RESUMEN
INTRODUCCIÓN: Una Reintervención Quirúrgica No Programada (RQNP) es aquella cirugía no planificada que se rea liza durante los primeros 30 días como consecuencia de una cirugía primaria. En Chile, el análisis y la tasa de RQNP son un indicador de calidad. OBJETIVO: describir y analizar las RQNP en pediatría. PACIENTES Y MÉTODO: Estudio observacional de corte transversal. Se revisaron los registros clínicos de los pacientes pediátricos sometidos a RQNP en el Hospital Carlos Van Buren en un período de 5 años. Se analizó su incidencia, indicaciones y causas que se clasificaron en 1) causas atribuibles a la técnica quirúrgica; 2) causas relacionadas al tratamiento; 3) patología propia del paciente y 4) otras causas. Se analizó además el cumplimiento de reuniones de análisis de RQNP. RESULTADOS: Se efectuaron 23 RQNP de un total de 5.503 cirugías en 5 años (0,42%). Hubo 11 RQNP de 3.434 cirugías electivas realizadas y 12 RQNP de 2069 cirugías de urgencia realizadas (0,32% v/s 0,58% respectivamente, p = NS). Hubo 2 RQNP en los 82 recién nacidos operados en el período (2,43%, p < 0,01). En todos los casos se realizaron reuniones de análisis de RQNP. En 18 de los 23 pacientes sometidos a RQNP se encontró una causa atribuible a la técnica o planificación quirúrgica. CONCLUSIONES: Las RQNP son poco frecuentes en pediatría excepto en el período neonatal. Se da total cumplimiento a la normativa nacional de reunión de análisis luego de una RQNP que indican que las causas son mayoritariamente atribuibles a la técnica o planificación quirúrgica.
INTRODUCTION: An Unplanned Return to the Operating Room (UROR) is an unplanned surgery performed during the first 30 days as a result of primary surgery. In Chile, the analysis and the UROR rate are quality indicators. OBJECTIVE: to describe and analyze UROR in a pediatrics. PATIENTS AND METHOD: Observa tional cross-sectional study. The clinical records of pediatric patients undergoing UROR at the Hos pital Carlos Van Buren over 5 years were reviewed. The incidence, indications, and causes of UROR were analyzed. The causes of UROR were classified as 1) causes attributable to surgical technique, 2) treatment-related causes, 3) the patient pathology, and 4) other causes. In addition, the observance of the case review meetings after an UROR was analyzed. RESULTS: 23 UROR out of 5,503 surgeries were performed in 5 years, (0.42%). There were 11 UROR out of 3,434 elective surgeries and 12 UROR out of 2,069 emergency ones (0.32% v/s 0.58% respectively, p=NS). There were 2 UROR out of 82 surgeries in newborns, (2.43%, p<0.01). After every UROR, a case review meeting was held. In 18 out of the 23 patients who underwent UROR (78%), the cause was attributable to the surgical technique or planning. CONCLUSIONS: UROR is rare in pediatric surgery, except for the newborn period. Case review meetings are held after every UROR case, according to the national guidelines. The causes of UROR are mostly attributable to the surgical technique or planning.
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Humanos , Masculino , Femenino , Recién Nacido , Lactante , Preescolar , Niño , Adolescente , Calidad de la Atención de Salud , Reoperación/estadística & datos numéricos , Atención a la Salud/normas , Chile , Estudios TransversalesRESUMEN
INTRODUCTION: An Unplanned Return to the Operating Room (UROR) is an unplanned surgery performed during the first 30 days as a result of primary surgery. In Chile, the analysis and the UROR rate are quality indicators. OBJECTIVE: to describe and analyze UROR in a pediatrics. PATIENTS AND METHOD: Observa tional cross-sectional study. The clinical records of pediatric patients undergoing UROR at the Hos pital Carlos Van Buren over 5 years were reviewed. The incidence, indications, and causes of UROR were analyzed. The causes of UROR were classified as 1) causes attributable to surgical technique, 2) treatment-related causes, 3) the patient pathology, and 4) other causes. In addition, the observance of the case review meetings after an UROR was analyzed. RESULTS: 23 UROR out of 5,503 surgeries were performed in 5 years, (0.42%). There were 11 UROR out of 3,434 elective surgeries and 12 UROR out of 2,069 emergency ones (0.32% v/s 0.58% respectively, p=NS). There were 2 UROR out of 82 surgeries in newborns, (2.43%, p<0.01). After every UROR, a case review meeting was held. In 18 out of the 23 patients who underwent UROR (78%), the cause was attributable to the surgical technique or planning. CONCLUSIONS: UROR is rare in pediatric surgery, except for the newborn period. Case review meetings are held after every UROR case, according to the national guidelines. The causes of UROR are mostly attributable to the surgical technique or planning.
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Atención a la Salud/normas , Calidad de la Atención de Salud , Reoperación/estadística & datos numéricos , Adolescente , Niño , Preescolar , Chile , Estudios Transversales , Femenino , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
Biliary atresia leads to cirrhosis in the vast majority of patients and constitutes the first cause of paediatric liver transplantation. Animal models allow us to understand the molecular basis and natural history of diseases. The aim of this study is to describe a surgically created animal model of biliary atresia with emphasis in long-term liver function. Forty-two 3-week-old Sprague-Dawley rats were randomly divided into two groups: bile duct ligature (BDL) and control. The animals were sacrificed on the 2nd, 4th, and 6th postoperative weeks. Blood samples were collected for liver function analysis. The spleen to body weight ratio was determined. Histopathological examination of liver tissue was performed by hematoxylin-eosin and Sirius red staining. Collagen quantification was determined by using colorimetric digital image analysis and was expressed as a percentage of total liver tissue area. Quantitative real-time polymerase chain reaction was performed to analyse gene expression levels of transforming growth factor-ß1 (Tgfb1) and apeline (Apln) genes. Statistical analysis was performed where P<0.05 was considered significant. Animals from BDL group developed increasing cholestasis with clinical and laboratory features. Splenomegaly was detected at 4th and 6th week (P<0.05). Histological evaluation of the liver showed ductular reaction, portal fibrosis and bile plugs. Collagen area to total liver tissue area had a median of 2.5% in the control group and 6.5 %, 14.3 % and 37.7 % in BDL rats at 2nd, 4th and 6th weeks respectively (P<0.001). Tgfb1 mRNA expression level was significantly higher at 6th week (P<0.001) in BDL group when compared to control. Apln mRNA expression level was significantly higher at 4th and 6th week (P<0.001) and showed a positive linear correlation (r = 0.975, P<0.05) in BDL group when compared to control. Bile duct ligature in young rats is an animal model that recreates clinical, laboratory, histological and molecular findings of biliary atresia. Bile duct ligature constitutes a good animal model to investigate therapeutic approaches for modifying the progression of liver fibrosis in biliary atresia.
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Conductos Biliares , Atresia Biliar/patología , Modelos Animales de Enfermedad , Amiloide , Animales , Conductos Biliares/patología , Conductos Biliares/cirugía , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Colágeno/genética , Colágeno/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Ligadura , Hígado/patología , Proteínas de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/patología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
PURPOSE: Intestinal dysganglionosis are a group of anomalies of the enteric nervous system that constitute infrequent but severe forms of constipation. Histochemical stainings are the gold standard diagnostic procedure for intestinal dysganglionosis. This study describes our experience with histochemistry in a large series of patients. METHODS: Between 1977 and 2010, 1,589 biopsies from children with persistent chronic constipation were studied. The specimens were snap frozen, sectioned and stained with acetylcholinesterase (AChE), acetylcholinesterase counterstained with hematoxilin and succinic dehydrogenase (SDH) histochemical stainings. RESULTS: Among the 1,589 biopsies, 946 (59.5%) were rectal biopsies, 242 (15.2%) were internal sphincter biopsies, 346 (21.8%) were intestinal mapping studies and 42 (2.7%) of them were colon specimens from surgical resections. From the rectal biopsy group, 544 (57.5%) patients were reported as normal. Hirschsprung disease was found in 163 (17.2%) patients with a median age at diagnosis of 8 months and a male to female ratio of 3:1. Intestinal neuronal dysplasia was found in 162 (17.2%) patients, hypoganglionosis in 3 (0.3%) of them and ganglioneuromatosis in 1 (0.1%). In 73 (7.7%) patients, the biopsy was not conclusive for different reasons. 34 out of the 42 resected colon specimens were Hirschsprung disease. Intestinal neuronal dysplasia was found in the proximal segment of the aganglionic bowel in 15 out of 34 (44%) patients. All the aganglionic resected colon specimens had a previous aganglionic rectal biopsy. There were no false positive results in this group. CONCLUSIONS: Histochemical stainings continue to be the gold standard in the diagnosis of intestinal dysganglionosis. The combination of two histochemical staining techniques provides a high level of accuracy in the diagnosis of intestinal dysganglionosis.
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Estreñimiento/metabolismo , Sistema Nervioso Entérico/anomalías , Enfermedades Intestinales/diagnóstico , Recto/patología , Niño , Preescolar , Enfermedad Crónica , Colon/metabolismo , Colon/patología , Estreñimiento/patología , Sistema Nervioso Entérico/patología , Femenino , Enfermedad de Hirschsprung/diagnóstico , Humanos , Hiperplasia , Inmunohistoquímica , Lactante , Masculino , Recto/metabolismo , Plexo Submucoso/patologíaRESUMEN
BACKGROUND: It has been reported that the smooth muscles in fetal airways exhibit spontaneous phasic contractions throughout gestation. However, the mechanism of these spontaneous contractions is unknown. In the bowel wall, interstitial cells of Cajal (ICCs), which are derived from c-kit positive precursor cells, play an important role as pacemaker cells responsible for the spontaneous, rhythmic activity in the smooth muscle cells. In this study, we investigated the spatial and temporal expression patterns of c-kit positive cells in the embryonic lung and its relationship to the smooth muscle cells surrounding the trachea and the bronchus. METHODS: Rat fetuses were removed from timed pregnant dams on embryonic days (E) 11.5, 13.5, 15.5, and 17.5. Immunohistochemical studies with anti c-kit antibody and anti α-SMA antibody were carried out using frozen sections. RESULTS: A small number of c-kit positive cells were observed in the mesenchyme of the lung bud on day E 11.5. They were markedly increased in number on day E 13.5. On day E 15.5 and on day E 17.5, strong c-kit expressions were observed on the vascular wall and moderate expressions in the mesenchyme. C-kit positive cells co-localized with α-SMA positive smooth muscle cells surrounding the airway epithelium. CONCLUSION: Co-localization of c-kit positive cells and airway smooth muscles in the fetal lung suggests that c-kit positive cells may play an important role in the spontaneous contractions of fetal airways. C-kit expressions in the fetal pulmonary vascular wall suggest that these cells may play an important role in vasculogenesis and angiogenesis of the embryonic lung.
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Células Intersticiales de Cajal/metabolismo , Pulmón/embriología , Preñez , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Animales , Femenino , Inmunohistoquímica , Pulmón/metabolismo , Masculino , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Rectal suction biopsy (RSB) is the gold standard diagnostic procedure for disorders of bowel motility. This study describes our experience with RSB stained with histochemistry as the first diagnostic approach in a large series of patients presenting with chronic constipation. Between 1993 and 2005, 766 children underwent RSB for persistent chronic constipation. The specimens were snap frozen, sectioned and stained with conventional hematoxylin and eosin (H&E) and with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and acetylcholinesterase (AChE) histochemical stainings. Adequate amount of submucosa was present in 655 (85.5%) out of 766 cases and formed the basis of this study. RSB in 540 (82%) patients were reported as normal. Hirschsprung's disease was found in 47 (7.2%) patients with characteristic features of absence of ganglion cells, increased AChE activity in the lamina propria and muscularis mucosae, thick nerve fibers in the submucosa, and a lack of NADPH-d-positive fibers in muscularis mucosae. RSB in 59 (9%) patients presented features of intestinal neuronal dysplasia such as submucosal hyperganglionosis, giant ganglia, ectopic ganglia and increased AChE activity in lamina propria. Hypoganglionosis was suspected in nine (1.3%) children because of sparse or absent ganglion cells and low AChE and NAPDH-d activity in muscularis mucosae. Three patients (0.4%) developed bleeding following RSB, requiring diathermy of the bleeding point. Thus, we conclude that RSB is a simple and safe method when used as the first diagnostic approach in patients with chronic constipation. The combination of two histochemical stainings techniques provides a high level of accuracy in the diagnosis of intestinal dysganglionosis.
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Acetilcolina/análisis , Biopsia con Aguja/métodos , Estreñimiento/patología , NADP/análisis , Recto/patología , Coloración y Etiquetado/métodos , Adolescente , Niño , Preescolar , Enfermedad Crónica , Estreñimiento/metabolismo , Diagnóstico Diferencial , Ganglios Simpáticos/química , Ganglios Simpáticos/patología , Humanos , Lactante , Recién Nacido , Músculo Liso/química , Músculo Liso/inervación , Músculo Liso/patología , Recto/química , Recto/inervación , Reproducibilidad de los Resultados , Estudios RetrospectivosRESUMEN
Congenital diaphragmatic hernia (CDH) is a major life-threatening cause of respiratory failure in the newborn. Recent data reveal the role of a retinoid-signaling pathway disruption in the pathogenesis of CDH. We describe the epidemiology and pathophysiology of human CDH, the metabolism of retinoids and the implications of retinoids in the development of the diaphragm and lung. Finally, we describe the existing evidence of a disruption of the retinoid-signaling pathway in CDH.
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Hernias Diafragmáticas Congénitas , Retinoides/metabolismo , Hernia Diafragmática/metabolismo , Humanos , Recién Nacido , Transducción de SeñalRESUMEN
BACKGROUND/PURPOSE: Severe pulmonary hypoplasia remains the main cause of the high mortality in newborn infants with congenital diaphragmatic hernia (CDH). Retinoids are a family of molecules derived from vitamin A, which play an important role in lung development. We hypothesized that retinoids promote alveologenesis at the end of gestation and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs in CDH. METHODS: Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation. Retinoic acid 5 mg/kg was given intraperitoneally on days 18, 19, and 20 of gestation and fetuses were recovered on day 21. We had 4 study groups: control (n = 24), control + retinoic acid (n = 22), CDH (n = 24), and CDH + retinoic acid (n = 19). Lungs from the 4 study groups were fixed, and the following stereological measurements were performed on vertical random sections: total lung volume, volume density of airspaces, volume density of air walls, gas exchange surface area, alveolar volume, and total number of alveoli per lung. Total DNA content of each lung was measured using a spectrophotometer. RESULTS: Total lung volume increased in CDH lungs after the addition of retinoic acid but remained the same in the control group. Gas exchange surface area was larger in CDH lungs after the addition of retinoic acid but remained unchanged in the control group. The total number of alveoli per lung was higher after the addition of retinoic acid. Total DNA content as well as total DNA content-lung weight ratio of the left lung increased significantly in the CDH group after the addition of retinoic acid compared with CDH without retinoic acid. CONCLUSIONS: Our results demonstrate that prenatal treatment with retinoic acid stimulates alveologenesis in hypoplastic lungs in CDH.
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Hernias Diafragmáticas Congénitas , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/embriología , Anomalías del Sistema Respiratorio/prevención & control , Tretinoina/administración & dosificación , Animales , Modelos Animales de Enfermedad , Femenino , Madurez de los Órganos Fetales/efectos de los fármacos , Inyecciones Intraperitoneales , Pulmón/efectos de los fármacos , Pulmón/embriología , Éteres Fenílicos , Embarazo , Preñez , Atención Prenatal , Probabilidad , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Anomalías del Sistema Respiratorio/tratamiento farmacológico , Sensibilidad y EspecificidadRESUMEN
PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results demonstrate that prenatal treatment with RA accelerates AEC-I proliferation in the hypoplastic lung in CDH.
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Proliferación Celular/efectos de los fármacos , Hernias Diafragmáticas Congénitas , Pulmón/anomalías , Alveolos Pulmonares/efectos de los fármacos , Anomalías del Sistema Respiratorio/tratamiento farmacológico , Tretinoina/farmacología , Animales , Modelos Animales de Enfermedad , Esquema de Medicación , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Hernia Diafragmática/fisiopatología , Inmunohistoquímica , Éteres Fenílicos , Embarazo , Preñez , Atención Prenatal , Probabilidad , Alveolos Pulmonares/citología , ARN Mensajero/análisis , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Anomalías del Sistema Respiratorio/patología , Anomalías del Sistema Respiratorio/prevención & control , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Retinoids play a key role in lung development. Recent studies suggest that retinoid signalling pathway may be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH), but the exact mechanism is not clearly understood. We hypothesized that nitrofen interferes with cellular uptake of retinol during lung morphogenesis and therefore designed this study to examine total retinol levels in lung, liver, and serum, and the gene expression of main components of the retinoid pathway in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to vehicle or 100 mg of nitrofen on day 9 of gestation. Term fetuses were divided in control and nitrofen with CDH and without CDH groups. Retinol levels in serum, lungs, and liver were measured using high-performance liquid chromatography. Reverse transcriptase-polymerase chain reaction was performed to evaluate the relative amount of cellular retinol-biding protein I, retinal dehydrogenase 1a2 and 1a3 (Aldh1a2 and Aldh1a3), retinoic acid receptors alpha and beta (RARalpha, RARbeta), and retinoid X receptor alpha (RXRalpha) expression in the lung. RESULTS: Total retinol levels in the lungs were significantly lower in both nitrofen with CDH (1.78 +/- 0.37 microg/g) and nitrofen without CDH (1.61 +/- 0.24 microg/g) groups compared with controls (2.43 +/- 0.31 microg/g) (P < .001), whereas serum retinol levels were significantly higher in nitrofen with and without CDH groups (0.77 +/- 0.13 and 0.75 +/- 0.11 microg/g, respectively) compared with controls (0.58 +/- 0.12 microg/g) (P < .001). There was no significant difference in liver retinol levels between the 3 groups. Relative expression of cellular retinol-biding protein I, Aldh1a3, RARalpha, RARbeta, and RXRalpha were significantly up-regulated in the lungs of the nitrofen with CDH group (0.70 +/- 0.15, 3.94 +/- 0.91, 2.15 +/- 0.47, 3.49 +/- 1.00, 1.88 +/- 0.42, respectively) and the nitrofen without CDH group (0.61 +/- 0.14, 3.72 +/- 0.31, 1.66 +/- 0.20, 3.28 +/- 1.02, 1.38 +/- 0.24, respectively) compared with controls (0.43 +/- 0.11, 2.71 +/- 0.47, 0.79 +/- 0.42, 1.85 +/- 0.69, 0.57 +/- 0.22, respectively) (P < .05). CONCLUSION: Our data clearly show that lung retinol storage is decreased in the nitrofen model of CDH. The associated increase in gene expressions of most downstream components of the retinoid signalling pathway may be a feedback reaction to the deficiency of lung retinol. These results suggest that nitrofen acts by interfering with the cellular uptake of retinol during lung morphogenesis resulting in pulmonary hypoplasia in this model.
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Enfermedades Pulmonares/embriología , Pulmón/anomalías , Preñez , Vitamina A/metabolismo , Animales , Biopsia con Aguja , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Femenino , Hernia Diafragmática/inducido químicamente , Pulmón/embriología , Enfermedades Pulmonares/fisiopatología , Morfogénesis/efectos de los fármacos , Morfogénesis/fisiología , Éteres Fenílicos/farmacología , Embarazo , ARN Mensajero , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Transducción de Señal , Vitamina A/análisisRESUMEN
PURPOSE: Pulmonary hypoplasia remains the principal cause of high morbidity and mortality in patients with congenital diaphragmatic hernia (CDH). The precise mechanisms causing lung hypoplasia remains unclear. Aquaporins (AQPs) are reported to constitute a family of water channels that facilitate membrane water permeability in various tissues of animals. Aquaporin 5 has been reported to be an important marker expressed in type I alveolar epithelial cells in late gestation and mediates water transport across the human airway epithelium. We hypothesized that AQP5 is reduced in hypoplastic lungs and therefore designed this study to determine AQP5 expression in normal and hypoplastic lungs. METHODS: Fetal rat lungs of control (n=23) and nitrofen-treated (n=37) dams were harvested on embryonic day (E) 15, E17, E19, and E21. The expression of the AQP5 was analyzed in each lung by real-time reverse transcriptase-polymerase chain reaction. Immunohistochemical studies were performed to evaluate the protein expression level of AQP5. RESULTS: Aquaporin 5 messenger RNA levels on E21 were significantly reduced in lungs from the nitrofen with CDH group (11.8 +/- 2.3) compared with normal controls (23.5 +/- 11.8) and nitrofen without CDH group (26.9 +/- 13.0) (P < .05). Aquaporin 5 immunohistochemistry demonstrated AQP5 strongly expressed at the apical membrane of type I alveolar epithelial cells in the normal and nitrofen without CDH groups. By contrast, the AQP5-positive cells were markedly reduced in hypoplastic lungs in the nitrofen with CDH group. CONCLUSION: Our results show that the expression of AQP5 is down-regulated in hypoplastic lungs with CDH. Down-regulation of AQP5 may result in abnormal pulmonary fluid metabolism in perinatal period and may be one of the mechanisms disturbing the pulmonary development in late stage in the CDH model.
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Animales Recién Nacidos , Acuaporina 5/genética , Hernia Diafragmática/metabolismo , Pulmón/anomalías , Animales , Acuaporina 5/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Feto/metabolismo , Regulación de la Expresión Génica , Marcadores Genéticos/genética , Hernias Diafragmáticas Congénitas , Inmunohistoquímica , Pulmón/embriología , Pulmón/metabolismo , Masculino , Éteres Fenílicos , Embarazo , Preñez , Probabilidad , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
PURPOSE: The pathogenesis of pulmonary hypoplasia associated with congenital diaphragmatic hernia is poorly understood. Recently, it has been reported that Wnt signaling pathway plays a critical role in branching lung morphogenesis. Mice lacking Wnt7b gene die soon after birth because of respiratory failure and display severe lung hypoplasia. Wnt2 gene is expressed in the distal airway during development. To test the hypothesis that Wnt-mediated signaling is altered in nitrofen-induced hypoplastic lungs, we examined the expression of Wnt genes and Wnt target gene, BMP4 in normal and nitrofen-treated lungs. MATERIALS AND METHODS: Fetal rat lungs of normal (n = 24) and nitrofen-treated (n = 24) dams were harvested on embryonic day (E)15, E17, E19, and E21. The expression of GATA6, the Wnt genes (Wnt7b, Wnt2), and BMP4 was analyzed in each lung by real-time reverse transcription polymerase chain reaction. RESULTS: The gene expression of Wnt7b, Wnt2, and BMP4 on E15 was significantly reduced (P < .05) in lungs from nitrofen-treated animals compared with normal lungs. The expression level of GATA6, which has been reported to transactivate Wnt7b expression, was also significantly reduced (P < .05) in lungs from the nitrofen group. CONCLUSION: Our results provide evidence for the first time that the Wnt signaling pathway is down-regulated in nitrofen-induced hypoplastic lungs in the early stages of lung development. Decreased expression of GATA6 may account for the down-regulation of Wnt signal pathway. These data suggest that the down-regulation of Wnt signaling pathway may disrupt branching lung morphogenesis, resulting in pulmonary hypoplasia in the nitrofen rat model of congenital diaphragmatic hernia.
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Factor de Transcripción GATA6/genética , Hernia Diafragmática/genética , Pulmón/anomalías , Transducción de Señal/genética , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Hernia Diafragmática/inducido químicamente , Pulmón/embriología , Éteres Fenílicos , Embarazo , Preñez , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
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.
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Diferenciación Celular , Células Epiteliales/patología , Hernia Diafragmática/complicaciones , Enfermedades Pulmonares/patología , Éteres Fenílicos/toxicidad , Alveolos Pulmonares/patología , Anomalías Inducidas por Medicamentos/patología , Animales , Biomarcadores , Femenino , Expresión Génica , Herbicidas/administración & dosificación , Herbicidas/toxicidad , Hernia Diafragmática/inducido químicamente , Hernias Diafragmáticas Congénitas , Inmunohistoquímica , Pulmón/anomalías , Pulmón/efectos de los fármacos , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/congénito , Éteres Fenílicos/administración & dosificación , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Retinoids are a group of molecules derived from vitamin A, which play an important role in lung development. Within the cell, retinol can either be oxidized to retinal or esterified to retinyl esters by lecithin : retinol acyltransferase (LRAT) for storage. Retinal is then oxidized to an active metabolite of vitamin A, retinoic acid (RA) by retinal dehydrogenase (RALDH). RA is the active metabolite of vitamin A. Cyp26 (a1,b1, and c1), which is a member of the cytochrome P450 family, acts by reducing the activity of RA. Cyp26 type b1 is the predominant subtype expressed in the murine lung. Several studies have suggested that nitrofen may interfere with the retinoid pathway resulting in congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia. Recently, it was reported that nitrofen may act by inhibiting RALDH2. The aim of this study was to examine the pulmonary expression of Cyp26b1, LRAT, and RALDH2, the key enzymes involved in the synthesis of RA, in order to understand the mechanisms underlying pulmonary hypoplasia in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation (D9). Fetal lungs were harvested at D15, D17, D19, and D21. D17, D19, and D21 lungs were divided into three groups: control, nitrofen without CDH and nitrofen with CDH, whereas D15 lungs were divided into only two groups; control and nitrofen as the diaphragm is not fully formed yet at this stage. Real- time PCR was performed to evaluate the relative level of Cyp26b1, LRAT, and RALDH2 expression in the lung. Relative levels of Cyp26b1 mRNA were significantly decreased in the lungs of nitrofen with CDH (D17;0.19 +/- 0.09, D19;0.70 +/- 0.20, D21;0.40 +/- 0.36) and nitrofen without CDH (D17;0.14 +/- 0.06, D19;0.54 +/- 0.42, D21;0.51 +/- 0.56) compared to controls (D17;0.35 +/- 0.16, D19;1.15 +/- 0.48, D21;1.28 +/- 0.78) (P < 0.05). LRAT expression was also significantly decreased in nitrofen with CDH (D17; 19.3 +/- 7.8, D19; 4.3 +/- 1.1, D21; 3.3 +/- 1.6) and nitrofen without CDH (D17; 21.2 +/- 11.1, D19; 4.5 +/- 3.6, D21; 4.1 +/- 1.6) compared to controls (D17; 153.7 +/- 29.8, D19; 26.8 +/- 16.8 D21; 10.1 +/- 3.8) (P < 0.05). There was no significant difference in the relative levels of Cyp26b1 and LRAT between nitrofen with CDH and nitrofen without CDH. There were no significant differences in RALDH2 expression among the groups at any stages. Down-regulation of Cryp26b1 and LRAT demonstrates that RA content is decreased in nitrofen induced hypoplastic lungs compared to controls. The finding that RALDH2 expression in the hypoplastic lung is not altered suggests that nitrofen may act by interfering with the retinoid metabolism during the early stage of the retinoid signaling pathway.
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Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Herbicidas/toxicidad , Hernia Diafragmática/inducido químicamente , Enfermedades Pulmonares/inducido químicamente , Pulmón/anomalías , Éteres Fenílicos/toxicidad , Tretinoina/metabolismo , Aciltransferasas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Femenino , Herbicidas/administración & dosificación , Hernia Diafragmática/enzimología , Hernias Diafragmáticas Congénitas , Pulmón/efectos de los fármacos , Pulmón/enzimología , Enfermedades Pulmonares/enzimología , Enfermedades Pulmonares/fisiopatología , Aceite de Oliva , Éteres Fenílicos/administración & dosificación , Aceites de Plantas/administración & dosificación , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Retinal-Deshidrogenasa/metabolismo , Ácido Retinoico 4-Hidroxilasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
BACKGROUND/PURPOSE: The relationship of the developing lung and kidney is not completely understood. Renal enlargement has been reported in association with pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Recent studies suggest that retinoids may be involved in the pathogenesis of CDH. The aims of this study were to investigate the effects of pulmonary hypoplasia on renal development and to evaluate retinoids status of kidneys in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 of gestation. Fetuses were recovered at term and divided into 3 groups: 1, control (n = 69); 2, nitrofen without CDH (n = 25); and 3, nitrofen with CDH (n = 40). Kidneys were dissected, weighed, and processed for biochemical measurements of DNA, proteins, total retinol content, and for immunohistochemical staining of proliferating cells. RESULTS: Kidneys were smaller in nitrofen-exposed animals vs control animals (group 3, 0.65 +/- 0.08; group 2, 0.62 +/- 0.09 vs group 1, 0.73 +/- 0.09% of body weight, P < .001), and there were no differences between right and left kidney weight in all the 3 groups. Regression of total kidney weight on body weight showed a linear direct correlation between them in all the groups. Total amount of DNA was significantly reduced in nitrofen-exposed animals vs controls (group 3, 80.58 +/- 35.65; group 2, 64.71 +/- 20.28 vs group 1, 110.34 +/- 42.15 microg, P < .01), but the DNA concentration remained the same in the 3 groups (group 3, 3.59 +/- 1.26; group 2, 3.06 +/- 1.19; group 1, 3.43 +/- 1.05 microg DNA/mg kidney). Total protein content (group 3, 1145.59 +/- 500.36; group 2, 993.2 +/- 276.62; group 1, 1287.48 +/- 312.52 microg), protein concentration (group 3, 49.76 +/- 11.12; group 2, 43.95 +/- 6.79; group 1, 47.38 +/- 6.93 microg protein/mg kidney), and protein-to-DNA ratio (group 3, 15.12 +/- 5.98; group 2, 16.22 +/- 6.85; group I, 16.16 +/- 7.02 microg/microg) were similar in all groups. Retinol concentration was significantly reduced in both nitrofen-exposed groups compared with the control group (group 3, 1.35 +/- 0.24; group 2, 1.28 +/- 0.11; group 1, 2.53+/-0.61 microg retinol/g kidney). Proliferation index was similar in all 3 groups (group 3, 50.43 +/- 8.81; group 2, 47.96 +/- 6.01; group 1, 47.64 +/- 5.76% of proliferating cells). CONCLUSIONS: Our data clearly show that renal enlargement in association with pulmonary hypoplasia is not seen in the nitrofen-induced CDH. These results rule out any possible relationship between lung and kidney development. Moreover, kidneys are hypoplastic in both nitrofen-exposed groups and have reduced retinol content, suggesting that a retinoid pathway disruption could be the common mechanism in the pathogenesis of lung and kidney hypoplasia in the nitrofen model of CDH.
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Hernia Diafragmática/inducido químicamente , Riñón/embriología , Enfermedades Pulmonares/inducido químicamente , Pulmón/anomalías , Retinoides/fisiología , Animales , Femenino , Hernia Diafragmática/fisiopatología , Hernias Diafragmáticas Congénitas , Enfermedades Renales/inducido químicamente , Enfermedades Renales/fisiopatología , Pulmón/embriología , Enfermedades Pulmonares/fisiopatología , Tamaño de los Órganos , Éteres Fenílicos/efectos adversos , Embarazo , Ratas , Ratas Sprague-Dawley , Toxinas Biológicas/efectos adversosRESUMEN
BACKGROUND/PURPOSE: As our understanding of the enteric nervous system improves, it becomes clear that it is no longer sufficient to simply determine whether enteric ganglion cells are present but also to determine whether correct number and types of ganglion cells are present. Nitric oxide is recognized as a potent mediator of inhibitory nerves responsible for the relaxation of the smooth muscle of the gastrointestinal tract. The aim of this study was to determine the normal nitrergic neuronal density and morphology in the submucosal plexus of the porcine distal bowel from fetal life to adulthood. METHODS: Distal large bowel specimens were obtained from porcine fetuses of gestational age E60 (n = 5), E90 (n = 5), 1-day-old piglets (n = 5), 4-week-old piglets (n = 5), 12-week-old piglets (n = 5), and adult pigs (n = 5). Whole-mount preparations of the submucosal plexus were made and stained with NADPH diaphorase histochemistry. The ganglia density, the number of ganglion cells per ganglia, and nucleus and cytoplasmic area were measured. RESULTS: Ganglia density decreased progressively and markedly with age until the adulthood (P < .001). On the contrary, ganglion cells increased their size over time predominantly because of increase in cytoplasm (P < .001). The number of ganglion cells per ganglia increased significantly during the fetal life. However, there was a significant reduction in the number of ganglion cells per ganglia during the period from birth to 4 weeks, remaining constant thereafter (P < .001). CONCLUSIONS: The quantitative and qualitative morphometric analysis of the colonic submucous plexus shows that significant developmental changes occur during fetal and postnatal life. These findings indicate that the age of the patient is of utmost importance during histopathologic evaluation of enteric nervous system disorders.
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Colon/crecimiento & desarrollo , Colon/inervación , Mucosa Intestinal/crecimiento & desarrollo , Mucosa Intestinal/inervación , Neuronas Nitrérgicas , Factores de Edad , Animales , Recuento de Células , Colon/citología , Colon/embriología , Mucosa Intestinal/citología , Mucosa Intestinal/embriología , PorcinosRESUMEN
PURPOSE: Changes in vascular structures as well as vascular endothelial growth factor (VEGF) downregulation have been reported in hypoplastic lungs associated with congenital diaphragmatic hernia. We hypothesized that VEGF may accelerate branching morphogenesis and thus may modulate lung growth in normal and nitrofen-induced pulmonary hypoplastic lungs. METHODS: A hypoplastic fetal lung model and a normal control lung model were induced by feeding pregnant rats with or without nitrofen, respectively. Fetal lungs harvested on day 13.5 were cultured at ambient oxygen tensions for 72 hours with 0, 25, 50, or 100 ng/mL of exogenous rat VEGF added daily in the serum-free medium. The rates of increase in bud count and airway contour were evaluated. Real-time polymerase chain reaction was carried out to evaluate the expression of surfactant protein C mRNA in the explants at the end of culture. RESULTS: Vascular endothelial growth factor accelerated the increase in bud count and airway contour in normal and hypoplastic lung explants compared to controls. Surfactant protein C mRNA expression was significantly increased at 50 ng/mL VEGF compared to controls in both normal and hypoplastic lung explants. CONCLUSION: These data suggest that VEGF plays an important role in lung morphogenesis and may accelerate lung growth in nitrofen-induced hypoplastic lung.
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Pulmón/anomalías , Pulmón/crecimiento & desarrollo , Morfogénesis , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Pulmón/embriología , Éteres Fenílicos , Ratas , Ratas Sprague-DawleyRESUMEN
There is increasing evidence to suggest that the retinoid pathway is involved in the pathogenesis of congenital diaphragmatic hernia (CDH). We hypothesised that retinoids are involved in the pathogenesis of associated pulmonary hypoplasia in CDH and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 of gestation. Foetal lungs were harvested on embryonic day 13.5 and were cultured for 96 h with or without exogenous retinoic acid (RA) (1 muM) added daily to the culture medium. Lungs were divided into four study groups: control (n=31); control + RA (n=19); nitrofen (n=19); and nitrofen + RA (n=12). Lung growth was assessed in each group by measuring branching morphogenesis, total DNA content and the proportion of proliferating cells stained by immunohistochemistry. One-way ANOVA test was used for statistical analysis. Retinoic acid significantly increased the growth of nitrofen-induced hypoplastic lungs, whilst growth of control lungs did not change. The number of lung buds and lung area of nitrofen-exposed hypoplastic lungs after 96 h of culture significantly increased after the addition of RA compared to the non-treated hypoplastic lungs (25.75+/-6.47 vs 15.11+/-3.29 and 0.98+/-0.18 mm(2) vs 0.65+/-0.13 mm(2), respectively; P<0.0001). Lung perimeter was also higher when RA was added to hypoplastic lungs compared to the non-treated ones, although it did not reach significance (12.51+/-2.53 mm vs 11.19+/-2.56 mm; P=0.17). Conversely, the addition of RA to control lungs did not affect the number of lung buds, lung area or lung perimeter after 96 h in culture compared to the non-treated ones (31.28+/-4.66 vs 31.81+/-6.67; 1.29+/-0.18(2) vs 1.29+/-0.23 mm(2) and 18.47+/-3.47 mm vs 17.89+/-2.94 mm, respectively; P=NS). Retinoic acid also increased the total DNA content and the proportion of proliferating cells in hypoplastic lungs compared to the non-treated ones (2.59+/-0.58 mug vs 1.96+/-0.31 mug and 57.89+/-9.46% vs 36.76+/-8.15%, respectively; P<0.001). The addition of RA did not affect either total DNA content or the proportion of proliferating cells in control lungs compared to the non-treated ones (4.04+/-0.64 mug vs 3.79+/-0.85 mug and 58.67+/-11.23% vs 56.03+/-10.36%, respectively; P=NS). This study demonstrates for the first time that RA rescues lung hypoplasia in nitrofen-induced hypoplastic lungs. These results suggest that retinoid pathway may be involved in the pathogenesis of associated pulmonary hypoplasia in CDH.
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Hernia Diafragmática/fisiopatología , Hernias Diafragmáticas Congénitas , Pulmón/anomalías , Pulmón/embriología , Retinoides/fisiología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Femenino , Pulmón/efectos de los fármacos , Embarazo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Tretinoina/farmacologíaRESUMEN
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.
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Anomalías Múltiples/embriología , Regulación del Desarrollo de la Expresión Génica , Hernia Diafragmática/embriología , Hernias Diafragmáticas Congénitas , Riñón/anomalías , Riñón/embriología , Sistema Renina-Angiotensina/genética , Animales , Regulación hacia Abajo , Femenino , Éteres Fenílicos , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Nitric oxide (NO) can accelerate branching morphogenesis of fetal rat lung explants in vitro, whereas its exact mechanism remains unclear. In this study, we investigate the effect of NO on the expression of fibroblast growth factor-10 (FGF10) and bone morphogenetic protein-4 (BMP4), which plays an important role in bud formation. METHODS: Fetal rat lungs harvested on day 13.5 of gestation were cultured in serum-free medium for 72 hours with 0, 50, 100, and 200 micromol/L of an NO donor, DETA NONOate (DETA/NO) (n = 4, 3, 6, and 5). The ratio of bud increment of each cultured lung was calculated, and the FGF10 and BMP4 mRNA expression levels were analyzed by real-time reverse transcription polymerase chain reaction. RESULTS: Bud increment ratio was significantly increased in 50, 100, and 200 micromol/L DETA/NO (3.3 +/- 0.2, 3.0 +/- 0.3, and 3.5 +/- 0.5) compared to controls (1.9 +/- 0.3) (P < .05). There was a significant increase in BMP4 mRNA expression in 100 micromol/L DETA/NO (190% +/- 20%) compared to controls (100% +/- 30%) (P < .05), whereas FGF10 mRNA expression was not significantly different between each DETA/NO group and controls. CONCLUSION: The NO donor not only promotes branching of fetal lung explants but also upregulates expression of BMP4, which is an important regulator of branching morphogenesis.