RESUMEN
OBJECTIVE: To examine the association of nursing overtime, nursing provision and unit occupancy rate with medical incident rates in the neonatal intensive care unit (NICU) and the risk of mortality or major morbidity among very preterm infants. STUDY DESIGN: Single center retrospective cohort study of infants born within 23 to 29 weeks of gestational age or birth weight <1000 g admitted at a 56 bed, level III NICU. Nursing overtime ratios (nursing overtime hours/total nursing hours), nursing provision ratios (nursing hours/recommended nursing hours based on patient dependency categories) and unit occupancy rates were pooled for all shifts during NICU hospitalization of each infant. Log-binomial models assessed their association with the composite outcome (mortality or major morbidity). RESULTS: Of the 257 infants that met the inclusion criteria, 131 (51%) developed the composite outcome. In the adjusted multivariable analyses, high (>3.4%) relative to low nursing overtime ratios (⩽3.4%) were not associated with the composite outcome (relative risk (RR): 0.93; 95% confidence interval (CI): 0.86 to 1.02). High nursing provision ratios (>1) were associated with a lower risk of the composite outcome relative to low ones (⩽1) (RR: 0.81; 95% CI: 0.74 to 0.90). NICU occupancy rates were not associated with the composite outcome (RR: 0.98; 95% CI: 0.89 to 1.07, high (>100%) vs low (⩽100%)). Days with high nursing provision ratios (>1) were also associated with lower risk of having medical incidents (RR: 0.91; 95% CI: 0.82 to 0.99). CONCLUSION: High nursing provision ratio during NICU hospitalization is associated with a lower risk of a composite adverse outcome in very preterm infants.
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Ocupación de Camas/estadística & datos numéricos , Enfermedades del Prematuro/epidemiología , Unidades de Cuidado Intensivo Neonatal/estadística & datos numéricos , Personal de Enfermería/organización & administración , Admisión y Programación de Personal , Femenino , Humanos , Lactante , Mortalidad Infantil/tendencias , Recien Nacido con Peso al Nacer Extremadamente Bajo , Recien Nacido Extremadamente Prematuro , Recién Nacido , Enfermedades del Prematuro/mortalidad , Masculino , Quebec , Análisis de Regresión , Estudios Retrospectivos , Recursos HumanosRESUMEN
OBJECTIVE: To evaluate the association between birth route and late-onset sepsis (LOS), and coagulase-negative Staphylococcal (CONS)-related LOS in preterm neonates. STUDY DESIGN: In this observational study, data from 20,038 infants born between 22 and 32 weeks' gestation and admitted to Canadian neonatal intensive care units between 2010 and 2014 were analyzed retrospectively. The impact of birth route on LOS was assessed using univariate analysis and multiple logistic regression. RESULTS: A total of 8218 neonates were born via vaginal route and 11,820 via cesarean section. Incidence rates of LOS for infants born vaginally and via a cesarean section were 13.1 and 13.2%, respectively, and there was no significant difference in odds of LOS between the groups (adjusted odds ratio (AOR): 0.99; 95% CI 0.87 to 1.12); however, the odds of CONS sepsis were higher in the cesarean group (AOR: 1.15; 95% CI: 1.01 to 1.32). CONCLUSION: Birth route did not have an impact on LOS, but was associated with CONS-related LOS.
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Parto Obstétrico/efectos adversos , Recien Nacido Extremadamente Prematuro , Enfermedades del Prematuro/epidemiología , Sepsis Neonatal/epidemiología , Parto Obstétrico/estadística & datos numéricos , Femenino , Edad Gestacional , Humanos , Lactante , Unidades de Cuidado Intensivo Neonatal/estadística & datos numéricos , Modelos Logísticos , Masculino , Oportunidad Relativa , Embarazo , Estudios Retrospectivos , Factores de Riesgo , Infecciones Estafilocócicas/epidemiologíaRESUMEN
The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2 ) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Animales , Artefactos , Genómica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Lugares Marcados de Secuencia , Transcripción GenéticaRESUMEN
Tracheal occlusion (TO) in fetal lambs induces pulmonary hyperplasia but has negative effects on type II cells. The purpose of this study was to determine whether antenatal steroids could reverse the adverse effects of TO on lung maturation in fetal lambs. Sixteen time-dated pregnant ewes (term, 145 d) and 24 of their fetuses were divided into six groups: 1) TO at 117 d gestation; 2) TO at 117 d with a single maternal intramuscular injection of 0.5 mg/kg betamethasone 24 h before delivery; 3) TO at 117 d and release of the occlusion 2 d before delivery; 4) TO and release of the occlusion with maternal steroids; 5) unoperated controls without antenatal steroid treatment; and 6) unoperated controls, littermates of groups 1-4, treated with antenatal steroids. All fetuses were killed at 137 d gestation. Outcome measurements consisted of lung weight-to-body weight ratio; lung morphometry determined by mean terminal bronchial density; and assessment of type II pneumocytes by in situ hybridization to the mRNA of surfactant proteins B and C. Lung weight-to-body weight ratio and mean terminal bronchial density were significantly different among groups with TO and controls, indicating increased lung growth and structural maturation. The density of type II pneumocytes was markedly decreased by TO. Release 2 d before sacrifice significantly increased the density and surfactant activity of type II pneumocytes, but to levels still far from controls. Steroids alone had an effect similar to release. An additive effect was noted with steroids and 2-d release resulting in type II cell density comparable to controls. After fetal TO, a single maternal intramuscular dose of 0.5 mg/kg of betamethasone 24 h before delivery allows partial recuperation of the type II pneumocytes, an effect that is potentiated by 2-d release.
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Modelos Animales de Enfermedad , Ovinos/embriología , Esteroides/uso terapéutico , Tráquea/efectos de los fármacos , Animales , Femenino , Hibridación in Situ , Embarazo , Proteolípidos/genética , Surfactantes Pulmonares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tráquea/metabolismo , Tráquea/patologíaRESUMEN
PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with thickened pulmonary arteries (PA) contributing to pulmonary hypertension. In the current study, the effects of antenatal glucocorticoids and reversible tracheal occlusion (TO) on PA structure were assessed in a hypoplastic lung model. METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation, TO at 108 days, and release of the occlusion (TR) at 129 days. All were given 1 dose of maternal glucocorticoids at 135 days. At 136 days (term, 145 days), the fetus was delivered by cesarian section. CDH (n = 7), CDH + TO (n = 6), CDH + TO + TR (n = 6), and unoperated twin controls (n = 16) were compared. Outcome measurements were (1) lung growth, represented by lung weight to body weight ratio (LW/BW), (2) lung structural maturation, which is inversely proportional to mean terminal bronchiole density (MTBD), (3) PA medial and adventitial areas (square micrometers), (4) lung capillary load, which is the ratio of vessel surface area (SA) to tissue SA ratio. RESULTS: CDH lungs were hypoplastic with a low LW/BW and high MTBD. The small PAs (<75 microm) of CDH had an increased medial area, indicating increased muscle mass and an increased adventitial area. CDH + TO +/- TR increased LW/BW and achieved normal structural lung maturity with a low MTBD. Only CDH + TO thinned the PA medial area closer to control values. The adventitial area remained thick in CDH +/- TO +/- TR when compared with controls. All 4 groups had similar capillary load. CONCLUSIONS: TO may be especially important for PA remodeling in the latter part of gestation, because TR 1 week before delivery prevents thinning of the small PAs in CDH. The shaping achieved by TO in terms of lung growth, structural maturity, and pulmonary artery medial area thinning may prove beneficial in lessening the severity of the associated pulmonary hypertension in CDH.
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Antiinflamatorios/uso terapéutico , Oclusión con Balón/métodos , Betametasona/uso terapéutico , Modelos Animales de Enfermedad , Enfermedades Fetales/terapia , Glucocorticoides/uso terapéutico , Hernia Diafragmática/terapia , Hernias Diafragmáticas Congénitas , Pulmón/anomalías , Pulmón/efectos de los fármacos , Síndrome de Circulación Fetal Persistente/terapia , Atención Prenatal/métodos , Arteria Pulmonar/anomalías , Arteria Pulmonar/efectos de los fármacos , Tráquea , Animales , Oclusión con Balón/instrumentación , Terapia Combinada , Evaluación Preclínica de Medicamentos , Enfermedades Fetales/mortalidad , Madurez de los Órganos Fetales , Edad Gestacional , Hernia Diafragmática/mortalidad , Humanos , Recién Nacido , Pulmón/crecimiento & desarrollo , Tamaño de los Órganos , Síndrome de Circulación Fetal Persistente/mortalidad , Arteria Pulmonar/crecimiento & desarrollo , Ovinos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND/PURPOSE: In normal lungs, fetal tracheal occlusion (TO) induces lung growth but decreases the number of type II cells; this is remedied if TO is released (TR) before delivery. In the current study, the effects of TO with or without TR on pulmonary structure and surfactant were assessed in the ovine model in which lung hypoplasia was induced by creation of a diaphragmatic hernia (CDH). METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation; TO was done at 108 days; and TR at 129 days. All ewes were given 1 dose of glucocorticoids at 135 days. At 136 days, the fetus was delivered. Lung weight to body weight ratio, mean terminal bronchiole density, type II cell density, bronchoalveolar lavage fluid (BAL) phosphatidylcholine (PC), BAL surfactant protein A (SP-A) and B (SP-B), and lung tissue SP-A and SP-B were assessed in CDH, CDH with TO, CDH with TO and TR, and controls. RESULTS: CDH lungs were hypoplastic and structurally immature, but had increased type II cell density. TO with or without TR caused lung growth with normalization of lung parenchymal architecture and type II cell density. Although the BAL SP-A and BAL SP-B were similar in all 4 groups, the BAL PC was low in CDH with or without TO or TR. Also, lung tissue SP-B levels were low in CDH with or without TO or TR. However, lung tissue SP-A levels were normal in CDH, but low in CDH with TO with or without TR. CONCLUSIONS: Despite the finding that lung morphology was improved in CDH with TO with or without TR animals, surfactant content and composition remained abnormal. Although surfactant secreted early by the fetus into alveolar spaces contained normal levels of BAL SP-A and BAL SP-B, the low levels of BAL PC and low lung tissue stores of SP-B indicate that these experimental lambs may experience respiratory insufficiency soon after birth. This implies that prophylactic surfactant at birth might be beneficial for CDH.
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Betametasona/farmacología , Glucocorticoides/farmacología , Hernia Diafragmática/fisiopatología , Pulmón/embriología , Pulmón/metabolismo , Surfactantes Pulmonares/metabolismo , Tráquea/cirugía , Análisis de Varianza , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Hernias Diafragmáticas Congénitas , Pulmón/citología , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Fosfatidilcolinas/metabolismo , Embarazo , OvinosRESUMEN
Hereditary surfactant protein B (SP-B) deficiency has been lethal in the first year of life without lung transplantation. We tested the hypothesis that SP-B gene mutations may result in milder phenotypes by investigating the mechanisms for lung disease in two children with less severe symptoms than have been previously observed in SP-B deficiency. Immunostaining patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indicated that both children were homozygous for a mutation in exon 5 that created an alternative splice site. Reverse transcriptase PCR and sequence analysis confirmed use of this splice site, which resulted in a frameshift and a premature termination codon in exon 7. The predominant reverse transcriptase PCR product, however, lacked exon 7, which restored the reading frame but would not allow translation of the exons that encode mature SP-B. Western blot analysis detected reduced amounts of mature SP-B as well as an aberrant SP-B proprotein that corresponded to the size expected from translation of the abnormal transcript. We conclude that a novel splicing mutation was the cause of lung disease in these children and that hereditary SP-B deficiency can be the cause of lung disease in older children.
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Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/genética , Mutación , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Recién Nacido , Enfermedades Pulmonares/metabolismo , Masculino , Empalme del ARNRESUMEN
Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.
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Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Estrés Oxidativo/fisiología , Oxígeno/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fase S/fisiología , Northern Blotting , Western Blotting , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , Humanos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
OBJECTIVE AND DESIGN: To test the hypothesis that glucocorticoid administration would diminish the lung expression of P-selectin mRNA in hyperoxia-exposed rats. ANIMALS: Adult male Sprague-Dawley rats were divided into 6 separate groups containing 10 to 13 animals per group. TREATMENT: Rats were dosed with 1 mg/kg of dexamethasone or vehicle only, ip. Immediately after dosing, animals were placed in > 95 % oxygen. Some animals were maintained in room air and are presented as 0 h of exposure to hyperoxia. Another group of animals was dosed with 10 mg/kg lipopolysaccharide (LPS) ip immediately after dosing with either dexamethasone or vehicle as above. METHODS: At 24 or 48 h, lung samples were obtained, and lung weight to body weight ratios calculated. In the LPS studies, samples were obtained 4 h after LPS dosing. In a subset of animals, lung sections were hybridized for P-selectin mRNA. All data except for hybridizations were analyzed with three-way ANOVA, with subsequent post-hoc testing. P-selectin hybridizations were quantified by counting the number of positive vessels per high-powered field, and subsequently analyzed by unpaired Student's t-test. Immunohistochemical analyses for P-selectin expression were also performed to determine whether changes in P-selectin mRNA were associated with differences in protein expression. All data are expressed as means +/- SEM. RESULTS: Rats dosed with dexamethasone had higher lung/body weight ratios after 24 and 48 h of exposure to hyperoxia than did similarly exposed rats dosed only with vehicle (at 48 h, 0.87 +/- 0.04 versus 0.65 +/- 0.06, respectively, P < 0.05). The higher ratios in hyperoxic animals dosed with dexamethasone were associated with much higher levels of lung expression for P-selectin mRNA than was observed in similarly exposed rats dosed with vehicle alone (at 48 h, 3.93 +/- 1.02, versus 0.20 +/- 0.06, respectively, P < 0.01). In contrast dexamethasone dosing lead to lower lung P-selectin mRNA expression in animals exposed to LPS (1.23 +/- 1.08 in dexamethasone dosed animals versus 6.80 +/- 0.92 in vehicle only dosed animals). Consistent with the mRNA data, P-selectin immunoreactivity increased as a function of hyperoxia-exposure time in animals dosed with dexamethasone, while immunoreactivity decreased as a function of hyperoxia-exposure time in animals dosed with vehicle only. CONCLUSIONS: Increased P-selectin mRNA combined with increased P-selectin protein expression in animals exposed to hyperoxia and dosed with dexamethasone suggests that enhanced expression of P-selectin may contribute to the greater lung injury and inflammation caused by hyperoxia in rats treated with dexamethasone.
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Antiinflamatorios/farmacología , Dexametasona/farmacología , Hiperoxia/metabolismo , Pulmón/metabolismo , Selectina-P/biosíntesis , ARN Mensajero/biosíntesis , Animales , Toxinas Bacterianas/toxicidad , Peso Corporal/efectos de los fármacos , Endotoxinas/toxicidad , Hemoglobinas/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Lipopolisacáridos/toxicidad , Pulmón/anatomía & histología , Pulmón/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Biosíntesis de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
Various stress proteins appear to play a role in injury and repair produced by inhaled pollutants. The present study examined the effect of inhaled endotoxin on the expression of the metallothionein and heme oxygenase genes. Rats were exposed to saline or endotoxin aerosols for 3 h and sacrificed up to 3 d following exposure. The significant induction of metallothionein mRNA in both the lung (fourfold increase) and liver (one-fold) were greatest at 3 h and returned to basal levels by 24 h after endotoxin exposure. Similarly, the increase in tissue metallothionein was greater in the lung. In situ hybridization in mice showed large increases in the relative abundance of metallothionein transcripts in epithelial cells of the conducting airways, in surrounding airway tissue, and in the nearby gas exchange region. While an endotoxin-induced significant increase in heme oxygenase mRNA followed a time course similar to that observed for metallo thionein, the relative magnitude was reversed for the lung and liver. Heme oxygenase mRNA was induced greater in the liver (twofold) than in the lung (60% above control). Our findings demonstrate that metallothionein and heme oxygenase are early response genes that are rapidly activated after inhalation of occupationally relevant concentrations of endotoxin.
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Hemo Oxigenasa (Desciclizante)/biosíntesis , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Metalotioneína/biosíntesis , Animales , Cobre/sangre , Inducción Enzimática , Hemo Oxigenasa (Desciclizante)/genética , Hibridación in Situ , Exposición por Inhalación , Hígado/enzimología , Pulmón/enzimología , Metalotioneína/genética , Ratones , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Zinc/sangreRESUMEN
Lung injury is a frequent consequence of oxygen (O2) therapy administered to newborns and adults with respiratory distress. Acute exposure to hyperoxia results in a well-described pathophysiologic response in the lungs. Because inflammation is an important component of pulmonary O2 toxicity, we have an interest in identifying the inflammatory mediators that increase during hyperoxia. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a member of the immunoglobulin superfamily that is expressed at the junctions between endothelial cells, is essential to the transendothelial migration of leukocytes. We hypothesized that increased expression of PECAM-1 occurs in pulmonary endothelial cells during hyperoxic lung injury. Adult mice were exposed to 100% O2 for up to 96 h. We analyzed PECAM-1 expression by RNA blot hybridization, in situ hybridization, and immunohistochemistry. A increase in PECAM-1 mRNA was seen as soon as 2 d of hyperoxia relative to unexposed control mice. PECAM-1 mRNA and protein were found in endothelial cells of both large and small arteries. The expression of PECAM-1 in capillary vessels was further confirmed using in situ hybridization at the electron microscope level. This increase in PECAM-1 expression coincided with the appearance of leukocytes in lung tissue. These observations suggest that PECAM-1 expression is a relatively early step in the inflammation cascade, and intervention at this phase may be critical to the prevention of further damage.
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Oxígeno/toxicidad , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Neumonía/inducido químicamente , Empalme Alternativo/inmunología , Animales , Western Blotting , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Endotelio/ultraestructura , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Hiperoxia/inmunología , Hiperoxia/fisiopatología , Hibridación in Situ , Pulmón/química , Pulmón/inmunología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C3H , Microscopía Inmunoelectrónica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Neumonía/inmunología , Neumonía/fisiopatología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.
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Hiperoxia/metabolismo , Interleucina-1/biosíntesis , Pulmón/metabolismo , Pulmón/patología , Animales , Hibridación in Situ , Interleucina-1/genética , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C3H , Oxígeno/toxicidad , Perfusión , ARN Mensajero/metabolismoRESUMEN
PURPOSE: The purpose of this study was to test the hypothesis that tracheal obstruction (plugging) in the fetal lamb model leads to a decrease in the absolute number of type II pneumocytes and that reversing the obstruction before birth (unplugging), allows the type II cells to recover while maintaining the beneficial effect on lung growth. METHODS: Nine time-dated pregnant ewes (term, 145 days), carrying 17 fetuses, were used in this surgical trial. The fetuses were divided into three experimental groups: group A underwent plugging at 93 days gestation, followed by unplugging at 110 days; group B animals had tracheal ligation at 93 days and group C consisted of unoperated controls. All fetuses were delivered by cesarean section at 136 days' gestation. The fetal trachea was obstructed with the tracheoscopically placed detachable balloon described by our group. Unplugging was performed by needle puncture of the balloon under tracheoscopic vision. Outcome measurements consisted of lung-to-body-weight ratio (LWBR), lung morphometry (mean terminal bronchial density [MTBD] and linear intercept [Lm]), and assessment of the number of type II pneumocytes. The latter was determined by in situ hybridization to the mRNA of surfactant protein-C, which is exclusively produced by type II cells. Statistics were calculated using a two-tailed unpaired t test and P less than .05 is considered significant. RESULTS: Seventeen animals are included in the results. All of them had lung samples analyzed for lung morphometry, whereas for type II cells analysis, three animals were studied in each group. Morphometric analyses were consistent with pulmonary hyperplasia for group B, whereas group A lungs showed more histological maturity than group C albeit not as marked as group B. In group A, there was a similar number of type II cells to that observed in group C (53.2 +/- 3.9 v 55.9 +/- 4.0, P = .66). However, for group B animals, the number of type II pneumocytes was markedly decreased compared with controls (4.7 +/- 0.1 v 55.9 +/- 4, P = .0003). CONCLUSIONS: The authors conclude that tracheal ligation until birth, although inducing pulmonary hyperplasia, significantly decreases the number of type II pneumocytes in the alveoli. After a temporary 15-day occlusion initiated at 95 days' gestation, there is complete normalization of the density of type II cells. These results bear importance on the duration of PLUG to treat the pulmonary hypoplasia seen in congenital diaphragmatic hernia. Temporary tracheal obstruction now needs to be tested in a hypoplastic lung model.
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Pulmón/embriología , Animales , Cateterismo , Recuento de Células , Femenino , Hernias Diafragmáticas Congénitas , Ligadura , Pulmón/citología , Embarazo , Proteolípidos/metabolismo , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Ovinos , Tráquea/embriologíaRESUMEN
UNLABELLED: Fetal tracheal occlusion (TO) has been shown to lead to lung hyperplasia in various animal models, and this procedure has already been carried out in human fetuses with congenital diaphragmatic hernia (CDH). However, the authors previously showed that TO caused a decrease in type II pneumocytes. PURPOSE: The aim of this study is to examine the effects of TO and release on type II pneumocytes. METHOD: To was carried out with a Swan Ganz or Fogarty catheter in fetal sheep at 116 to 118 days of gestation. TO was maintained for 2 weeks followed by deflation of the balloon for 1 week before delivery, in group 1; in group 2, TO was maintained for 19 days and released 2 days before delivery. Group 3 consisted of previously reported animals who had TO maintained until birth. Unoperated twins served as controls. All specimens were analyzed using the surfactant protein C (SP-C) mRNA as a specific marker for type II pneumocytes. We used Northern Blot and in situ hybridization techniques to quantify total SP-C and the density of type II cells. Electron microscopy (EM) was also used to evaluate and quantitate type II cells. RESULTS: TO resulted in significant lung growth in all groups. In situ hybridization and Northern Blot analysis showed that there was a complete recovery of type II cells in group 1 versus controls. Quantitative EM analysis confirmed these findings. In group 2 the number of type II cells was decreased but there was an increase in SP-C content per type II cell versus group 3. CONCLUSION: Lung growth after TO appears to occur at the expense of type II cell differentiation. This effect is reversible with the release of TO before birth in this animal model.
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Obstrucción de las Vías Aéreas/patología , Enfermedades Fetales/patología , Pulmón/embriología , Obstrucción de las Vías Aéreas/metabolismo , Análisis de Varianza , Animales , Biomarcadores , Diferenciación Celular , Modelos Animales de Enfermedad , Enfermedades Fetales/metabolismo , Hernia Diafragmática/embriología , Humanos , Fenotipo , Surfactantes Pulmonares/genética , ARN Mensajero/genética , Ovinos , TráqueaRESUMEN
It was previously shown that tracheal obstruction accelerated fetal lung growth and eventually reversed the pulmonary hypoplasia in experimental diaphragmatic hernia. We have successfully developed a reversible tracheal obstruction technique in fetal sheep using balloon occlusion and showed that 3 wk of obstruction induced significant lung growth of the same magnitude as the tracheal ligation. The purpose of this study was to examine the effects of 1 and 3 wk of tracheal occlusion on the alveolar cell population with specific attention to the type II pneumocytes. We first showed that 1 wk of occlusion induced a significant increase in lung weight and in alveolar surface area. We then used the surfactant protein C (SP-C) mRNA as a specific marker of differentiated type II pneumocytes. Total RNA was isolated from fetal sheep lung with or without tracheal occlusion, and Northern blots were hybridized with a cDNA probe specific for the sheep SP-C. The results show a dramatic decrease in SP-C mRNA expression (8.8-fold, p < 0.01). In situ hybridization showed a marked decrease in the density of cells expressing SP-C, as well as the amount of SP-C mRNA expressed by the cells. The effect was present as early as 1 wk of occlusion. The sparseness of type II pneumocytes was further confirmed by electron microscopy. We thus conclude that tracheal obstruction causes a profound decrease in the number of type II pneumocytes in the lungs. Given the crucial role of type II pneumocytes in surfactant production, we could speculate that, if tracheal occlusion is able to accelerate lung growth, the final product is probably surfactant-deficient.
Asunto(s)
Obstrucción de las Vías Aéreas/patología , Enfermedades Fetales/patología , Alveolos Pulmonares/patología , Obstrucción de las Vías Aéreas/metabolismo , Animales , Biomarcadores , Diferenciación Celular/fisiología , Enfermedades Fetales/metabolismo , Fenotipo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/genética , ARN Mensajero/biosíntesis , Ovinos , TráqueaRESUMEN
Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.
RESUMEN
Cell-to-cell communication is often disrupted when tissue damage occurs, triggering new signals to cope with the injury. The expression of intercellular adhesion molecule (ICAM-1), a protein involved in the migration, binding, and activation of leukocytes, is markedly increased in mouse lungs damaged by acute hyperoxic exposure. Type I alveolar epithelial cells are sensitive to hyperoxic lung injury, and must be removed from the air spaces following their destruction. In contrast, type II pneumocytes are relatively resistant to hyperoxia and may have a role in the removal process. Two reports demonstrate increased ICAM-1 in alveoli after hyperoxia (Welty et al., 1993, AJRCMB 9:393-400; and Kang et al., 1993, AJRCMB 9:350-355), but the cellular site(s) of ICAM-1 synthesis were not determined. We hypothesized that during in vivo exposure to 100% oxygen (O2), type II pneumocytes synthesize and secrete ICAM-1, an important step in attracting inflammatory cells to the site of injury. Adult mice were exposed to 100% O2 for up to 72 h. To determine whether type II cells express ICAM-1, tissue sections were studied by electron microscopy single-label in situ hybridization or light microscopy dual-label in situ hybridization, using radiolabeled and nonradiolabeled probes. In the lungs of unexposed animals, ICAM-1 mRNA was detected in many cells-including type I pneumocytes-but not in type II cells. After hyperoxia, ICAM-1 transcripts were detected in bona fide, surfactant protein C mRNA-containing, type II alveolar epithelial cells. This observation suggests that type II cells play an important and previously unrecognized role in pulmonary inflammation from O2 toxicity and emphasizes the importance of type II pneumocytes in alveolar repair after injury.
Asunto(s)
Hiperoxia/fisiopatología , Molécula 1 de Adhesión Intercelular/genética , Alveolos Pulmonares/citología , Animales , Epitelio/patología , Epitelio/fisiología , Epitelio/ultraestructura , Hiperoxia/patología , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C3H , Microscopía Inmunoelectrónica , Alveolos Pulmonares/ultraestructura , ARN Mensajero/análisisRESUMEN
Fibrosis, characterized by the accumulation of collagen, is a consequence of a chronic inflammatory response. The purpose of this study was to determine if tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA expression are altered acutely after irradiation, during the so-called "latent" phase of pulmonary injury, and to examine if these alterations persist through the development of pneumonitis and fibrosis. Further, we wished to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by slot blotting and hybridized with radiolabeled cDNA probes encoding for TNF-alpha, IL-1 alpha and IL-1 beta. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. It was found that TNF-alpha mRNA levels were increased in C57BL/6 mice at days 1 and 7 postirradiation after 5 Gy and day 14 postirradiation after both 5 and 12.5 Gy, and IL-1 alpha mRNA levels were increased in C57BL/6 mice at days 56, 112 and 182 postirradiation after both 5 and 12.5 Gy, and IL-1 beta mRNA levels in the C3H/HeJ mice were increased at days 56 and 182 postirradiation after 12.5 Gy. In summary, these studies demonstrated early and persistent alterations in TNF-alpha, IL-1 alpha and IL-1 beta mRNA levels even at the lower dose (5 Gy). The temporal relationship between the elevation of these cytokines and the strain-dependent variation in fibrosis response suggests that IL-1 alpha and TNF-alpha contribute to the radiation-induced component of pulmonary fibrosis, whereas IL-1 beta may have a protective function.
Asunto(s)
Interleucina-1/biosíntesis , Fibrosis Pulmonar/inmunología , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de la radiación , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Radioisótopos de Cesio , Susceptibilidad a Enfermedades , Femenino , Rayos gamma , Inmunidad Innata , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Tórax , Factores de TiempoRESUMEN
While treatment with supplemental oxygen is often essential in patients with lung disease, prolonged therapy may cause lung injury by itself. Although the mechanisms responsible for initiating hyperoxic lung damage almost certainly involve primary oxidative transformations, the possible contributions of inflammation to the tissue injury have been attracting increasing research activity. Increases in intercellular adhesion molecule-1 (ICAM-1) coincide with the inflammation, but in other models of inflammation transient adhesion mediated by members of the Selectin gene family was found to be essential before ICAM-1/beta 2 interactions could occur. We, therefore, wondered whether a similar sequence of initial transient adhesion followed by subsequent responses would be observed in hyperoxic lung inflammation. We, therefore, determined the effects of hyperoxia exposure on lung mRNA for P- and E-Selectin in mouse lungs. We found that there was no detectable mRNA for E-Selectin through 72 h of hyperoxia exposure by Northern blotting, but that mRNA for P-Selectin was detectable as early as 48 h after initiation of hyperoxia. To determine the location of P-Selectin upregulation we examined hyperoxia-exposed mouse lungs by in situ hybridization and found that the upregulation of P-Selectin at 48 h was localized to large muscularized vessels, at 72 h expression was detected in some medium size muscularized vessels, and at 96 h abundant expression was observed also on nonmuscularized small vessels. In conclusion, increases in mRNA for P-Selectin early in the course of hyperoxia exposure suggest that P-Selectin expression in hyperoxic lungs increases in parallel with upregulation of ICAM-1, leading to the accumulation of neutrophils in hyperoxic lungs, and that interventions targeting these two adhesion molecules may lead to a diminution in hyperoxic lung inflammation and lung injury.