Nitrofen-induced Diaphragmatic Hernia Research Articles

Decreased Rac1 Cardiac Expression in Nitrofen-Induced Diaphragmatic Hernia.

The high incidence of cardiac malformations in humans and animal models with congenital diaphragmatic hernia (CDH) is well known. The hypoplasia of left heart is common among fetuses with CDH and has been identified as a poor prognostic factor. However, the precise mechanisms underlying cardiac maldevelopment in CDH are not fully understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) plays a key role in cardiomyocyte polarity and embryonic heart development. Deficiency of Rac1 is reported to impair elongation and cytoskeletal organization of cardiomyocytes, resulting in congenital cardiac defects. We designed this study to test the hypothesis that Rac1 expression is downregulated in the developing hearts of rats with nitrofen-induced CDH. Following ethical approval (REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D18 and D21 and divided into CDH and control (CTRL) (n = 6 for each group and time point). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and confocal-immunofluorescence microscopy were performed to detect cardiac gene and protein expression of Rac1. qRT-PCR and Western blot analysis revealed that Rac1 expression was significantly decreased in the CDH group compared with controls (p < 0.05). Confocal-immunofluorescence microscopy revealed that Rac1 cardiac expression was markedly decreased in the CDH group compared with controls. Decreased cardiac Rac1 expression in the nitrofen-induced CDH suggests that Rac1 deficiency during morphogenesis may impair structural cardiac remodeling, resulting in congenital cardiac defects.

Congenital Diaphragmatic Hernia: Not Quite There Yet

Congenital Diaphragmatic Hernia: Not Quite There Yet

A longer tracheal occlusion period results in increased lung growth in the nitrofen rat model

Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with severe congenital diaphragmatic hernia. Limited data are available regarding the effect of duration of TO on lung development. Our objective was to evaluate the effects of long (2 and 2.5 days) versus short (1 day) TO on lung development in rats with nitrofen-induced diaphragmatic hernia. Nitrofen was administered on embryonic day (ED) 9 and fetal TO performed either on ED18.5, 19 or 20 (term = 22 days). Sham-operated and untouched littermates served as controls. On ED21, lungs were harvested and only fetuses with a left-sided diaphragmatic defect were included in further analyses. Lung-body-weight ratio incrementally increased with the duration of TO. Increased proliferation following long TO was confirmed by immunohistochemistry and qRT-PCR for the proliferation marker Ki-67. Irrespective of duration, TO induced more complex airway architecture. Medial wall thickness of pulmonary arteries was thinner after long rather than short TO. In the nitrofen rat model of congenital diaphragmatic hernia, a longer period of TO leads to enhanced lung growth and less muscularized pulmonary arteries.

The role of primary myogenic regulatory factors in the developing diaphragmatic muscle in the nitrofen-induced diaphragmatic hernia

The nitrofen model of congenital diaphragmatic hernia (CDH) is widely used to investigate the pathogenesis of CDH. However, the exact pathomechanism of the diaphragmatic defect is still unclear. Diaphragmatic muscularization represents the last stage of diaphragmatic development. Myogenic differentiation 1 (MyoD) and myogenic factor 5 (Myf5) play a crucial role in muscularization. MyoD(-/-) : Myf5(+/-) mutant mice show reduced diaphragmatic size, whereas MyoD(+/-) : Myf5(-/-) mutants have normal diaphragms. We designed this study to investigate diaphragmatic gene expression of MyoD and Myf5 in the nitrofen CDH model. Pregnant rats received nitrofen or vehicle on day 9 of gestation (D9), followed by cesarean section on D18 and D21. Fetal diaphragms (n = 40) were micro-dissected and divided into CDH group and controls. MyoD and Myf5 mRNA-expression were determined using Real-time PCR. Immunohistochemistry was performed to evaluate protein expression of MyoD and Myf5. Relative diaphragmatic mRNA expression levels and immunoreactivity of MyoD were decreased in the CDH group on D18 and D21. Myf 5 mRNA and protein expression were not altered in the CDH group. This is the first study showing that MyoD expression is selectively decreased in the diaphragm muscle in the nitrofen model of CDH.

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.

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.

Altered expression of angiotensin II receptor subtypes and transforming growth factor-? in the heart of nitrofen-induced diaphragmatic hernia in rats

The renin-angitensin system (RAS) plays an important role as a growth factor in cardiac development. Angiotensin converting enzyme is involved in converting angiotensin I to angiotensin II (Ag-II). The effects of Ag-II are mediated by two primary receptors, type 1 (AT1) and type 2 (AT2). Ag-II stimulates transforming growth factor-beta1(TGF-beta1) and acts as a potent stimulant of myocyte growth and fetal contractile protein gene transcription. The aim of this study was to determine the expression of Ag-II receptor subtypes and TGF-beta1 in the hypoplastic heart of nitrofen-induced congenital diaphragmatic hernia (CDH). CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5. The fetuses were divided into three groups: normal controls (n=16), nitrofen-treated without CDH (n=16), and nitrofen-induced CDH (n=16). Reverse transcriptase-polymerase chain reaction was performed to evaluate mRNA expression of AT1, AT2, and TGF-beta1. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin. AT1 and AT2 mRNA expressions were significantly decreased in CDH heart compared with controls (0.43+/-0.33 vs. 1.0+/-0.48 and 0.62+/-0.23 vs. 1.4+/-0.43, respectively). TGF-beta1 mRNA expressions were also significantly decreased in CDH heart compared with controls (0.38+/-0.17 vs. 0.72+/-0.26). No significant difference was found between the hearts of controls and nitrofen-treated rats without CDH. The decreased expression of AT1, AT2, and TGF-beta1 mRNA in the hypoplastic heart suggests that the downregulation of RAS may be involved in the pathogenesis of cardiac hypoplasia in nitrofen-induced CDH.

Expression of vasoactive mediators during mechanical ventilation in nitrofen-induced diaphragmatic hernia in rats

The high mortality in patients with congenital diaphragmatic hernia (CDH) has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PPH). Endothelin-1 (ET-1), nitric oxide (NO), and calcitonin gene-related peptide (CGRP) have been reported to be important vasoactive mediators in the perinatal pulmonary circulation. The exact mechanism by which these vasoactive mediators interact to regulate the perinatal pulmonary vascular tone in CDH with PPH is not fully understood. We hypothesized that the altered pulmonary vascular reactivity in CDH is due to imbalance in vasoactive mediators. This study was designed to investigate mRNA expression of ET-1, eNOS, and CGRP in CDH lung in the perinatal period. A CDH model was induced in pregnant rats following administration of nitrofen. In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The newborn rats were intubated and ventilated, and ventilation was continued for 1-6 h. Left lungs were collected from both groups at 0, 1, and 6 h after ventilation (n=8 in each group). Reverse transcriptase-polymerase chain reaction on lung tissue was performed to evaluate the relative level of ET-1, eNOS, and CGRP mRNA expression. The results showed a significant increase in ET-1 mRNA in CDH lung at 1 and 6 h after ventilation compared with controls. In CDH lung, eNOS mRNA and CGRP mRNA levels were significantly increased at 1 h but were similar to control values at 6 h after ventilation. The increased expression of vasoconstrictor ET-1 mRNA and vasodilators eNOS mRNA and CGRP mRNA in the CDH lung at 1 h after ventilation suggests that pulmonary vascular tone is rapidly changing after birth. An imbalance in the production of vasoconstrictors and vasodilators by the CDH lung may contribute to high pulmonary vascular resistance.

Reductions in the incidence of nitrofen-induced diaphragmatic hernia by vitamin A and retinoic acid.

Congenital diaphragmatic hernia (CDH) is a serious medical condition in which the developing diaphragm forms incompletely, leaving a hole through which the abdominal contents can enter the thoracic space and interfere with lung growth. A perturbation of the retinoid system has been linked to the etiology of CDH. This includes findings that nitrofen, which induces CDH in rodents, inhibits the key enzyme for retinoic acid (RA) production, retinaldehyde dehydrogenase-2 (RALDH2) in vitro. Published studies indicate that antenatal vitamin A administration on gestational day (D) 12 in the nitrofen model of CDH reduced the severity and incidence of right-sided defects and lung hypoplasia. In this study, we administered nitrofen on D8, to include the induction of clinically more prevalent left-sided defects, and examined the efficacy of several vitamin A administration paradigms to gain insights into the developmental stage of susceptibility. Furthermore, we tested the hypothesis that administration of RA, the product of RALDH2 activity, is more potent than administering the substrate, vitamin A, in reducing the incidence of CDH. The incidence of CDH was reduced from approximately 54% (nitrofen alone) to approximately 32% with vitamin A treatment. The efficacy of RA treatment was very marked, with a reduction in the incidence of CDH to approximately 15%. Administration of vitamin A or RA on approximately D10 was most effective. These data lend further support for the potential involvement of retinoid signaling pathways and the etiology of CDH and support data from in vitro studies demonstrating a nitrofen-induced suppression of RALDH2.

Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats

Background/purpose Newborns with congenital diaphragmatic hernia (CDH) still have a high mortality rate, which has been attributed to pulmonary hypoplasia and pulmonary hypertension. Fibroblast growth factors (FGFs) are essential components of the gene network that regulates lung development. Recent studies suggest that the new member of FGF family, FGF-10, plays a fundamental role in branching morphogenesis and is essential for lung formation. FGF-10-deficient mice exhibit complete absence of lungs. FGF-7 promotes epithelial proliferation and expansion leading to the formation of cystlike structures. The aim of this study was to determine the gene level expression of FGF-10 and FGF-7 in the lung of nitrofen-induced CDH. Methods Congenital diaphragmatic hernia (CDH) was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: normal controls (n = 16), nitrofen induced without CDH (n = 16), and nitrofen-induced CDH (n = 16). Total RNA and DNA were extracted from the lung in each group and measured. mRNA was extracted from total RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expressions of FGF-10 and FGF-7. Levels of mRNA were expressed as a ratio of the band density divided by that of β-actin, a house-keeping gene. Results FGF-10 mRNA expression was decreased significantly in CDH lung (2.914 ± 0.320) compared with controls (4.062 ± 0.307; P < .05) and nitrofen induced without CDH lung (3.923 ± 0.250; P < .01). FGF-7 mRNA expression was decreased significantly in CDH lung (0.777 ± 0.097) compared with controls (1.028 ± 0.093; P < .01). Conclusions Decreased gene expression of FGF-10 and FGF-7 in the hypoplastic lung suggests that pulmonary hypoplasia in nitrofen-induced CDH rat may be caused by reduced synthesis of FGF-10 and FGF-7 during lung morphogenesis.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1: expression in the lung of fetal rats with nitrofen-induced diaphragmatic hernia.

The surrounding extracellular matrix of airway wall tissues changes in response to mechanical stresses and hypoxia. The presence of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), is correlated with collagen degradation and tissue repair in lung disorders. The aim of this study was to evaluate the expression of MMP-9 and TIMP-1 in the lung of fetal rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Administering 100 mg of nitrofen dissolved in 1 ml olive oil to pregnant Wistar rats on day 9 of gestation induced left-sided CDH in fetal rats. In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into two groups: normal controls (n = 10) and nitrofen-induced left-sided CDH (n = 10). Immunoreactivity of the staining for MMP-9 and TIMP-1 in the lung tissues was semiquantitatively analyzed using the staining scores. The relative amount of MMP-9 or TIMP-1 divided by the amount of beta-actin for each lung sample was measured by using the real-time reverse-transcriptase polymerase chain reaction. The immunoreactivity of MMP-9 was significantly increased in the CDH group (n = 5) compared with the control group (n = 5) (p = 0.031). On the other hand, the immunoreactivity of TIMP-1 in the two groups was not significantly different (n = 0.134). The relative amount of MMP-9 (or TIMP-1) in the CDH group (n = 5) does not differ significantly from that in the control group (n = 5) (p = 0.059, 0.596, respectively), but the relative amount of MMP-9 is higher in the CDH group, although it is not significantly higher. On the other hand, the ratios of MMP-9 to TIMP-1 were significantly higher in the CDH group (p = 0.028). In conclusion, fetal rats with nitrofen-induced CDH, a model of respiratory disorders, manifested the excess of MMP-9 activity due to the absence of TIMP-1 that would suggest a trend toward disruption of the extracellular matrix in the CDH lung tissues.

Effect of mechanical ventilation on the pulmonary expression and production of elastin in nitrofen-induced diaphragmatic hernia in rats

Background/Purpose: Hypolastic lung in congenital diaphragmatic hernia (CDH) shows markedly thickened alveolar walls, increased interstitial tissue, and markedly diminished alveolar air space, showing morphologic immaturity. Decrease in lung compliance and distensibility often is seen in human CDH as well as experimentally produced CDH. Collagen and elastin, critical components of the lung connective tissue, have been suggested to have important influence on lung compliance and maximal expansion. The barotrauma caused by mechanical ventilation is known to produce structural changes in the pulmonary architecture. The aim of this study was to investigate the expression and production of elastin in the lung in newborn rats with CDH during mechanical ventilation. Methods: CDH was induced in rat embryos after administration of nitrofen to pregnant dams on day 9.5 of gestation. Cesarean section was performed on day 21 of gestation. The newborn rats were intubated using a 24-gauge Teflon catheter. After ligation of the umbilical cord, the intubated animals were transferred immediately to a warm plate and connected to a modified ventilator. Ventilation was continued for a maximum of 6 hours. The relative amount of soluble elastin in the lung was assessed using an enzyme-linked immunosorbent assay (ELISA) technique. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of tropoelastin mRNA expression in the lung. Results: Elastin mRNA in the CDH lung was increased significantly (P <.01) at one hour after ventilation compared with ventilated controls. Elastin protein significantly increased in the CDH lung at one hour (P <.01) and 6 hours (P <.01) after starting ventilation compared with controls. Conclusion: The data show that during mechanical ventilation, elastin production is increased significantly in the CDH lung, and this may further affect lung compliance. J Pediatr Surg 37:1253-1257. Copyright 2002, Elsevier Science (USA). All rights reserved.

Altered gene level expression of thyroid hormone receptors α-1 and β-1 in the lung of nitrofen-induced diaphragmatic hernia

Purpose: Thyroid receptor alpha-1 (TR-α1) and thyroid receptor beta-1 (TR-β1) are thought to be essential for the fetal and postnatal development of the lung. The authors investigated gene level expression of TR-α1 and TR-β1 in the lung of nitrofen-induced congenital diaphragmatic hernia (CDH) using reverse transcription polymerase chain reaction (RT-PCR). Methods: CDH was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: normal controls (n = 16), nitrofen-induced CDH (n = 16), and nitrofen-treated without CDH (n = 16). mRNA was extracted from the left lung in each group. RT-PCR was performed to evaluate mRNA expressions of TR-α1 and TR-β1. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene. Results: TR-α1 mRNA expression was decreased significantly in CDH lung (1.618 ± 0.148) compared with controls (2.658 ± 0.251; P < .01) and nitrofen-treated without CDH lung (2.232 ± 0.193; (P < .05). TR-β1 mRNA expression also was significantly decreased in CDH lung (2.223 ± 0.270) compared with controls (3.569 ± 0.262; P < .01) and nitrofen-treated without CDH lung (3.235 ± 0.299; P < .05). Conclusion: These data suggest that the downregulation of thyroid hormone signaling pathway through altered expression of TR-α1 and TR-β1 during lung morphogenesis may be a contributory factor in the pathogenesis of pulmonary hypoplasia in nitrofen-induced CDH. J Pediatr Surg 36:1675-1678. Copyright © 2001 by W.B. Saunders Company.

Gene expression of insulin-like growth factor-1 and epidermal growth factor is downregulated in the heart of rats with nitrofen-induced diaphragmatic hernia.

Newborns with congenital diaphragmatic hernia (CDH) still have high mortality. Recently, a possible role of cardiac maldevelopment has been suggested. Human and experimental studies have demonstrated that heart weight is significantly reduced in the presence of CDH. Recent studies have suggested an important role for insulin-like growth factor-I (IGF-I) in the regulation of cardiac growth, structure, and function. Administration of IGF-I to normal rats has been shown to cause cardiac hypertrophy. Epidermal growth factor (EGF) plays an important role in cardiac differentiation and development. The aim of this study was to determine the gene-level expression of IGF-I and EGF in the hearts of rats with nitrofen-induced CDH using the reverse-transcription polymerase chain reaction technique (RT-PCR). CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5 of gestation (term 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into three groups: normal controls (n = 8), nitrofen without CDH (n = 8), and nitrofen-induced CDH (n = 8). Total RNA was extracted from the hearts in each group and measured. mRNA was extracted from total RNA. RT-PCR was performed to evaluate mRNA expressions of IGF-I and EGF. Levels of mRNA were expressed as a ratio of band density divided by that of beta-actin, a housekeeping gene known to be expressed at a constant level. IGF-I mRNA expression was significantly decreased in CDH hearts (0.177 +/- 0.109) compared to controls (0.393 +/- 0.138) (P < 0.01) and nitrofen hearts without CDH (0.321 +/- 0.088) (P < 0.05). EGF mRNA expression was significantly decreased in CDH hearts (0.218 +/- 0.118) compared to controls (0.534 +/- 0.196) (P < 0.01) and nitrofen hearts without CDH (0.383 +/- 0.136) (P < 0.05). Decreased cardiac gene expression of IGF-I and EGF in the hypoplastic heart suggests that cardiac hypoplasia in nitrofen-induced rat CDH may be due to reduced synthesis of IGF-I and EGF by myocytes in the developing heart.

Effect of hyperoxia on surfactant protein gene expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Recently, the role of oxygen (O2) as a regulator of pulmonary surfactant-associated protein (SP) gene expression has been reported. The mRNA level of SP has been demonstrated to be increased in the lungs of animals exposed to hyperoxia. The aim of this study was to investigate SP mRNA expression in hypoplastic CDH lung in rats during mechanical ventilation in order to determine the effect of O2 on SP synthesis in CDH. A CDH model was induced in pregnant rats following administration of nitrofen. The newborn rats with CDH and controls were intubated and ventilated. Ventilation was continued for 6 h under 100% oxygen. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amounts of mRNA expression of SP-A, SP-B, SP-C, and SP-D. Relative amounts of SP-A, SP-B, and SP-D mRNA expression in CDH lung were significantly decreased compared to controls at birth and 6 h after ventilation. There was no significant difference in SP-C mRNA expression between CDH animals and controls. Upregulated mRNA expression of SP-A, SP-B, and SP-D in lungs of control animals at 6 h after ventilation suggests that oxygenation accelerates postnatal SP synthesis in normal lungs. The inability of O2 to increase SP mRNA expression in hypoplastic CDH lung suggests that the hypoplastic lung is not responsive to increased oxygenation for the synthesis of SP.

Antenatal dexamethasone enhances surfactant protein synthesis in the hypoplastic lung of nitrofen-induced diaphragmatic hernia in rats

Background/Purpose: Pulmonary hypoplasia is one of the main causes for the high mortality rate in patients with congenital diaphragmatic hernia (CDH). The expression of surfactant protein A in the hypoplastic CDH lung is reduced, and its concentration is decreased in the amniotic fluid of pregnancies complicated by CDH. In a CDH experimental model, prenatal glucocorticoid treatment has proved its efficacy in correcting the parameters of pulmonary biochemical and morphologic immaturity. The aim of this study was to investigate whether maternal administration of dexamethasone has any effect on the expression of surfactant protein A and surfactant protein B in nitrofen-induced experimental CDH rat model. Methods: CDH was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (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 = 16); group II, nitrofen-induced CDH (n = 16); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 16). Indirect immunohistochemistry was performed using alkaline-phosphatase-coagulated streptavidin using anti-SP-A and anti-SP-B polyclonal antibodies. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate relative amount of SP-A and SP-B mRNA expression. Results: In the CDH lung (group II) we observed a markedly reduced number of type II pneumocytes positive for SP-A, and SP-B was increased to a level close to that of the control group. The relative amount of SP-A and SP-B was reduced significantly in group II compared with controls (P <.05) and significantly increased in group III compared with group II animals (P <.01). Conclusion: These results suggest that antenatal glucocorticoid treatment increases the production of surfactant proteins in the CDH hypoplastic lung. J Pediatr Surg 35:1468-1473. Copyright © 2000 by W.B. Saunders Company.

Antenatal dexamethasone improves atrial natriuretic peptide receptors in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats.

Atrial natriuretic peptide (ANP) plays a major role in electrolyte and volume homeostasis through potent biological effects including vasorelaxation, bronchorelaxation, lung permeability, and clearance. There are two distinct biochemical and functional classes of ANP receptors, guanylate cyclase receptor (GC-R) and clearance receptors (clearance-R). Two subtypes of GC-R have been described, GCA-R and GCB-R. Antenatal glucocorticoid therapy (AGT) has been demonstrated to improve pulmonary immaturity and abnormal structure of pulmonary arteries in animal models of congenital diaphragmatic hernia (CDH). The aim of this study was to investigate the effect of antenatal glucocorticoid administration on the ANP system in nitrofen-induced CDH hypoplastic lung in rats. A CDH model was induced in pregnant rats following administration of nitrofen on day 9.5 of gestation. Dexamethasone (Dex) was given intraperitoneally on days 18.5 and 19.5; cesarean section was performed on day 21. Reverse transcription polymerase chain reaction was performed to evaluate the relative amounts of GCA-R, GCB-R and clearance-R mRNA expression. The mRNA expression of GCA-R, GCB-R, and clearance-R was significantly increased in CDH compared to control lung. ANP receptor mRNA expression was significantly decreased in CDH lung with compared to without Dex treatment. Our finding of increased ANP receptor mRNA expression in CDH lung suggests that the hypoplastic lung has high sensitivity for ANP. Decreased mRNA expression of ANP receptors in CDH lung after Dex treatment suggests that AGT may improve pulmonary physiological function of ANP in hypoplastic CDH lung.

Antenatal dexamethasone enhances endothelin receptorB expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats

Background/Purpose: The hypoplastic lung and persistent pulmonary hypertension (PPH) are the principle causes of high mortality and morbility in infants with congenital diaphragumatic hernia (CDH). Endothelin-1 (ET 1), which is produced by vascular endothelial cells and some leukocytes, plays a key role in modulating pulmonary vascular tone in PPH. Two different receptors (ETA and ETB) for ET-1 have been characterized. Binding of ET-1 to ETA, which is present on smooth muscle cells in fetal lung, results in vasoconstriction. However, binding of ET-1 to ETB which is present on endothelial cells results in vasodilation mediated by endogenous nitric oxide. Antenatal glucocorticoid therapy has been shown to prevent abnormal pulmonary arterial structural changes in animal model with CDH. The aim of this study was to investigate the effect of antenatal glucocorticoid administration on ET-1 system in nitrofen-induced CDH hypoplastic lung in rats.Methods: A CDH model was induced in pregnant rats after administration of nitrofen on day 9.5 of gestation. Dexamethazone (Dex) was given intraperitoneally on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. Rat ET-1 protein expression was measured in solubilized lung tissue extracts, by sandwich type enzyme-linked immunosorbent assay (ELISA) analysis. Reverse transcription polymerase chain reaction was performed to evaluate the relative amount of ET-1, ET., and ETB mRNA expression.Results: The ET-1 protein and mRNA expression of ET-1 and both receptors were increased significantly in CDH lung compared with controls. Although there was no significant difference in ETA mRNA expression between CDH lung with Dex treatment and without Dex treatment, ETB mRNA expression was elevated significantly in CDH lung with Dex treatment compared with CDH lung without Dex treatment.Conclusion: These findings suggest that antenatal glucocorticoid therapy may modulate pulmonary vascular tone in CDH hypoplastic lung by selectively upregulating local expression of ETB

Antenatal dexamethasone suppresses tumor necrosis factor-alpha expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats.

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Tumor necrosis factor-alpha (TNF-alpha) drastically decreases surfactant phospholipids synthesis by isolated human type II pneumocytes. Recently, it was shown that TNF-alpha mRNA expression is increased in human hypoplastic CDH lung. Antenatal glucocorticoid therapy demonstrates improved surfactant biochemical immaturity in an animal CDH model. The aim of this study was to investigate the effect of antenatal dexamethasone (Dex) on TNF-alpha protein and gene expression in nitrofen-induced CDH hypoplastic lung in rats. A CDH model was induced in pregnant rats after the administration of nitrofen on d 9.5 of gestation. Dex was given intraperitoneally on d 18.5 and 19.5. Cesarean section was performed on d 21. In situ hybridization was performed with a rat TNF-alpha-specific and digoxigenin-labeled oligonucleotide probe. TNF-alpha level was measured in solubilized lung tissue extracts by ELISA. In control lung, TNF-alpha mRNA expression was weak or absent. In contrast, strong TNF-alpha mRNA expression was demonstrated in type II pneumocytes and bronchiolar epithelium in CDH lung. In Dex-treated CDH lung, TNF-alpha mRNA expression was weak in both type II pneumocytes and the bronchiolar epithelium. The level of TNF-alpha was elevated significantly in CDH lung compared with levels in control lung extracts (p < 0.01). In Dex-treated CDH lung, TNF-alpha protein was significantly decreased compared with CDH lung (p < 0.05). Our findings suggest that the reduction in the local production of TNF-alpha may be one contributing mechanism by which antenatal glucocorticoid therapy improves pulmonary parenchymal immaturity, including surfactant.

Pulmonary neuroendocrine cells in nitrofen-induced diaphragmatic hernia and the effect of prenatal glucocorticoids.

The high mortality associated with congenital diaphragmatic hernia (CDH) is due to pulmonary hypoplasia and hypertension, structural and functional abnormalities which can to some extent be ameliorated by prenatal administration of glucocorticoids. In the hypoplastic, hypertensive lungs of neonatal rats in which CDH has been induced by nitrofen, those pulmonary neuroendocrine cells (PNCs) containing calcitonin gene-related peptide (CGRP) increase in number, and it has been suggested that this might be due to inhibition of secretion of the peptide, the consequent decrease in its vasodilatory effects contributing to the hypertension. Whether this increase affects the entire population of PNCs, however, and how these cells are affected by administration of prenatal glucocorticoids, is unknown. As revealed by immunolabelling for protein gene product (PGP) 9.5, a general marker of NCs and expressed per cm2 tissue section, the total PNC population in rats with nitrofen-induced CDH was significantly greater than in controls receiving only olive oil (672 vs 375/cm2, P = 0.03) and was further increased (824 per cm2) in animals treated prenatally with dexamethasone (n = 8 in all groups). The increase in the total PNC population in rats with CDH is similar in magnitude to that described for the CGRP-containing subpopulation. Since the major role of the products of PNCs is now thought to be the regulation of development of pulmonary tissues and their response to injury, it is probable that the expansion of their population in the abnormal lungs associated with CDH is an adaptive response to pulmonary maldevelopment, a response possibly augmented by exogenous corticosteroids.