Congenital Diaphragmatic Hernia Model Research Articles

Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia

BackgroundThe severity of pulmonary hypoplasia is a main determinant of outcome for babies with congenital diaphragmatic hernia (CDH). Antenatal administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) has been shown to rescue morphological features of lung development in the rat nitrofen model of CDH. Herein, we evaluated whether AFSC-EV administration to fetal rats with CDH is associated with neonatal improvement in lung function. MethodsAFSC-EVs were isolated by ultracentrifugation and characterized by size, morphology, and canonical marker expression. At embryonic (E) day 9.5, dams were gavaged with olive oil (control) or nitrofen to induce CDH. At E18.5, fetuses received an intra-amniotic injection of either saline or AFSC-EVs. At E21.5, rats were delivered and subjected to a tracheostomy for mechanical ventilation (flexiVent system). Groups were compared for lung compliance, resistance, Newtonian resistance, tissue damping and elastance. Lungs were evaluated for branching morphogenesis and collagen quantification. ResultsCompared to healthy control, saline-treated pups with CDH had fewer airspaces, more collagen deposition, and functionally exhibited reduced compliance and increased airway resistance, elastance, and tissue damping. Conversely, AFSC-EV administration resulted in improvement of lung mechanics (compliance, resistance, tissue damping, elastance) as well as lung branching morphogenesis and collagen deposition. ConclusionsOur studies show that the rat nitrofen model reproduces lung function impairment similar to that of human babies with CDH. Antenatal administration of AFSC-EVs improves lung morphology and function in neonatal rats with CDH. Level of EvidenceN/A (animal and laboratory study).

Rescuing lung development through embryonic inhibition of histone acetylation.

A major barrier to the impact of genomic diagnosis in patients with congenital malformations is the lack of understanding regarding how sequence variants contribute to disease pathogenesis and whether this information could be used to generate patient-specific therapies. Congenital diaphragmatic hernia (CDH) is among the most common and severe of all structural malformations; however, its underlying mechanisms are unclear. We identified loss-of-function sequence variants in the epigenomic regulator gene SIN3A in two patients with complex CDH. Tissue-specific deletion of Sin3a in mice resulted in defects in diaphragm development, lung hypoplasia, and pulmonary hypertension, the cardinal features of CDH and major causes of CDH-associated mortality. Loss of SIN3A in the lung mesenchyme resulted in reduced cellular differentiation, impaired cell proliferation, and increased DNA damage. Treatment of embryonic Sin3a mutant mice with anacardic acid, an inhibitor of histone acetyltransferase, reduced DNA damage, increased cell proliferation and differentiation, improved lung and pulmonary vascular development, and reduced pulmonary hypertension. These findings demonstrate that restoring the balance of histone acetylation can improve lung development in the Sin3a mouse model of CDH.

Development of a 3D-printed neonatal congenital diaphragmatic hernia model and standardisation of intra-operative measurement

IntroductionThree-dimensional (3D) printing is frequently used for surgical simulation and training, however, no widely available model exists for neonatal congenital diaphragmatic hernia (CDH). The aim of this study was to develop a 3D-printed model of CDH and test interobserver variability in the simulated model for obtaining measurements of the diaphragmatic defect and ipsilateral diaphragm.MethodsA term fetal MRI (3.5 kg) of thorax, diaphragm and defect (15 mm × 5 mm) were delineated and segmented after parental consent to produce 3D-printed models. Consultant and trainee paediatric surgeons were invited to measure the posterior-lateral diaphragmatic defect and ipsilateral diaphragm. Mean measurement error was calculated (millimetres). Data are presented as median (range) and number/total (%).ResultsAn abdominal and thoracoscopic model were produced and tested by 52 participants (20 consultants and 32 trainees). Diaphragmatic defect via laparotomy measured 15 (10–20) mm (AP) × 16 (10–25) mm (ML) and thoracoscopically 14 (11–19) mm (AP) × 15 (11–22) mm (ML). Mean error per measurement was 4 (1–17) mm via laparotomy vs. 3 (0.5–9.5) mm thoracoscopically. Mean error was similar between consultants and trainees via laparotomy (4.3 vs. 3.9 mm, p = 0.70) and thoracoscopically (3 vs. 3 mm, p = 0.79). Error did not correlate with experience as operating surgeon via laparotomy (β = 13.0 [95% CI − 55.9 to 82.0], p = 0.71) or thoracoscopically (β = 1.4[95% CI − 6.4 to 9.2], p = 0.73.ConclusionsWe have designed and built simulation models for CDH repair via laparotomy and thoracoscopically. Operators can reliably measure the diaphragmatic defect and ipsilateral diaphragm, regardless of surgical experience and operative approach.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Prenatal treprostinil improves pulmonary arteriolar hypermuscularization in the rabbit model of congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a congenital malformation characterized by pulmonary hypoplasia, pulmonary hypertension, and cardiac dysfunction. Pulmonary hypertension represents the major cause of neonatal mortality and morbidity. Prenatal diagnosis allows assessment of severity and selection of foetal surgery candidates. We have shown that treprostinil, a prostacyclin analogue with an anti-remodelling effect, attenuates the relative hypermuscularization of the pulmonary vasculature in rats with nitrofen-induced CDH. Here we confirm these observations in a large animal model of surgically-created CDH. In the rabbit model, subcutaneous maternal administration of treprostinil at 150 ng/kg/min consistently reached target foetal concentrations without demonstrable detrimental foetal or maternal adverse effects. In pups with CDH, prenatal treprostinil reduced pulmonary arteriolar proportional medial wall thickness and downregulated inflammation and myogenesis pathways. No effect on alveolar morphometry or lung mechanics was observed. These findings provide further support towards clinical translation of prenatal treprostinil for CDH.

Lung proliferation is dependent on the duration not thetimepoint of tracheal occlusion in nitrofen rats with diaphragmatic hernia.

Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with congenital diaphragmatic hernia (CDH). Limited data are available regarding the effect of duration versus timepoint of TO. Our objective was to document the impact of TO on lung development in the near-term period in rats with nitrofen-induced CDH. Nitrofen was administered on embryonic day (ED)9 and fetal TO was performed on ED18.5, 19, or 20 (term=ED22). Sham-operated and untouched littermates served as controls. Lungs were harvested in 0.5-day steps and only fetuses with a left-sided CDH were included in further analyses. Healthy fetuses provided a reference for normal near-term lung development. Duration of TO in the nitrofen rat model for CDH predicts lung growth in terms of lung-body-weight ratio as well as an increased mRNA level of the proliferation marker Ki67. Longer TO also induced a more complex airway architecture. The timepoint of TO was not predictive of lung growth. In the nitrofen rat model of CDH, a longer period of TO leads to enhanced lung growth and more refined airway architecture.

Overactivated Epithelial NF-κB Disrupts Lung Development in Congenital Diaphragmatic Hernia.

Abnormal lung development is the main cause of morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH), a common birth defect (1:2500) of largely unknown pathobiology. Recent studies discovered that inflammatory processes, and specifically NF-κB associated pathways are enriched in human and experimental CDH. However, the molecular signaling of NF-κB in abnormal CDH lung development and its potential as a therapeutic target requires further investigation. Using sections and hypoplastic lung explant cultures from the nitrofen rat model of CDH and human fetal CDH lungs, we demonstrate that NF-κB and its downstream transcriptional targets are hyperactive during abnormal lung formation in CDH. NF-κB activity was especially elevated in the airway epithelium of nitrofen and human CDH lungs at different developmental stages. Fetal rat lung explants had impaired pseudoglandular airway branching after exposure to nitrofen, together with increased phosphorylation and transcriptional activity of NF-κB. Dexamethasone, the broad and clinically applicable anti-inflammatory NF-κB antagonist, rescued lung branching and normalized NF-κB signaling in hypoplastic lung explants. Moreover, specific NF-κB inhibition with curcumenol similarly rescued ex vivo lung hypoplasia and restored NF-κB signaling. Lastly, we showed that prenatal intraperitoneal dexamethasone administration to pregnant rat dams carrying fetuses with hypoplastic lungs, significantly improves lung branching and normalizes NF-κB in vivo. Our results indicate that NF-κB is aberrantly activated in human and nitrofen CDH lungs. Anti-inflammatory treatment with dexamethasone and/ or specific NF-κB inhibition should be investigated further as a therapeutic avenue to target lung hypoplasia in CDH.

Single-cell transcriptomic profiling of microvascular endothelial cell heterogeneity in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a neonatal anomaly that includes pulmonary hypoplasia and hypertension. We hypothesized that microvascular endothelial cell (EC) heterogeneity is different in CDH lungs and related to lung underdevelopment and remodeling. To test this, we evaluated rat fetuses at E21.5 in a nitrofen model of CDH to compare lung transcriptomes among healthy controls (2HC), nitrofen-exposed controls (NC) and nitrofen-exposed subjects with CDH. Single-cell RNA sequencing with unbiased clustering revealed 3 distinct microvascular EC clusters: a general population (mvEC), a proliferative population and a population high in hemoglobin. Only the CDH mvEC cluster had a distinct inflammatory transcriptomic signature as compared to the 2HC and NC endothelial cells, e.g. greater activation and adhesion of inflammatory cells and production of reactive oxygen species. Furthermore, CDH mvECs had downregulated Ca4, Apln and Ednrb gene expression. Those genes are markers for ECs important to lung development, gas exchange and alveolar repair (mvCa4+). mvCa4+ ECs were reduced in CDH (2HC [22.6%], NC [13.1%] and CDH [5.3%], p < 0.0001). Overall, these findings identify transcriptionally distinct microvascular endothelial cell clusters in CDH, including the distinctly inflammatory mvEC cluster and the depleted group of mvCa4+ ECs, which together may contribute to pathogenesis.

In utero delivery of miRNA induces epigenetic alterations and corrects pulmonary pathology in congenital diaphragmatic hernia

Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy in utero as gas exchange is managed by the placenta, but impaired lung function results in critical illness from the time a baby takes its first breath. MicroRNA (miR) 200b and its downstream targets in the TGF-β pathway are critically involved in lung branching morphogenesis. Here, we characterize the expression of miR200b and the TGF-β pathway at different gestational times using a rat model of CDH. Fetal rats with CDH are deficient in miR200b at gestational day 18. We demonstrate that novel polymeric nanoparticles loaded with miR200b, delivered in utero via vitelline vein injection to fetal rats with CDH results in changes in the TGF-β pathway as measured by qRT-PCR; these epigenetic changes improve lung size and lung morphology, and lead to favorable pulmonary vascular remodeling on histology. This is the first demonstration of in utero epigenetic therapy to improve lung growth and development in a pre-clinical model. With refinement, this technique could be applied to fetal cases of CDH or other forms of impaired lung development in a minimally invasive fashion.

Fetal lung hypoxia and energetic cell failure in the nitrofen-induced congenital diaphragmatic hernia rat model.

Congenital diaphragmatic hernia (CDH) pathogenesis is poorly understood. We hypothesize that fetal CDH lungs are chronically hypoxic because of lung hypoplasia and tissue compression, affecting the cell bioenergetics as a possible explanation for abnormal lung development. To investigate this theory, we conducted a study using the rat nitrofen model of CDH. We evaluated the bioenergetics status using H1 Nuclear magnetic resonance and studied the expression of enzymes involved in energy production, the hypoxia-inducible factor 1α, and the glucose transporter 1. The nitrofen-exposed lungs have increased levels of hypoxia-inducible factor 1α and the main fetal glucose transporter, more evident in the CDH lungs. We also found imbalanced AMP:ATP and ADP:ATP ratios, and a depleted energy cellular charge. Subsequent transcription levels and protein expression of the enzymes involved in bioenergetics confirm the attempt to prevent the energy collapse with the increase in lactate dehydrogenase C, pyruvate dehydrogenase kinase 1 and 2, adenosine monophosphate deaminase, AMP-activated protein kinase, calcium/calmodulin-dependent protein kinase 2, and liver kinase B1, while decreasing ATP synthase. Our study suggests that changes in energy production could play a role in CDH pathogenesis. If confirmed in other animal models and humans, this could lead to the development of novel therapies targeting the mitochondria to improve outcomes.

Congenital diaphragmatic hernia increases the sensitivity of pulmonary arteries to nitric oxide

Left congenital diaphragmatic hernia (CDH) can lead to pulmonary arteries abnormalities in the contralateral and ipsilateral sides of the diaphragm. Nitric oxide (NO) is the main therapy used to attenuate the vascular effects of CDH, but it is not always effective. We hypothesized that the left and right pulmonary arteries do not respond similarly to NO donors during CDH. Therefore, vasorelaxant responses of the left and right pulmonary arteries to sodium nitroprusside (SNP, a NO donor) were determined in a rabbit experimental model of left CDH. CDH was surgically induced in the fetuses of rabbits on the 25th day of pregnancy. On the 30th day of pregnancy, a midline laparotomy was performed to access the fetuses. The fetuses’ left and right pulmonary arteries were isolated and mounted in myograph chambers. Vasodilation was evaluated by cumulative concentration-effect curves to SNP. Protein expression of guanylate cyclase isoforms (GCα, GCβ) and the α isoform of cGMP-dependent protein kinase 1 (PKG1α), and the concentration of NO and cGMP were determined in the pulmonary arteries. The left and right pulmonary arteries of newborns with CDH exhibited increased vasorelaxant responses to SNP (i.e. the potency of SNP was increased) compared to the control group. GCα, GCβ, and PKG1α expression were decreased, while NO and cGMP concentrations were increased in the pulmonary arteries of newborns with CDH compared to the control group. The increased cGMP mobilization may be responsible for the increased vasorelaxant responses to the SNP in the pulmonary arteries during left CDH.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation invitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs invitro and in nitrofen rat tracheas invivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

Upregulated TSP1 and CD47 expression in the lung in nitrofen-induced congenital diaphragmatic hernia.

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). The secreted glycoprotein thrombospondin-1 (TSP1), a ligand for receptor CD47, is widely expressed on both systemic and pulmonary vascular cells. TSP1-CD47 signaling has been reported to be one of the pathogeneses of pulmonary hypertension (PH). After creating a nitrofen-induced CDH rat model, fetuses were sacrificed on D17, D19 and D21 and divided into a control group and a CDH group. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression of TSP1, CD47 and Runx3 (a regulator of TSP1). An immunofluorescence study was performed to evaluate the expression and localization of TSP1, CD47 and Runx3. The relative mRNA expression of pulmonary TSP1, CD47 and Runx3 on D21 was significantly increased in the CDH group (p=0.005, p=0.001, p=0.046, and p=0.002, respectively). The immunofluorescence study also confirmed the overexpression of TSP1, CD47 and Runx3 in the CDH group. Our results provide evidence that TSP1-CD47 signaling is involved in the pathogenesis of PH in a nitrofen-induced CDH model. Our data suggest that anti-CD47 antibodies can be novel therapeutic targets for the treatment of PH in CDH.

Neonatal rodent ventilation and clinical correlation in congenital diaphragmatic hernia.

Ventilator management is a critical part of managing congenital diaphragmatic hernia (CDH). We aimed to use a murine model and patient data to study CDH-associated differences in oxygenation, airway resistance, and pulmonary mechanics by disease severity. We used the nitrofen model of CDH. For control and CDH rodents, data were collected within the first hour of life. Oxygen saturations (SpO2 ) were collected using MouseOx, and large airway resistance and inspiratory capacities were collected using flexiVent. A single-center, retrospective review of term CDH infants from 2014 to 2020 was performed. Tidal volumes were collected every 6 h for the first 48 h of life or until the patient was taken off conventional ventilation. Newborns that were mechanically ventilated but had no pulmonary pathology were used as controls. CDH severity was defined using theCDH Study Group (CDHSG) classification system. Control rodents had a median SpO2 of 94% (IQR: 88%-98%); CDH pups had a median SpO2 of 27.9% (IQR: 22%-30%) (p < 0.01). CDH rodents had lower inspiratory capacity than controls (median:110 μl, IQR:70-170 vs. median:267 μl, IQR: 216-352;p < 0.01). CDH infants had a lower initial SpO2 than control infants. Overall, CDH infants had lower tidal volumes than control infants (median: 4.2 ml/kg, IQR: 3.3-5.0 vs. 5.4 ml/kg, IQR: 4.7-6.2;p = 0.03). Tidal volumes varied by CDHSG stage. Newborns with CDH have lower SpO2 and lower, CDHSG stage specific, tidal volumes than control infants. The nitrofen model of CDH reflects these differences. Rodent models may be useful in studying therapeutic ventilatory strategies for CDH infants.

The fetal lamb model of congenital diaphragmatic hernia shows altered cerebral perfusion using contrast enhanced ultrasound

BackgroundNeurodevelopmental impairment is common in survivors of congenital diaphragmatic hernia (CDH). Altered cerebral perfusion in utero may contribute to abnormal brain development in CDH patients. Methods5 fetal lambs with surgical left-CDH and 5 controls underwent transuterine cranial Doppler and contrast enhanced ultrasound (CEUS). Global and regional perfusion metrics were obtained. Biometric and perfusion data were compared between groups via nonparametric Mann Whitney U test and Spearman's rank order correlation. ResultsNo significant differences in cerebral Doppler measurements were identified between groups. By CEUS, CDH animals demonstrated significantly decreased global brain perfusion and increased transit time. With focal regions-of-interest (ROIs), there was a tendency towards decreased perfusion in the central/thalamic region in CDH but not in the peripheral brain parenchyma. Transit time was significantly increased in both ROIs in CDH, whereas flux rate was decreased in the central/thalamic region but not the peripheral brain parenchyma. Biometric CDH severity was correlated to perfusion deficit. There was no difference in cardiomyocyte histology. ConclusionThe fetal lamb model of CDH shows altered cerebral perfusion as measured by CEUS, correlating to disease severity. This suggests a physiological abnormality in fetal cerebrovascular perfusion that may contribute to abnormal brain development and neurodevelopmental impairment in survivors.

Liver pathological alterations in fetal rabbit model of congenital diaphragmatic hernia.

To date, fetal liver implication is not a well-understood phenomenon in congenital diaphragmatic hernia (CDH). We evaluated the fetal morphologic changes on liver growth after surgical procedure in CDH experimental model. A diaphragmatic defect at gestational day E25 and tracheal occlusion (TO) at E27 were surgically created in rabbit fetuses. Five experimental groups were assessed: control group, left CDH, right CDH, CDH + TO, and TO alone. Body and organ growth were measured. For histological evaluation of the CDH effect, liver sections were collected. Left-CDH group had livers with increased leukocyte infiltration in comparison with controls (p= 0.02). Increased capillary sinusoid congestion and hepatocyte vacuolation were greater in left-CDH compared with the right-CDH group (p= 0.05). Capillary sinusoid congestion and interstitial edema were more evident in the left-CDH compared with CDH + TO group (p= 0.05). Increases in sinusoid congestion, hepatocyte vacuolation, and interstitial edema were also greater in the CDH + TO compared with controls (p≤ 0.02). Intrathoracic liver weight was higher in right-CDH compared with left-CDH group (p< 0.001). Total lung weights (TLW) were significantly lower in both left-CDH compared with controls (p< 0.001), CDH + TO (p= 0.01), and TO (p< 0.01) and in right-CDH compared with CDH + TO (p< 0.01) and TO (p< 0.01). Decreased kidney and heart weights were also recorded. Hemodynamics and structural fetal liver changes in laterality were noted in CDH model. Regulation of intrathoracic liver weights seems to be disturbed by the absence of diaphragmic contact. Pulmonary injury is supported by the effect of a first hit, while the growth of internal organs suggests a multisystemic remodeling related to the fetal adaptation.

Nitrofen rat model of congenital diaphragmatic hernia demonstrates increased lung stiffness using shear wave elastography

Patients with congenital diaphragmatic hernia (CDH) have intrinsic lower lung compliance. Shear wave elastography (SWE) is an emerging noninvasive ultrasound technology to evaluate fetal lung stiffness with the potential to correlate with histopathologic analyses and postnatal pulmonary function. We aimed to evaluate lung elastography measurements in a nitrofen rat model of CDH.

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats.

Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Defective mesothelium and limited physical space are drivers of dysregulated lung development in a genetic model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a developmental disorder associated with diaphragm defects and lung hypoplasia. The etiology of CDH is complex and its clinical presentation is variable. We investigated the role of the pulmonary mesothelium in dysregulated lung growth noted in the Wt1 knockout mouse model of CDH. Loss of WT1 leads to intrafetal effusions, altered lung growth, and branching defects prior to normal closure of the diaphragm. We found significant differences in key genes; however, when Wt1 null lungs were cultured ex vivo, growth and branching were indistinguishable from wild-type littermates. Micro-CT imaging of embryos in situ within the uterus revealed a near absence of space in the dorsal chest cavity, but no difference in total chest cavity volume in Wt1 null embryos, indicating a redistribution of pleural space. The altered space and normal ex vivo growth suggest that physical constraints are contributing to the CDH lung phenotype observed in this mouse model. These studies emphasize the importance of examining the mesothelium and chest cavity as a whole, rather than focusing on single organs in isolation to understand early CDH etiology.

Stereological Lung Parameters After Fetoscopic Abdominal Decompression of Congenital Diaphragmatic Hernia in an Ovine Model: A Pilot Study.

Background: Fetoscopic endoluminal tracheal occlusion (FETO) improves lung maturation in severe cases of congenital diaphragmatic hernia (CDH) but it does not ameliorate lung compression by herniated abdominal organs. Surgically opening the fetal abdomen (abdominal decompression [AD]) reduces the intrathoracic pressure by diverting the abdominal organs into the amniotic cavity-a probable causal therapy for lung hypoplasia and pulmonary hypertension in CDH. Open surgical abdominal decompression has been reported: we describe a minimally invasive approach in an ovine model of CDH as a probable fetoscopic intervention. Materials and Methods: Eight fetuses were included, 2 were kept as HEALTHY controls. A CDH (left side, liver down) was created by open fetal surgery at midgestation in 6 fetuses, 2 were taken as CDH controls. Fetoscopic abdominal decompression (fAD) was performed 21 days later in 4 animals. The fetuses were retrieved at the end of gestation and evaluated by lung stereology. Results: fAD led to a near total evacuation of the thoracic cavity in 2 of the 4 animals. Fetuses with CDH had a lower total volume and fraction of alveolar air space, a lower volume fraction of the parenchyma, and an increase of the volume fraction of the alveolar septa and atelectasis, as well as an increased mean thickness of alveolar septa compared with HEALTHY fetuses. Fetuses treated with abdominal decompression showed an improvement of stereological parameters. Conclusions: In spite of relevant limitations (pilot study, small groups, spontaneous closure of the abdominal incision) we were able to demonstrate that abdominal decompression for CDH can be performed by fetoscopy. Our results support the hypothesis of causally improving lung development by abdominal decompression, thus implying increased survival in extreme cases of CDH. A refinement of the fetoscopic techniques and direct comparison to FETO appears warranted.