesin family member 7 (Kif7) plays a crucial role in diaphragmatic and pulmonary morphogenesis by controlling proliferation of mesenchymal cells. Loss of Kif7 has been reported to result in diaphragmatic defects and PH. We hypothesized that diaphragmatic and pulmonary Kif7 expression is decreased in the nitrofen-induced CDH model. METHODS: Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms and lungs were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Kif7 were analyzed by qPCR. Immunohistochemical staining was performed to evaluate Kif7 protein expression. RESULTS: Relative mRNA expression of Kif7 was significantly reduced in pleuroperitoneal folds (D13), developing diaphragms and lungs (D15), and fully muscularized diaphragms and differentiated lungs (D18) of nitrofen-exposed fetuses compared to controls. Immunoreactivity/immunofluorescence of Kif7 was markedly decreased in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. CONCLUSION: Decreased Kif7 expression during diaphragmatic development may interfere with mesenchymal cell proliferation, leading to defective pleuroperitoneal folds, and resulting in diaphragmatic defects and associated PH in the nitrofen-induced CDH model.

Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia.

PURPOSE: Signal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. RESULTS: Pulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. CONCLUSION: Markedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH.

Increased pulmonary vascular expression of receptor for advanced glycation end products (RAGE) in experimental congenital diaphragmatic hernia.

AIM OF THE STUDY: Persistent pulmonary hypertension (PH) continues to be a major cause of high mortality in congenital diaphragmatic hernia (CDH). The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin protein family. Recently, RAGE has been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodeling in experimental PH. RAGE has been reported to be highly upregulated in lung tissue of patients with severe PH. We designed this study to investigate the hypothesis that RAGE expression is 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 polymerase chain reaction, Western blotting, and confocal immunofluorescence were performed. MAIN RESULTS: Pulmonary RAGE gene expression levels were significantly increased in nitrofen-induced CDH compared to controls (p<0.003). Western blotting and confocal microscopy revealed increased pulmonary RAGE protein expression in CDH compared to controls. CONCLUSION: This study provides striking evidence of increased gene and protein expression of RAGE in the pulmonary vasculature of nitrofen-induced CDH, suggesting that increased expression of RAGE may play a role in the pathogenesis of PH in nitrofen-induced CDH.