Altered gene level expression of thyroid hormone receptors alpha-1 and beta-1 in the lung of nitrofen-induced diaphragmatic hernia.

PURPOSE: Thyroid receptor alpha-1 (TR-alpha1) and thyroid receptor beta-1 (TR-beta1) are thought to be essential for the fetal and postnatal development of the lung. The authors investigated gene level expression of TR-alpha1 and TR-beta1 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-alpha1 and TR-beta1. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene. RESULTS: TR-alpha1 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-beta1 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-alpha1 and TR-beta1 during lung morphogenesis may be a contributory factor in the pathogenesis of pulmonary hypoplasia in nitrofen-induced CDH.

Cardiac gene expression and synthesis of atrial natriuretic peptide in the nitrofen model of congenital diaphragmatic hernia in rats: effect of prenatal dexamethazone treatment.

BACKGROUND/PURPOSE: The aim of this study was to determine the gene and protein levels of atrial natriuretic peptide (ANP) in the heart of nitrofen-induced congenital diaphragmatic hernia (CDH) in rats and to evaluate the effect of antenatal dexamethazone (Dex) treatment. METHODS: CDH model was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, day 22). Dexamethazone (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 = 10); group II, nitrofen-induced CDH (n = 10); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 10). ANP protein was measured using enzyme-linked immunosorbent assay (ELISA) technique. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of atrial natriuretic peptide (ANP) mRNA expression. RESULTS: There was a significant reduction in ANP mRNA (P <.05) and protein (P <.01) levels in heart of group II (CDH) compared with group I. Antenatal Dex treatment significantly increased both ANP mRNA and protein levels in the heart of CDH animals (P <.05). CONCLUSIONS: The reduced cardiac ANP gene expression and ANP synthesis indicates that the heart in CDH is functionally immature and may be unable to respond to hemodynamic load accompanying persistent pulmonary hypertension (PPH). ANP or drugs such as steroids, which raise endogenous ANP levels, may have a therapeutic application in CDH complicated by PPH.

Selective neurotrophin deficiency in infantile hypertrophic pyloric stenosis.

BACKGROUND/PURPOSE: Increasing evidence suggests that the enteric nervous system is under the control of neurotrophins. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), promote differentiation, growth, and survival of various central and peripheral nervous system neurons. The biological effects of neurotrophins are mediated by the interactions with high-affinity tyrosine kinase receptors (TrkA, TrkB, TrkC). Recently, abnormalities of intramuscular innervation have been reported in infantile hypertrophic pyloric stenosis (IHPS). To further understand the reported abnormalities in pyloric innervation in IHPS, the authors analyzed the expression of Trk receptors and the neurotrophins content in IHPS. METHODS: Full-thickness muscle biopsy specimens were obtained from 8 IHPS patients (age range, 23 to 41 days) at pyloromyotomy and from 8 age-matched controls without gastrointestinal disease at autopsy performed within 12 hours after death. Indirect immunohistochemistry was performed using ABC (Avidin Biotin peroxidase Complex) method with anti-Trk specific antibodies (A,B,C). Quantitative analysis was performed using sandwich-type ELISA for NGF, BDNF, NT-3, and NT-4/5. RESULTS: The intensity of staining of the myenteric plexus for TrkA, TrkB, and TrkC was similar among IHPS and controls. There was a lack of TrkA-positive nerve fibers in IHPS compared with controls. The quantity of total NGF, NT-3, and BDNF in IHPS was significantly lower than in controls. CONCLUSIONS: The reduced production of neurotrophins in IHPS may be responsible for the delay in the functional and structural maturation of pyloric innervation in IHPS. The lack of TrkA-positive nerve fibers in pyloric muscle may explain the abnormal intramuscular innervation in IHPS.

Structural immaturity of the heart in congenital diaphragmatic hernia in rats.

PURPOSE: The form and function of the heart are the final result of an integration of cells, tissues, and extracellular material. The extracellular matrix (ECM) is a complex array of different molecular components, and it plays an important role for the transfer of mechanical force in both contraction and relaxation phases in the cardiac cycle. ECM plays also a significant role in the development of the heart. The aim of this study was to evaluate the expression of important ECM components in the heart of rats with induced CDH to test the hypothesis that an alteration of ECM may contribute to the cardiac maldevelopment, which recently has been identified as a contributive factor for the high mortality rate in babies with congenital diaphragmatic hernia (CDH). METHODS: CDH model 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 2 groups: normal control (n = 10) and nitrofen-induced CDH (n = 10). Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of tropoelastin and alpha1 (I) procollagen mRNA expression. Elastin protein content was measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: There was a reduction in tropoelastin mRNA (P <.05) and procollagen mRNA (P <.05) in CDH compared with controls. The cardiac alpha-elastin content also was reduced in CDH (P <.01). CONCLUSIONS: The reduced cardiac tropoelastin and procollagen gene expression and the reduced alpha-elastin content indicate that the heart in CDH structurally is immature. The reduced production of cardiac ECM may contribute to a contractile dysfunction, which makes the heart unable to respond to the hemodynamic load accompanying persistent pulmonary hypertension (PPH).

Antenatal dexamethasone administration increases fetal lung DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia in rats.

Antenatal glucocorticoids treatment has been shown to correct pulmonary immaturity. The thymidine analog bromodeoxyuridine (BrdU) is incorporated into S-phase cells and used as a marker of DNA synthesis. In this study, we investigated the effect of antenatal glucocorticoid administration on DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia (CDH) in rats to better understand the effect of antenatal glucocorticoids on CDH lung. The CDH model was induced in pregnant rats using nitrofen. Dexamethasone (0.25 mg/kg) was given on d 18.5 and 19.5 of gestation (term = 22 d). BrdU was administered 1 h before fetuses were killed on d 21, and detected by immunohistochemistry. DNA synthesis was evaluated by percentage of BrdU-incorporated nuclei (BrdU labeling index). Total RNA and soluble protein were extracted from another set of left lungs to measure RNA and protein content. BrdU labeling index and total RNA content were significantly decreased in CDH lung compared with control rats. Antenatal dexamethasone treatment significantly increased BrdU labeling index and RNA and protein content in the left CDH lung. Our findings of decreased DNA synthesis and decreased RNA and protein content in CDH lung suggest that lung growth and development are suppressed in hypoplastic CDH lung. Increased DNA synthesis and increased RNA and protein content in dexamethasone-treated CDH lung suggest that antenatal glucocorticoids may accelerate fetal lung growth and development in CDH.

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.

The hypoplastic heart in congenital diaphragmatic hernia: reduced expression of basic fibroblast growth factor and platelet-derived growth factor.

Newborn infants with congenital diaphragmatic hernia (CDH) still have high mortality. Recently, the possible role of a cardiac maldevelopment in the high mortality has been suggested. Human and animal studies have demonstrated that heart weight is significantly reduced in the presence of CDH. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are pleiotropic regulatory peptides that are expressed in myocardium in precise developmental and spatial programs. PDGF and bFGF both stimulate cardiac growth by inducing cell proliferation and stimulating the synthesis of extracellular matrix. The aim of this study was to investigate the presence of heart hypoplasia in nitrofen-induced CDH in rats and the role of specific tissue growth factors (bFGF and PDGF) in its genesis. 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 in two groups: normal controls (n = 8) and nitrofen-induced CDH (n = 8). Total RNA, DNA, and soluble proteins were extracted from the heart in each group and measured. mRNA was extracted from total RNA and a reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression of bFGF and PDGF. The heart/body weight ratio (HBWR) and DNA content were significantly decreased (P < 0.01) in CDH animals compared to controls. RNA and protein content were also reduced in CDH. The expression of bFGF and PDGF mRNA was significantly reduced in the CDH group compared to controls (P < 0.01). The decreased HBWR, DNA, RNA, and protein content in the CDH heart indicates that the heart is hypoplastic in nitrofen-induced left CDH. The downregulation of bFGF and PDGF gene expression in the CDH heart suggests that these regulating peptides may play an important role in the genesis of cardiac hypoplasia in CDH.

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.

Structural immaturity of the pylorus muscle in infantile hypertrophic pyloric stenosis.

Recent reports indicate that extracellular matrix and cytoskeleton plasmalemmal elements are altered in infantile hypertrophic pyloric stenosis (IHPS). Desmin is a cytoskeletal protein that is important for the organization and function of muscular fibers. It has been found to be increased in the smooth muscle in chronic intestinal pseudo-obstruction and in skeletal muscle in some forms of myopathies as well as in unexplained hypertrophic cardiomyopathies. The aim of this study was to analyze the expression of desmin in IHPS. Full-thickness muscle-biopsy specimens were obtained from 8 IHPS patients (age range 23 to 41 days) at pyloromyotomy, from 8 age-matched controls without evidence of gastrointestinal (GI) disease at autopsy, and from 2 stillborns who died at 27 and 30 weeks of gestation without evidence of GI disease. Indirect immunohistochemistry was performed using the avidin-biotin-peroxidase complex method with anti-desmin and visualized by development with 3-diaminobenzidine tetrahydrochloride. Pyloric muscle in IHPS demonstrated strong desmin immunoreactivity. The expression of desmin was also strong in the muscular layers of fetal pylorus. In the age-matched controls absent or weak desmin immunoreactivity was seen in the pyloric muscle layer. The increased amount of desmin in hypertrophied pyloric muscle in IHPS may result in inco-ordination of contraction and relaxation of the pylorus, thus causing motility dysfunction. The similar pattern of desmin expression in IHPS and fetal pylorus suggests that the organization of intermediate filaments in IHPS is in a fetal stage of development.

Glial-derived growth factor signaling pathway in infantile hypertrophic pyloric stenosis.

BACKGROUND/PURPOSE: Glial-derived growth factor (GDNF), which is the ligand of RET is reported to be essential for the development of enteric nervous system. A GDNF knockout mouse model has shown that the gastric region is a critical passing site between GDNF-RET-independent neuroblasts (colonizing the esophagus) and GDNF-RET-dependent neuroblasts (colonizing the small and large bowel). The earliest GDNF site of production is the mesenchyme and the outer smooth muscle cell (SMC) layer of the developing bowel. In the mature gastrointestinal tract the presence of GDNF is restricted to enteric glial cells. The aim of this study was to investigate the expression of GDNF and RET in infantile hypertrophic pyloric stenosis (IHPS). METHODS: Full-thickness muscle biopsy specimens were obtained from 8 IHPS patients at pyloromyotomy and from 8 age-matched controls without gastrointestinal disease. Indirect immunohistochemistry was performed using avidin-biotin-peroxidase complex method with anti-GDNF and anti-RET antibodies. Quantitative analysis was performed using sandwich-type enzyme-linked immunosorbent assay (ELISA) for GDNF. RESULTS: GDNF- and RET-positive nerve fibers were absent or markedly reduced in IHPS compared with controls. GDNF was expressed strongly by smooth muscle cells of both muscular layers in IHPS, whereas no GDNF expression was detected in pyloric muscle of controls. The quantity of total GDNF in IHPS was significantly higher than in controls (P < .01). CONCLUSIONS: The lack or markedly decreased number of GDNF-positive nerve fibers in IHPS supports the hypothesis of a selective immaturity of the enteric glia in the muscular layers in IHPS. The strong expression of GDNF in smooth muscle cells in IHPS and the increased levels of GDNF in IHPS suggest a compensatory mechanism by which the smooth muscle cells continue to produce GDNF until maturation of the enteric glial cells occurs.

Gastroesophageal reflux in neurologically impaired children: partial or total fundoplication?

BACKGROUND: It is difficult to give guidelines when approaching gastroesophageal disease in neurologically impaired children. Indication for surgery has been increasing over recent years, but there is no consensus on the surgical technique of choice. Nothing has been written specifically comparing the results of different procedures in these patients, so far. STUDY DESIGN: We retrospectively compare the short- and long-term results of two different types of fundoplication in a series of children operated on for documented gastroesophageal reflux disease at our institution. RESULTS: One group (group A) of 27 patients, operated on between 1977 and 1993, underwent Nissen fundoplication, the other (group B), formed of 20 patients all of whom were operated on between 1993 and 1995, underwent Thai fundoplication. We compared the results in terms of positive outcome (recovery) and negative outcome (minor and major complication), computing the relative odds of group A versus group B in terms of risk of complication, and we compared the mean operative time and the length of hospital stay by means of a student's t-test analysis. CONCLUSIONS: Our results show that there is no statistical difference between the two procedures in terms of relative risk of complication and success rate. The duration of surgery and hospital stay were significantly shorter in group B. The Thal procedure can, therefore, be proposed as first choice in the management of these patients.