Transuterine Fetal Tracheal Occlusion Model in Mice.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

When primary repair is not enough: a comparison of synthetic patch and muscle flap closure in congenital diaphragmatic hernia?

PURPOSE: Primary closure is often inadequate for large congenital diaphragmatic hernia (CDH) and necessitates repair by prosthetic patch or autologous muscle flap. Our aim was to evaluate outcomes of open patch versus flap repair, specifically diaphragmatic reherniation. METHODS: A retrospective review (IRB #2017-6361) was performed on all CDH patients repaired from 2005 to 2016 at a single academic children's hospital. Patients were excluded from final analysis if they had primary or minimally invasive repair, expired, or were lost to follow-up. RESULTS: Of 171 patients, 151 (88.3%) survived to discharge, 9 expired after discharge and 11 were lost to follow up, leaving 131 (86.8%) long-term survivors. Median follow-up was 5 years. Open repair was performed in 119 (90.8%) of which 28 (23.5%) underwent primary repair, 34 (28.6%) patch repair, and 57 (47.9%) flap repair. Overall, 6/119 (5%) patients reherniated, 1/28 (3.6%) in the primary group, 3/34 (8.8%) in the patch group, and 2/57 (3.5%) in the flap group. Comparing prosthetic patch to muscle flap repair, there was no significant difference in the number of patients who recurred nor time to reherniation (3 vs. 2, p = 0.295; 5.5 ± 0.00 months vs. 53.75 ± 71.06 months, p = 0.288). One patient in the patch group recurred twice. CONCLUSIONS: Both muscle flap and patch repair of large CDH are feasible and durable with a relatively low risk of recurrence.

Left congenital diaphragmatic hernia-associated musculoskeletal deformities.

AIM: With the advancement in the treatment strategies of congenital diaphragmatic hernia (CDH), there is an increase in the survival rates. This fact leads to an increase in the morbidity and extrapulmonary complications in the long term such as failure to thrive, hernia recurrence, neurodevelopmental delay, gastrointestinal problems, and musculoskeletal anomalies. Herein, we aim to investigate the association between the long-term musculoskeletal complications in CDH patients regarding the defect size, repair type, and perinatal parameters. METHODS: After Institutional Review Board approval was obtained (2017-6361), a retrospective chart review was performed on CDH patients from 2003 to 2016. Patients who were operated due to left-sided isolated congenital diaphragmatic hernia and survived to date were included in the study. Data were collected on demographics, preoperative characteristics, operative interventions, and postoperative outcomes. Statistical analysis was performed with IBM SPSS Statistics 20.0.0 (Chicago, IL). RESULTS: There were 98 patients with left CDH of whom 33 (33.7%) had primary repair, 25 (25.5%) had patch repair, and 40 (40.8%) had muscle flap repair. The median age of the patients was 6.00 ± 3.83 years. 45 patients (45.9%) had large diaphragmatic defects, 28 patients (28.6%) had at least one type of musculoskeletal deformities, 2 of which were pectus carinatum, 16 were pectus excavatum, and 18 were scoliosis. CDH patients who had small diaphragmatic defects and repaired with a patch were less likely develop musculoskeletal deformities while who had primary abdominal closure after ventral hernia significantly have more pectus excavatum. CONCLUSION: Although there was a trend towards an increased risk of the pectus deformity and scoliosis in patients repaired with muscle flap, it did not reach statistical significance. There is a correlation between musculoskeletal deformities and the severity of the CDH.

Proteomic profiling of tracheal fluid in an ovine model of congenital diaphragmatic hernia and fetal tracheal occlusion.

Congenital diaphragmatic hernia (CDH) occurs in ~1:2,000 pregnancies and is associated with substantial morbidity and mortality. Fetal tracheal occlusion (TO) is an emerging therapy that improves lung growth and reduces mortality, although substantial respiratory compromise persists in survivors. In this study, we used tracheal fluid in a fetal sheep model of CDH with TO for proteomic analysis with subsequent validation of findings in sheep lung tissue. We found that the proteomic profiles of CDH tracheal fluid was most similar to control lung and CDH/TO lung most similar to TO lung. Among 118 proteins altered in CDH, only 11 were reciprocally regulated in CDH/TO. The most significantly altered pathways and processes were cell proliferation, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling, inflammation, and microtubule dynamics. CDH suppressed and TO promoted cell proliferation and AKT-related signaling cascades. By Western blot analysis and immunohistochemistry, epithelial PCNA and phosphorylated AKT were decreased in CDH and increased in TO and CDH/TO lungs. The Wnt target Axin2 was decreased threefold in CDH lung compared with control without a significant increase in CDH/TO lung. Cilia-related pathways were among the most dysregulated with CDH lung having a nearly twofold increase in acetylated α-tubulin and a relative increase in the number of ciliated cells. While TO improves lung growth and patient survival in CDH, the procedure substantially alters many processes important in lung development and cell differentiation. Further elucidation of these changes will be critical to improving lung health in infants with CDH treated with TO.

Fetal tracheal occlusion in mice: a novel transuterine method.

BACKGROUND: Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. MATERIALS AND METHODS: Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. RESULTS: Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. CONCLUSIONS: Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.

Role of intraluminal bowel echogenicity on prenatal ultrasounds to determine the anatomical level of intestinal atresia.

OBJECTIVE: To evaluate the correlation between different degrees of bowel intraluminal echogenicity showed by prenatal ultrasounds and the anatomic level of intestinal atresia. METHODS: We report three cases of intestinal atresia at different intestinal levels verified during the neonatal surgery with specific ultrasonographic prenatal features. Intensity of sonolucency was analyzed using the image-processing program ImageJ for quantitative measurements based on the gray-scale intensity values. RESULTS: A total of three cases are reported, a jejunal, an ileal and a colonic atresia. All cases showed intestinal dilatation. Both, jejunal and ileal atresia, showed two degrees of hypoechoic intestinal content, while colonic atresia showed hyperechogenic content dilated loop at prenatal ultrasound scan. CONCLUSIONS: We propose the use of prenatal ultrasounds echogenicity of intestinal dilated loop fluid content to help in determining the level of obstruction in bowel atresia. These are initial results, to be confirmed by a multicentric research with more cases.

Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

OBJECTIVES: Necrotizing enterocolitis is a severe multifactorial intestinal disorder that primarily affects preterm newborns, causing 20-40% mortality and morbidity. Intestinal fatty acid-binding protein has been reported to be a biomarker for the detection of intestinal injuries. Our aim was to assess intestinal tissue injury and the molecular expression of intestinal fatty acid-binding protein over time in a necrotizing enterocolitis model. METHODS: A total of 144 Newborn rats were divided into two groups: 1) Control, which received breastfeeding (n=72) and 2) Necrotizing Enterocolitis, which received formula feeding and underwent hypoxia and hypothermia (n=72). A total of six time points of ischemia (2 times a day for 3 days; 12 pups for each time point) were examined. Samples were collected for analysis of body weight, morphological and histological characteristics, intestinal weight, intestinal weight/body weight ratio, injury grade, and intestinal fatty acid-binding protein levels. RESULTS: Body and intestinal weights were lower in the Necrotizing Enterocolitis group than in the Control group (p<0.005 and p<0.0005, respectively). The intestinal weight/body weight ratio was higher in the Necrotizing Enterocolitis group than in the Control group (p<0.005) only at the sixth ischemia time point. The Necrotizing Enterocolitis group displayed higher expression of intestinal fatty acid-binding protein (p<0.0005) and showed greater tissue damage than the Control group. CONCLUSION: Intestinal fatty acid-binding protein was an efficient marker of ischemic injury to the intestine and a good correlation was demonstrated between the time of ischemic injury and the grade of intestinal injury.

Combined antenatal therapy with retinoic acid and tracheal occlusion in a rat model of congenital diaphragmatic hernia.

AIM: To investigate the effect of combined prenatal treatment with retinoic acid (RA) and tracheal occlusion (TO) on the pulmonary vascular morphology and expression of vascular endothelial growth factors (VEGF) and its receptors in a rat model of congenital diaphragmatic hernia (CDH). MATERIAL AND METHODS: Rats were given nitrofen at 9 days of gestation followed by no treatment (CDH), RA (CDH + RA), TO (CDH + TO), or both (CDH + RA + TO) (n = 16). We measured the median wall thickness of the pulmonary arterioles (MWT) and analyzed the expression of VEGF and its receptors (VEGFR1 and VEGFR2). RESULTS: Compared to control animals, CDH had increased MWT (44 ± 15 vs. 58 ± 7; p < 0.05) and decreased expression of VEGF, VEGFR1, and VEGFR2 (p < 0.05). Treatment with RA or TO alone, and RA + TO reduced the MWT (46 ± 9, 42 ± 11, 46 ± 8, respectively) and improved the expression of VEGF, VEGFR1, and VEGFR2 compared to CDH (p < 0.05). However, the combination of RA + TO did not confer additional benefit in the reduction of the MWT or in increasing the VEGF and its receptors compared to either treatment alone. CONCLUSION: Antenatal treatment with either RA or TO improved the MWT and expression of VEGF and its receptors in a CDH rat model. However, combined treatment with RA + TO was not superior to either treatment alone.

Myocardial effects of fetal endoscopic tracheal occlusion in lambs with CDH.

INTRODUCTION: Fetal endoscopic tracheal occlusion in congenital diaphragmatic hernia (CDH) may reduce pulmonary hypertension and ameliorate postnatal cardiac output. The effects of sustained early (ETO) and late (LTO) tracheal occlusion on left ventricular (LV) cells in the lamb model have not been described. MATERIALS AND METHODS: CDH was created in lambs at 70 days' gestation (term = 145 days). ETO (85 days) or LTO (105 days) was sustained till term. After cesarean section (140 days) fetuses were euthanized and hearts harvested. LV myocardial cells were studied by histological and immunofluorescence (TGF-beta 1, endothelin-1) assays in CDH, ETO, LTO, and the control group (two subjects per group). Small intramyocardial arteries were evaluated by traditional histology. RESULTS: LV myocardial histology in CDH and LTO was similar. ETO-induced LV myocardial cell enlargement and increased endothelin-1 and TGF-beta 1 staining; a weaker immunofluorescence signal was observed in LTO compared with ETO. Myocardial vascular wall thickness was greater in CDH than in controls. ETO was associated with a vascular wall thickness within the range of controls. CONCLUSION: With only two fetuses in each group, only an explorative evaluation was possible. The time point at which TO is performed seems to have an effect on cardiac morphology. Functional studies as well as confirmation in clinical samples are mandatory.

Prematurity and fetal lung response after tracheal occlusion in fetuses with severe congenital diaphragmatic hernia.

OBJECTIVE: To evaluate the independent association of fetal pulmonary response and prematurity to postnatal outcomes after fetal tracheal occlusion for congenital diaphragmatic hernia. METHODS: Fetal pulmonary response, prematurity (<37 weeks at delivery) and extreme prematurity (<32 weeks at delivery) were evaluated and compared between survivors and non-survivors at 6 months of life. Multivariable analysis was conducted with generalized linear mixed models for variables significantly associated with survival in univariate analysis. RESULTS: Eighty-four infants were included, of whom 40 survived (47.6%) and 44 died (52.4%). Univariate analysis demonstrated that survival was associated with greater lung response (p=0.006), and the absence of extreme preterm delivery (p=0.044). In multivariable analysis, greater pulmonary response after FETO was an independent predictor of survival (aOR 1.87, 95% CI 1.08-3.33, p=0.023), whereas the presence of extreme prematurity was not statistically associated with mortality after controlling for fetal pulmonary response (aOR 0.52, 95% CI 0.12-2.30, p=0.367). CONCLUSION: Fetal pulmonary response after FETO is the most important factor associated with survival, independently from the gestational age at delivery.

Effect of corticosteroids and lung ventilation in the VEGF and NO pathways in congenital diaphragmatic hernia in rats.

PURPOSE: The use of dexamethasone (Dx) stimulates growth, fetal lung maturation and can improve pulmonary hypertension in congenital diaphragmatic hernia (CDH). Our aim was to evaluate the effect of Dx on the lung after fetal pulmonary ventilation in the CDH rat model. METHODS: Some groups underwent prenatal treatment with dexamethasone (0.4 mg/kg) that was given at 18.5 gestational day (GD). Sprague-Dawley rat fetuses were divided into groups: control (C); ventilated control (CV); control exposed to dexamethasone (CDx); ventilated control exposed to dexamethasone (CVDx); congenital diaphragmatic hernia (CDH), ventilated CDH (CDHV), CDH exposed to dexamethasone (CDHDx) and ventilated CDH exposed to dexamethasone (CDHVDx). At 21.5 GD fetuses were delivered by C-section, weighed and ventilated for 30 min. We analyzed the lung morphometry by Masson's Trichrome stain, and VEGF, VEGFR1, VEGFR2 and NOS3 expression by immunohistochemistry. RESULTS: All fetuses with CDH, with or without prenatal dexamethasone showed lung and body weight lower than control fetuses (p < 0.05). All groups that received dexamethasone showed a decrease in the medial muscular layer of arterioles, the internal diameter of the air spaces (Lma) and length of parenchymal transection/airspace ratio (p < 0.05). In the immunohistochemistry, VEGF decreased more in CDHDV group (p < 0.05). VEGFR1 showed no difference, whereas VEGFR2 decreased significantly in the CDHDV group (p < 0.05). NOS3 increased in the group CDHDV (p < 0.05). CONCLUSION: The use of prenatal dexamethasone added to ventilation alters the VEGF and NO pathways.

Arabin cervical pessary to prevent preterm birth in severe twin-to-twin transfusion syndrome treated by laser surgery.

OBJECTIVES: To describe the outcome of patients with twin-to-twin transfusion syndrome and cervical length ≤ 25 mm, treated with laser and an Arabin cervical pessary. METHODS: Retrospective analysis of a consecutive series of all cases with severe twin-to-twin transfusion syndrome who underwent laser surgery: a group with cervical length above 25 mm (group A) and two groups who had a cervical length of 25 mm or less prior to the procedure. The first 8 cases (group B) were managed expectantly and the next 8 cases had a cervical pessary inserted immediately after laser surgery (group C). Gestational age at birth was the primary outcome. The secondary outcome was a composite one encompassing severe neonatal morbidity. RESULTS: The median gestational age at laser surgery was 20 weeks in all groups but the median gestational age at delivery was significantly higher in group C versus B (28 vs 32 weeks, p = 0.01). Severe neonatal morbidity was present in 18% in group C and 70% in group B (p < 0.01). CONCLUSION: Early results suggest a potential role for pessary use in prolonging gestation in cases with shortened cervix at the time of laser. A randomized trial to test this hypothesis should be performed.

Tracheobronchomegaly following intrauterine tracheal occlusion for congenital diaphragmatic hernia.

BACKGROUND: Fetuses with severe congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia may benefit from fetal endoluminal tracheal occlusion (FETO). Enlargement of the main bronchi and trachea appears to be a common complication of FETO. OBJECTIVE: To retrospectively evaluate the trachea and main bronchi of infants who underwent FETO for CDH and compare diameters with age-matched references. MATERIAL AND METHODS: Postnatal and follow-up chest radiographs were performed in seven children with unilateral CDH treated by FETO. Additional CT was performed in six of these (one neonate died before CT could be performed). Images were acquired from 3 days to 23 months of age. For each child, radiographs and CT images with optimal visualisation of the airways were selected for retrospective analysis. Tracheal and bronchial morphology was assessed by two experienced paediatric radiologists, and the diameters of these structures measured and compared with age-matched references. RESULTS: Mean diameters of the trachea and main bronchi were above the age-matched normal range in all patients, regardless of the side of the hernia or the degree of lung hypoplasia. CONCLUSION: Enlargement of the trachea and main bronchi appears following FETO and persists at least to the age of 5 years.

Congenital tumours involving the head, neck and central nervous system.

Congenital intracranial tumours are uncommon and differ from those occurring in older children in clinical presentation, imaging characteristics and prognosis. These tumours are often detected incidentally on routine prenatal US and/or fetal MRI. Hence, the paediatric radiologist should be familiar with the features of those lesions that should be included in the differential diagnosis. In general, the prognosis of these conditions is poor owing to large tumour size and the limitations of adjuvant therapy at such a young age. Congenital lesions involving the head and neck region require a meticulous imaging approach using both US and MRI techniques to better guide prenatal planning and fetal or neonatal surgical procedures.

[EXIT procedure in the management of severe foetal airway obstruction. the paediatric otolaryngologist's perspective]. / Procedimiento EXIT en el manejo de fetos con severo compromiso respiratorio. La perspectiva del otorrinolaringólogo pediátrico.

The ex-utero intrapartum treatment (EXIT) procedure is a technique designed to allow partial foetal delivery via caesarean section with establishment of a safe foetal airway by either intubation, bronchoscopy, or tracheostomy while foetal oxygenation is maintained through utero-placental circulation. The most common indication for the EXIT procedure is the presence of foetal airway obstruction, which is usually caused by a prenatal diagnosed neck mass. We report three cases of head and neck tumours with airway obstruction treated by means of EXIT and with different solutions in the management of the airway. With the involvement of Paediatric Otolaryngologists in EXIT, new indications and select variations from the standard EXIT protocol should be considered.