Congenital Diaphragmatic Hernia Animals Research Articles

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.

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

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 β (TGFβ) signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodelling. Genetic studies have linked TGFβ and Eng mutations to human arterial PH and other cardiovascular syndromes. Eng interacts with the TGFβ receptors 1 and 2 (Tgfβr1, Tgfβr2). 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 Tgfβr1 and Tgfβr2. 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, Tgfβr1 and Tgfβr2 were assessed via qRT-PCR and western blotting. Immunofluorescence staining for Eng was combined with CD34 to evaluate Eng expression in the pulmonary vasculature. Relative mRNA levels of Eng, Tgfβr1 and Tgfβr2 were significantly downregulated in CDH lungs compared to controls (Eng CDH 0.341±0.022, Eng Ctrl 0.471±0.031, p=0.0015; Tgfβr1 CDH 0.161±0.008, Tgfβr1 Ctrl 0.194±0.01, p=0.0114; Tgfβr2 CDH 0.896±0.099, Tgfβr2 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. This study demonstrates for the first time a reduced expression of Endoglin in the pulmonary vasculature of nitrofen-induced CDH. Abnormal Eng/Tgfβr1/Tgfβr2 signalling may contribute to impaired vascular remodelling and development of PH in this CDH animal model.

Early neonatal echocardiographic findings in an experimental rabbit model of congenital diaphragmatic hernia.

This study aimed to demonstrate that congenital diaphragmatic hernia (CDH) results in vascular abnormalities that are directly associated with the severity of pulmonary hypoplasia and hypertension. These events increase right ventricle (RV) afterload and may adversely affect disease management and patient survival. Our objective was to investigate cardiac function, specifically right ventricular changes, immediately after birth and relate them to myocardial histological findings in a CDH model. Pregnant New Zealand rabbits underwent the surgical procedure at 25 days of gestation (n=14). CDH was created in one fetus per horn (n=16), and the other fetuses were used as controls (n=20). At term (30 days), fetuses were removed, immediately dried and weighed before undergoing four-parameter echocardiography. The lungs and the heart were removed, weighed, and histologically analyzed. CDH animals had smaller total lung weight (P<0.005), left lung weight (P<0.005), and lung-to-body ratio (P<0.005). Echocardiography revealed a smaller left-to-right ventricle ratio (LV/RV, P<0.005) and larger diastolic right ventricle size (DRVS, P<0.007). Histologic analysis revealed a larger number of myocytes undergoing mitotic division (186 vs 132, P<0.05) in CDH hearts. Immediate RV dilation of CDH hearts is related to myocyte mitosis increase. This information may aid the design of future strategies to address pulmonary hypertension in CDH.

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia

Background Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3′-5′-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3′-5′-guanylosine monophosphate. Methods Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. Results In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5′hydroxymethyl-2′furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1–induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. Conclusions Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

Expression and function of phosphodiesterases in nitrofen‐induced congenital diaphragmatic hernia in rats

Congenital diaphragmatic hernia (CDH) is an anomaly associated with pulmonary hypoplasia and pulmonary hypertension (PH). The limited efficacy of current approaches to treat PH in CDH, including inhaled nitric oxide (NO), drives the search for other therapies. Phosphodiesterases (PDEs) degrade cyclic nucleotide second messenger cAMP and cGMP downstream of NO thereby limiting the vasodilatory response to NO. To identify therapeutic targets by cataloguing the expression and function of PDE isoforms in the pulmonary vasculature in nitrofen-induced CDH in fetal rats. Quantitative RT-PCR revealed PDE1-5 and PDE9 mRNA expression in pulmonary arteries (PAs) of control and nitrofen-induced CDH term fetal rats. In this order of potency, the PDE inhibitors Sildenafil (PDE5) > EHNA (PDE2) > Rolipram (PDE4) > Cilostamide (PDE3) all dilated isolated third generation PA after pre-constriction with the thromboxane analog U46619. Hyperoxic pre-incubation of PAs significantly attenuated vasodilatation induced by the PDE5 inhibitor Sildenafil (65% vs. 33%, P < 0.004). CDH PAs dilated significantly less to PDE2 inhibitor EHNA compared to control (51% vs. 72%, P < 0.05). Subsequently PDE2 protein expression was higher in PAs of CDH animals. Most PDE isoforms exist in the PAs of fetal rats and their inhibition causes pulmonary vasodilatation. PDE5 inhibition was the most potent vasodilator, however, there were no differences between groups. PDE5-induced vasodilatation was attenuated by hyperoxic pre-incubation. PDE inhibitors might be considered therapeutic targets in combination with iNO in neonates with CDH.

The Myenteric Plexus of the Esophagus is Abnormal in an Experimental Congenital Diaphragmatic Hernia Model

Infants surviving congenital diaphragmatic hernia (CDH) suffer from anatomical and functional esophageal abnormalities. Previous work in the nitrofen animal model of CDH demonstrated malformations in neural crest-derived structures, including the vagus and recurrent laryngeal nerves. The aim of the present study was to assess whether the esophageal myenteric plexus is abnormal in rats with CDH. We used the nitrofen-induced CDH fetal rat model. Two sections of the proximal, medium and distal esophagus from both groups were processed for immunohistochemical staining with anti-neuron specific enolase and anti-S-100 antibodies; the number of stained areas was recorded for each group. Whole-mount preparations of the entire esophagus of Control and CDH animals were histochemically stained for acetylcholinesterase; the density and area of the ganglia and the number of cells/ganglia were determined. Comparisons between groups were made by standard statistical methods. The number of immunohistochemically stained areas in transversal sections were decreased in CDH animals for anti-enolase (11.5+/-6.06 vs. 1.93+/-1.49, control vs. CDH, p<0.001) and anti S-100 antibodies (8.57+/-4.1 vs. 4.06+/-2.82, p<0.001). In whole-mount preparations the number of ganglia per high power field (35.16+/-6.57 vs. 29.29+/-10.26, p<0.05), the number of cells per ganglia (11.85+/-3.52 vs. 2.28+/-4.61, p<0.0001) and the relative area of the ganglia (0.35+/-0.32 vs. 0.18+/-0.42%, p<0.001), were also significantly decreased in CDH animals compared with Controls. Esophageal intrinsic innervation is defective in rat fetuses with CDH. If patients with CDH bear the same anomalies, this may explain some of their esophageal motility disorders. Finally, these findings support the concept of neural crest involvement in the pathogenic pathways of CDH.

Prenatal tetrandrine treatment can reverse the abnormal conditions in the lung of newborn with congenital diaphragmatic hernia

Pulmonary hypoplasia and persistent pulmonary hypertension are the most important reasons for the high morbidity and mortality of congenital diaphragmatic hernia (CDH). Despite surgical advances and advances in neonatal intensive care, the mortality still remains high. Then the research on how to improve prenatal fetal lung growth has become a focus. Some researches involved in fetal surgery, tracheal occlusion, prenatal use of corticosteroids etc., have been carried out in CDH animal models and humans. But the results either showed no benefit for the outcome of CDH or were unproved. Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra. It has been used in traditional Chinese medicine for several decades to treat patients with silicosis, asthma and pulmonary hypertension etc. Some researches showed that prenatal tetrandrine administration can improve the lung development in CDH rat models. We hypothesize that prenatal treatment with tetrandrine can reverse the abnormal condition in the lung of newborn with CDH, and thus decrease the mortality.

Downregulation of GATA4 and GATA6 in the heart of rats with nitrofen-induced diaphragmatic hernia

The high incidence of cardiac malformations in humans and animal models with congenital diaphragmatic hernia (CDH) is well known. The precise molecular mechanisms underlying cardiac maldevelopment in CDH are still unclear. It has been reported that GATA4 and GATA6, members of the GATA transcription factor family, act cooperatively to regulate cardiovascular development, and the levels of cardiac GATA4 and GATA6 are important regulators of cardiomyocyte proliferation, cardiac morphogenesis, and embryo survival. In addition, the GATA4/GATA6 double heterozygous mutant embryo model displayed a spectrum of cardiovascular malformations similar to those seen in human CDH and nitrofen-induced animal models, including ventricular and aortopulmonary septal defects and thin ventricular myocardium. To test the hypothesis that expression of GATA4 and GATA6 is reduced in early stages of gestation in a CDH hypoplastic heart, we investigated the expression of GATA4 and GATA6 in the hearts of nitrofen-treated rats in early gestation. Wnt2, bone morphogenetic protein 4 (BMP4), and myocyte enhancer factor 2C (MEF2C) were also investigated as GATA4/6 target genes involved in cardiogenesis. Fetal rat hearts of normal (n = 7) and nitrofen-treated (n = 7) dams were harvested on embryonic day 13. The expression of GATA4, GATA6, Wnt2, BMP4, and MEF2C was analyzed in each heart by real-time reverse transcription-polymerase reaction. The gene expression of GATA4, GATA6, Wnt2, BMP4, and MEF2C on embryonic day 13 were significantly reduced (P < .05) in the hearts of nitrofen-treated animals compared with normal hearts of equivalent age. Decreased expression of GATA4 and GATA6 and their target genes in the developing fetal heart may perturb the delicate regulation of cardiovascular development, resulting in cardiovascular malformations in the nitrofen rat model.

Prenatal treatment with retinoic acid accelerates type 1 alveolar cell proliferation of the hypoplastic lung in the nitrofen model of congenital diaphragmatic hernia

Retinoids play an important role in lung development. A recent study has demonstrated that prenatal treatment with retinoic acid (RA) stimulates alveologenesis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia (CDH). Furthermore, it has also been demonstrated that the differentiation from alveolar epithelial cells type II (AECs-II) into alveolar epithelial cells type I (AECs-I), which is the key process in lung development, is disturbed in this model. We hypothesized that retinoids promote alveologenesis by stimulating differentiation of AECs-II to AECs-I at the end of gestation; and therefore, we investigated the effect of RA on the pulmonary expression of intercellular adhesion molecule 1 (ICAM-1), a marker for AECs-I, and thyroid transcription factor 1 (Ttf-1), a marker for AECs-II, in nitrofen-induced hypoplastic lungs. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day of gestation (D) 9. Five milligrams per kilogram of RA was given intraperitoneally on D18, D19, and D20; and fetuses were recovered on D21. We had 4 study groups: control (n = 7), control + RA (n = 7), CDH (n = 6), and CDH + RA (n = 6). The expression of ICAM-1 and Ttf-1 was analysed in each lung by real-time reverse transcription polymerase chain reaction and immunohistochemistry. One-way analysis of variance test was used for statistical analysis. Expression levels of ICAM-1 were significantly reduced in CDH lungs compared with normal controls, whereas levels increased significantly in CDH group after the addition of RA (P < .05). Expression levels of Ttf-1 were significantly decreased in lungs from RA-treated CDH animals compared with CDH without RA (P < .05). The ICAM-1 and Ttf-1 immunoreactivity demonstrated similar pattern of expression in various groups. Our results demonstrate that prenatal treatment with RA accelerates AEC-I proliferation in the hypoplastic lung in CDH.

Surfactant Maturation Is Not Delayed in Human Fetuses with Diaphragmatic Hernia

BackgroundPulmonary hypoplasia and persistent pulmonary hypertension account for significant mortality and morbidity in neonates with congenital diaphragmatic hernia (CDH). Global lung immaturity and studies in animal models suggest the presence of surfactant deficiency that may further complicate the pathophysiology of CDH. However, data about surfactant status in human fetuses with CDH at birth are contradictory. The lack of a chronological study of surfactant content in late pregnancy has been a significant limitation. The appropriateness of administering surfactant supplements to neonates with CDH is therefore a debated question.Methods and FindingsWe investigated surfactant content in human fetuses with CDH compared to age-matched fetuses with nonpulmonary diseases used as controls. Concentrations of disaturated phosphatidylcholine and surfactant proteins were found to be similar at a given stage of pregnancy, with both components showing a similar pattern of increase with progressing pregnancy in fetuses with CDH and in control fetuses. Thyroid transcription factor 1, a critical regulator of surfactant protein transcription, similarly displayed no difference in abundance. Finally, we examined the expression of three glucocorticoid-regulated diffusible mediators involved in lung epithelial maturation, namely: keratinocyte growth factor (KGF), leptin, and neuregulin 1 beta 1 (NRG1-β1). KGF expression decreased slightly with time in control fetuses, but remained unchanged in fetuses with CDH. Leptin and NRG1-β1 similarly increased in late pregnancy in control and CDH lungs. These maturation factors were also determined in the sheep fetus with surgical diaphragmatic hernia, in which surfactant deficiency has been reported previously. In contrast to the findings in humans, surgical diaphragmatic hernia in the sheep fetus was associated with decreased KGF and neuregulin expression. Fetoscopic endoluminal tracheal occlusion performed in the sheep model to correct lung hypoplasia increased leptin expression, partially restored KGF expression, and fully restored neuregulin expression.ConclusionsOur results indicate that CDH does not impair surfactant storage in human fetuses. CDH lungs exhibited no trend toward a decrease in contents, or a delay in developmental changes for any of the studied surfactant components and surfactant maturation factors. Surfactant amounts are likely to be appropriate to lung size. These findings therefore do not support the use of surfactant therapy for infants with CDH. Moreover, they raise the question of the relevance of CDH animal models to explore lung biochemical maturity.

Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics

Congenital diaphragmatic hernia (CDH) is a common birth defect with high mortality and morbidity. Two hundred seventy CDH patients were ascertained, carefully phenotyped, and classified as isolated (diaphragm defects alone) or complex (with additional anomalies) cases. We established different strategies to reveal CDH-critical chromosome loci and genes in humans. Candidate genes for sequencing analyses were selected from CDH animal models, genetic intervals of recurrent chromosomal aberration in humans, such as 15q26.1-q26.2 or 1q41-q42.12, as well as genes in the retinoic acid and related pathways and those known to be involved in embryonic lung development. For instance, FOG2, GATA4, and COUP-TFII are all needed for both normal diaphragm and lung development and are likely all in the same genetic and molecular pathway. Linkage analysis was applied first in a large inbred family and then in four multiplex families with Donnai-Barrow syndrome (DBS) associated with CDH. 10K SNP chip and microsatellite markers revealed a DBS locus on chromosome 2q23.3-q31.1. We applied array-based comparative genomic hybridization (aCGH) techniques to over 30, mostly complex, CDH patients and found a de novo microdeletion in a patient with Fryns syndrome related to CDH. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) techniques allowed us to further define the deletion interval. Our aim is to identify genetic intervals and, in those, to prioritize genes that might reveal molecular pathways, mutations in any step of which, might contribute to the same phenotype. More important, the elucidation of pathways may ultimately provide clues to treatment strategies.

Impaired alveolar epithelial cell differentiation in the hypoplastic lung in nitrofen-induced congenital diaphragmatic hernia

Pulmonary hypoplasia is the principal cause of morbidity and mortality in infants with congenital diaphragmatic hernia (CDH). Still, relatively little is known about the mechanisms causing lung hypoplasia associated with CDH. The differentiation from alveolar epithelial cells type II (AECs-II) into alveolar epithelial cells type I (AECs-I) is one of the key processes in lung development in late gestation. It is well known that increased lung expansion promotes differentiation into AECs-I phenotype, whereas reduced lung expansion promotes AECs-II phenotype. The recent availability of cell-specific molecule markers for AECs-I and AECs-II has provided an opportunity to study the various characteristics of these two cell types. To test the hypothesis that the differentiation of AECs-II to AECs-I is impaired in the CDH hypoplastic lung, we investigated molecular markers for AECs-I [ICAM-1, T1alpha, aquaporin 5 (AQP5)] and molecular markers for AECs-II [thyroid transcription factor-1 (Ttf-1), surfactant protein (SP)-B and C] in the nitrofen-induced CDH lung. Fetal rat lungs of normal (n = 7) and nitrofen-treated (n = 14) dams were harvested on embryonic day 21. The expression of the ICAM1, T1alpha, AQP5, SP-B, C and Ttf-1 was analyzed in each lung by real-time reverse transcription polymerase chain reaction. Immunohistochemical studies were performed to evaluate the protein expression level of ICAM1 and Ttf1. Expression levels of ICAM-1, T1alpha and AQP5 were significantly reduced (P < 0.05) in the lungs from nitrofen-treated CDH animals compared to normal controls. ICAM-1 and AQP5 immunohistochemistry showed a diffuse pattern of expression in the alveolar cells in normal lungs. By contrast, the ICAM-1 and AQP5 positive cells were markedly reduced in hypoplastic lungs with CDH. On the other hand, the expression levels of Ttf-1, SP-B and C were significantly (P < 0.05) increased in the lungs from nitrofen-treated CDH animals compared to normal controls. The population of Ttf-1 positive cells was slightly increased in the lungs from nitrofen-treated animals in immunohistochemical study. Our results demonstrate that there is significant reduction in the proportion of AECs-I and increase in the proportion of AECs-II in the hypoplastic lung in the nitrofen-induced CDH. This data provides the first evidence to support the hypothesis that AEC differentiation is impaired in CDH hypoplastic lung.

Role of ATP-Dependent Potassium Channels in Pulmonary Vascular Tone of Fetal Lambs With Congenital Diaphragmatic Hernia

High mortality in newborn babies with congenital diaphragmatic hernia (CDH) is principally due to persistent pulmonary hypertension. ATP-dependent potassium (K(ATP)) channels might modulate pulmonary vascular tone. We have assessed the effects of Pinacidil, a K(ATP) channel opener, and glibenclamide (GLI), a K(ATP) channel blocker, in near full-term lambs with and without CDH. In vivo, pulmonary hemodynamics were assessed by means of pressure and blood flow catheters. In vitro, we used isolated pulmonary vessels and immunohistochemistry to detect the presence of K(ATP) channels in pulmonary tissue. In vivo, pinacidil (2 mg) significantly reduced pulmonary vascular resistance (PVR) in both controls and CDH animals. GLI (30 mg) significantly increased pulmonary arterial pressure (PAP) and PVR in control animals only. In vitro, pinacidil (10 microM) relaxed, precontracted arteries from lambs with and without CDH. GLI (10(-5) microM) did not raise the basal tone of vessels. We conclude that activation of K(ATP) channels could be of interest to reduce pulmonary vascular tone in fetal lambs with CDH, a condition often associated with persistent pulmonary hypertension of the newborn.

Altered renin–angiotensin system gene expression causes renal hypoplasia in the rats with nitrofen-induced diaphragmatic hernia

The association between renal hypoplasia and pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) has become recently appreciated. However, the underlying mechanisms responsible for this association are still unknown. Renin-angiotensin system (RAS) plays an important role in renal and somatic growth, angiogenesis and reproduction. We hypothesized that abnormal expression of RAS components may be responsible for renal hypoplasia in CDH. We therefore designed this study to examine the gene expression of main components of RAS in the kidney of nitrofen-induced CDH in the rat. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 of gestation. Foetuses were recovered at term and divided into three groups: control (n=8), nitrofen without CDH (n=8) and CDH (n=8). Reverse transcription polymerase chain reaction was performed to evaluate the relative amount of angiotensinogen (AGT), angiotensin II type 1 receptor with 1a and 1b subtypes (AT(1a)R and AT(1b)R), angiotensin II type 2 receptor (AT(2)R), angiotensin-converting enzyme (ACE) and renin expression in the kidney. AT(1a)R, AT(1b)R, AT(2)R, AGT and renin levels were significantly decreased in the kidney of CDH rats compared with controls. We did not find a significant difference in ACE between CDH animals and controls. Our data show that the downregulation of RAS may play an important role in the pathogenesis of renal hypoplasia in the nitrofen-induced CDH.

Congenital diaphragmatic hernia: searching for answers

Background Pulmonary hypoplasia and hypertension are the primary causes of morbidity and mortality in infants with congenital diaphragmatic hernia (CDH). At present, the origin of CDH and the causes of pulmonary hypoplasia and hypertension are unknown. Data Sources This article reviews the available published data regarding the origin of CDH and the pathogenesis of the associated pulmonary hypertension and hypoplasia. These investigations have employed human tissues as well as two types of CDH animal models. Conclusions Investigations performed to date have not yet provided definitive answers regarding the pathogenesis of CDH. However, they have yielded many new and exciting discoveries and several opportunities for intervention. Ongoing research should open new possibilities to improve the outcome for these unfortunate babies with CDH.

The adrenal cortex in experimental congenital diaphragmatic hernia

Background/Purpose: Adrenal cortical malfunction was found recently in patients with severe congenital diaphragmatic hernia (CDH). The current study tests the hypothesis that the development and function of the adrenal cortex could be abnormal in an experimental model of CDH. Methods: Pregnant rats were exposed on day 9.5 of gestation to 100 mg of 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofen) diluted in olive oil. The sham group was treated only with oil. Fetuses were recovered on the 21st day, bled, and examined for the presence or absence of CDH. Adrenal glands from sham and CDH animals were dissected, weighed, and prepared for histologic, biochemical, and immunohistochemical studies (ki-67) aimed at measuring total DNA, total protein, and the proportion of proliferating cells. Serum corticosterone levels were assayed. The results in both groups were compared with parametric tests with a significance level of P <.05. Results: The adrenal weight was not different in CDH animals versus controls (0.049 ± 0.014 v 0.052 ± 0.012% of body weight; not significant). Total DNA was reduced significantly (1.180 ± 0.481 v 1.909 ± 0.893 μg; P <.05) with unchanged DNA to protein ratio. Proliferation index in both groups was 20.1 ± 3.1% and 26.5 ± 7.5%, respectively (not significant), and the proliferating cells were mainly located in the glomerular areas of the glands. Corticosterone levels were similar in both groups. Conclusions: Nitrofen induces very slight changes in the development of adrenal glands of fetal rats, expressed by reduced cell proliferation especially in glomerular areas, reduced total DNA with preservation of cell sizes (constant DNA to protein ratio), with no change in function because corticosterone levels remained unchanged. It is doubtful that primary adrenal malformation/malfunction contributes to the severity of CDH in this model. J Pediatr Surg 38:682-684. © 2003 Elsevier Inc. All rights reserved.

Antenatal dexamethasone enhances endothelin-1 synthesis and gene expression in the heart in congenital diaphragmatic hernia in rats

Background/Purpose: Although high levels of endothelin-1 (ET-1) in plasma may be relevant in certain pathophysiologic states, such as pulmonary hypertension accompanying congenital diaphragmatic hernia (CDH), experimental evidence favors a local, paracrine, or autocrine role for ET-1 in most tissues. Evidence of ET-1 production has been documented in fetal heart tissue where it exerts growth-enhancing and mitogenic effects. ET-1 also has a potent positive inotrope action on cardiac muscle. ET-1 −/− homozygous mice display a wide variety of cardiac anomalies, which also are features of the human and of the experimental CDH. Autopsy reports have shown that total heart weight is reduced significantly in the presence of CDH, and animal models have documented the presence of cardiac hypoplasia associated with CDH. Experimental and clinical studies have shown that prenatal exposure to corticosteroids improves cardiovascular function in the immediate newborn period. The aim of this study was to determine cardiac gene expression of ET-1 and of its receptor ETA and the cardiac ET-1 content in the heart of nitrofen-induced CDH in rats and to evaluate the effect of antenatal Dexamethasone (Dex) treatment. Methods: A CDH model was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dex (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: group I, control (n = 8); group II, nitrofen-induced CDH (n = 8); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 8). ET-1 protein was measured using ELISA. RT-PCR was performed to evaluate the relative amount of ET-1 and ETA mRNA expression. Results: There was a reduction in ET-1 mRNA (P <.05) and in ETA mRNA (P <.01) in the heart of CDH group compared with controls. ET-1 protein level also was reduced in heart of CDH compared with controls. Antenatal Dex treatment increased significantly both ET-1 mRNA and protein levels in the heart of CDH animals (P <.05 and P <.01, respectively). Conclusions: The reduced cardiac ET-1 gene expression and ET-1 synthesis may be responsible for the heart hypoplasia associated with CDH. Prenatal corticosteroids increase the cardiac production of ET-1, and this may enhance heart growth and cardiac inotropism at birth. J Pediatr Surg 37:1563-1567. Copyright 2002, Elsevier Science (USA). All rights reserved.

Altered guanylyl-cyclase activity in vitro of pulmonary arteries from fetal lambs with congenital diaphragmatic hernia.

Nitric oxide (NO) plays a major role in the modulation of perinatal pulmonary vascular tone. Congenital diaphragmatic hernia (CDH), a major cause of severe persistent pulmonary hypertension of the newborn (PPHN), is often refractory to inhaled NO. Alterations in NO/cyclic guanosine 3',5' monophosphate (cGMP)-mediated pulmonary vasodilatation may contribute to PPHN in CDH. We assessed NO/cGMP-mediated pulmonary vasorelaxation in vitro in 140-d gestational lamb fetuses with surgically created left CDH (term = 147 d) to age-matched controls. Relaxation of fourth generation intralobar pulmonary artery rings in response to the endothelium-dependent vasodilator, acetylcholine (ACh), and to the specific inhibitor of cGMP-phosphodiesterase (PDE), zaprinast, did not differ between the two groups. By contrast, relaxation in response to the calcium ionophore A23187 was impaired in CDH as compared with control animals. Relaxation in response to the NO donor sodium nitroprusside (SNP) (a direct activator of soluble guanylyl cyclase [sGC]) was also impaired in CDH animals as compared with controls. Repeating the challenge increased vasorelaxation in response to SNP in CDH as compared with control animals. Immunohistochemistry revealed the presence of endothelial NO-synthase in the endothelium of pulmonary arteries from both control and CDH animals. We conclude that endothelium-dependent vasodilatation in response to ACh and A23187 was differently affected in the fetal surgical CDH-lamb model. Furthermore, activity of sGC but not that of PDE was impaired in CDH animals. PPHN and decreased inhaled NO responsiveness in CDH may involve decreased sGC activity.

Pulmonary effects of in utero tracheal occlusion are dependent on gestational age in a rabbit model of diaphragmatic hernia

Purpose: The authors investigated the effect of gestational age on lung development and maturation after in utero tracheal occlusion (TO) in a rabbit model of congenital diaphragmatic hernia (CDH). Methods: In 46 fetal rabbits, CDH was created at 23 days' gestational age (GA; term, 31 days), corresponding to the pseudoglandular phase of lung development. A second intervention was performed at either 26, 27, or 28 days on 6 fetuses in each GA group. At that time, either TO (CDH + TO), or a sham operation (CDH + sham) was performed. Nonoperated littermates served as internal normal controls (CTR). All fetuses were delivered by cesarean section at 30 days GA to assess lung response by lung-to-body-weight ratio, pulmonary morphometry, and the density of type II pneumocytes. Results: After TO, the lungs were significantly larger than in CDH animals; their weight was proportional to the duration of TO. Pulmonary morphometry in TO fetuses was comparable with that of controls. The density of type II cells was inversely related to the gestational age at which TO was performed, with normal values with TO at GA at 28 days. Conclusion: Timing of TO is critical to subsequent pulmonary development: early in gestation TO leads to pulmonary overgrowth and type II pneumocyte depletion, whereas normal values are obtained when TO is delayed till 28 of 32 days. J Pediatr Surg 37:11-17. Copyright © 2002 by W.B. Saunders Company.

Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia–Associated pulmonary hypoplasia

Background/Purpose: The mechanisms that cause pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). Methods: ERK-1 activities were measured using immune-complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil–treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, activated) forms of the enzymes. Results: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil–treated controls (P < .02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P = .003; dp-ERK-2, P = .04), whereas ERK-1 and ERK-2 protein levels were unchanged. Conclusions: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are reduced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes. J Pediatr Surg 36:1490-1496. Copyright © 2001 by W.B. Saunders Company.