Lungs Of Lambs Research Articles

Immunohistochemical Evaluation of Inflammatory Infiltrate in the Skin and Lung of Lambs Naturally Infected with Sheeppox Virus

The present study describes immunophenotypic characteristics of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus (SPV). Three lambs revealed typical cutaneous and pulmonary lesions of sheeppox. Histologically, cutaneous and pulmonary lesions consisted of hyperplastic and/or degenerative changes in the epithelium with mononuclear cells, neutrophils, and typical sheeppox cells (SPCs), which had a vacuolated nucleus and marginated chromatin with occasional granular intracytoplasmic inclusions. The inflammatory infiltrate in pox lesions in both skin and lung was characterized by the presence of MHC II+ dendritic cells, CD4+, CD8+, gammadelta+ T cells, IgM+ cells, and CD21+ cells. Loss of expression of MHC I and MHC II antigens was observed in the affected areas of skin and lung. SPCs, stained with anti-SPV antibody, were also positive for CD14 and CD172A, antigens expressed on monocytes and macrophages. CD14 and CD172A negative SPCs were considered to be SPV infected degenerated epithelial cells or fibroblasts.

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.

Transcription Factors NF-κB and C/EBPδ and IL-1-Induced Expression of Surfactant Protein A in Lung Explants during the Perinatal Period

Interleukin-1 (IL-1) increases the expression of surfactant protein A (SP-A) in rabbit, lamb and human fetal lung. The upregulation disappears towards term. Among the transcription factors, IL-1 activates nuclear factor ĸB (NF-ĸB) and CCAAT/enhancer-binding protein δ (C/EBPδ). NF-ĸB presumably has a role in IL-1-induced upregulation of SP-A. Also, C/EBPδ may regulate SP-A expression. The aim was to study the role of these transcription factors in the induced effect of IL-1 on SP-A expression. Explants from fetal and neonatal rabbit lung were cultured in vitro followed by studies using immunohistochemistry, electrophoretic mobility shift assay and Northern analysis. We found gestation-dependent changes in IL-1-induced immunoreactivities of NF-ĸB and C/EBPδ in the nuclei of alveolar cells. This increase in nuclear transcription factors correlated with IL-1-induced SP-A expression levels. As studied in the explants from fetal and newborn lung, the SP-A mRNA expression additionally associated with C/EBPδ mRNA and with the binding of nuclear extracts from the lung explants to the C/EBP consensus probe. On the basis of the present and previous studies, we propose that NF-ĸB and C/EBPδ have potential mediator roles in IL-1-induced upregulation of SP-A in immature lung.

Chronic endotoxin exposure does not cause sustained structural abnormalities in the fetal sheep lungs

Chronic early gestational chorioamnionitis is associated with development of bronchopulmonary dysplasia in preterm infants. A single intra-amniotic exposure to endotoxin decreased alveolarization and reduced expression of endothelial proteins in 125-day gestational age preterm lambs. We hypothesized that prolonged exposure to intra-amniotic endotoxin would cause progressive lung inflammation and inhibit alveolar and pulmonary vascular development. Endotoxin (1 mg/day) or saline was administered via an intra-amniotic osmotic pump from 80 to 108 days of gestational age (continuous pump) or by four weekly 10-mg intra-amniotic endotoxin injections starting at 100 days of gestational age (multiple dose). Lung morphometry, lung inflammation, vascular effects, and lung maturation were measured at delivery. The continuous pump lambs delivered at 100 days (approximately 70% of total endotoxin exposure) had lung inflammation, fewer saccules, and decreased endothelial proteins endothelial nitric oxide synthase and VEGF receptor 2 expression compared with controls. The continuous pump (delivered at 138 days) and multiple dose lambs (delivered at 130 and 145 days) had mild persistent lung inflammation and no significant differences in lung morphometry or expression of endothelial proteins compared with controls. Surfactant saturated phosphatidylcholine pool sizes were increased in all endotoxin-exposed groups, but lung function was not changed relative to controls. Contrary to our hypothesis, a prolonged fetal exposure to intra-amniotic endotoxin caused mild persistent inflammation but did not lead to progressive structural abnormalities in lungs of near-term gestation lambs.

EFFECT OF TEMPORARY TRACHEAL OCCLUSION ON THE ENDOTHELIN SYSTEM IN EXPERIMENTAL CASES OF DIAPHRAGMATIC HERNIA

Previously, the authors have shown that tracheal occlusion (TO) partially reverses the onset of congenital diaphragmatic hernia (CDH)-induced pulmonary hypertension (PH) and abnormal pulmonary vascular development whereas release of the occlusion (TR) abolishes these clinical benefits. As a consequence of their mitogenic and vasoactive properties, the authors hypothesize that the expression of endothelin (ET)-1 and ET receptor (ETA) genes is increased in lungs of CDH lambs, and that this increase is abolished partially in CDH + TO but not in CDH + TO + TR. A surgical left-sided CDH was created in fetal lambs at 80 days of gestation (gd), followed by TO at 108 gd, and by TR at 129 gd. Four groups were compared: CDH, CDH + TO, CDH + TO + TR, and nonoperated controls (C). Assessment of mRNA expression by Northern blot showed significantly lower ET-1 and ETA levels in the CDH group than in the CDH + TO±TR groups (P < .05). Endothelin protein expression levels were lower in CDH±TO±TR groups when compared with controls for airways and vessels (P < .05) with the exception of endothelial cells. In contrast, ETA protein expression levels were higher in CDH±TO±TR groups compared with controls for airways and blood vessels smooth muscles (P < .05). These results suggest that involvement of the endothelin system in the pulmonary hypertension associated with CDH is limited. However, the endothelin system appears to be modulated during development.

Endothelin B Receptor Blockade Attenuates Pulmonary Vasodilation in Oxygen-Ventilated Fetal Lambs

Background: Endothelin-1 (ET-1) contributes to the regulation of pulmonary vascular tone in the normal ovine fetus and in models of perinatal pulmonary hypertension. In the fetal lamb lung, the effects of ET-1 depend on the balance of at least two endothelin receptor subtypes: ET_A and ET_B. ET_A receptors are located on smooth muscle cells and mediate vasoconstriction and smooth muscle proliferation. Stimulation of endothelial ET_B receptors causes vasodilation through release of nitric oxide and also functions to remove ET-1 from the circulation. However, whether activation of ET_B receptors contributes to the fall in pulmonary vascular tone at birth is unknown. Objective and Methods: To determine the role of acute ET_B receptor blockade in pulmonary vasodilation in response to birth-related stimuli, we studied the hemodynamic effects of selective ET_B receptor blockade with BQ-788 during mechanical ventilation with low (<10%) and high FiO₂ (100%) in near-term fetal sheep. Results: Intrapulmonary infusion of BQ-788 did not change left pulmonary artery (LPA) blood flow and pulmonary vascular resistance (PVR) at baseline. In comparison with controls, BQ-788 treatment attenuated the rise in LPA flow with low and high FiO₂ ventilation (p < 0.001 vs. control for each FiO₂ concentration). PVR progressively decreased during mechanical ventilation with low and high FiO₂ in both groups, but PVR remained higher after BQ-788 treatment throughout the study period (p < 0.001). Conclusions: We conclude that selective ET_B receptor blockade attenuates pulmonary vasodilation at birth. We speculate that ET_B receptor stimulation contributes to pulmonary vasodilation at birth in the ovine fetus.

Vascular changes after intra-amniotic endotoxin in preterm lamb lungs.

Chorioamnionitis is associated with preterm delivery and bronchopulmonary dysplasia (BPD), characterized by impaired alveolar and pulmonary vascular development and vascular dysfunction. To study the vascular effects in a model of chorioamnionitis, preterm lambs were exposed to 20 mg of intra-amniotic endotoxin or saline for 1, 2, 4, or 7 days and delivered at 122 days gestational age (term = 150 days). This intra-amniotic endotoxin dose was previously shown to induce lung maturation. The effect of intra-amniotic endotoxin on expression of endothelial proteins was evaluated. Muscularization of the media and collagen deposition in adventitia of small pulmonary arteries was used to assess vascular remodeling. Compared with controls, bronchoalveolar lavage fluid protein content was increased 2 days after intra-amniotic endotoxin exposure. Vascular endothelial growth factor (VEGF) 165 isoform mRNA decreased 2-4 days after intra-amniotic endotoxin. VEGF, VEGF receptor-2, endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, and Tie-2 protein expression in the lung coordinately decreased 1-7 days after intra-amniotic endotoxin. Intra-amniotic endotoxin appeared to selectively decrease eNOS expression in small pulmonary vessels compared with large vessels. Medial smooth muscle hypertrophy and increased adventitial fibrosis were observed 4 and 7 days after intra-amniotic endotoxin. These results demonstrate that, in the preterm lamb lung, antenatal inflammation inhibits endothelial cell protein expression followed by vascular remodeling changes in small pulmonary arteries. Exposure to antenatal inflammation may cause vascular remodeling and contribute to the development of BPD.

P09.02: Accuracy of magnetic resonance imaging for measuring fetal lamb lungs and organs

Congenital diaphragmatic hernia (CDH) is associated with a high mortality rate (over 50%). Prediction is more or less correlated to lung volume at 26 weeks, currently based on lung-to-head ratio (LHR) (Metkus, J Ped Surg 1996; 31: 148–152). Alternatively, lung volume measurement is performed by magnetic resonance imaging (MRI). (Mahieu-Caputo BJOG 2001; 108: 863–868). We conducted an experimental study to determine agreement and precision of MRI fetal organs volume determination versus volume determination at macroscopy (necropsy). In a total of 9 sheeps under general anaesthesia, 14 fetal lambs underwent MRI. Lungs where measured in T2 weighted images in several planes (axial, coronal and sagittal), livers and kidneys where measured in one plane. Lungs where divided in hypoplastic lungs (DH was created at a mean gestational age (GA) of 73 days, n = 10), and normal lungs (n = 18). Necropsy was done less then 24 hours after MRI (at a mean GA of 120 days). At necropsy, organ volumes where determined by the volume displacement method. Ranges volume at necropsy, means ± SD, bias (mean of the difference necropsy volume-MRI volume) and precision (SD of the difference), are represented. Analysis is made according to Bland and Altman (The Lancet 1986; i: 307–310). There was a better agreement in small volumes (Kidney, bias = 2.01) comparing to bigger volumes (Liver, bias = − 35.49) and when lung volumes where measured in the axial plane (bias = − 15.58) comparing to coronal (bias = − 20.22) or sagittal planes (bias = − 26.62). Normal lung volumes in axial planes showed a better agreement comparing to CDH lungs, but this was not the case in the coronal or the sagittal planes. Bland and Altman analyses showed no clinically important systematic errors in the MRI measurements. MRI performed in the axial plane is reliable in predicting CDH as well as normal lung volumes in fetal lamb.

Variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry.

Antenatal exposure to intra-amniotic (i.a.) endotoxin initiates a complex series of events, including an inflammatory cascade, increased surfactant production, and alterations to lung structure. Using the low frequency forced oscillation technique as a sensitive tool for measurement of respiratory impedance, we aimed to determine which factors contributed most to measured changes in lung mechanics. Respiratory impedance data obtained from sedated preterm lambs exposed to either i.a. injection with saline or 20 mg of endotoxin 1, 2, 4, and 15 days before delivery at 125 days gestation were studied, and association with indexes of standard lung morphometry, inflammatory response, and alveolar surfactant-saturated phosphatidylcholine (Sat PC) pool size was demonstrated. Reduction in tissue impedance with increasing interval between exposure and delivery was evident as early as 4 days after i.a. endotoxin injection, coinciding with resolution of inflammatory reaction, increased alveolar surfactant pools, and contribution of alveolar ducts to the parenchymal fraction, and a later decrease in the tissue component of the parenchymal fraction. Decreases in tissue damping (resistance) were more marked than decreases in tissue elastance. Log alveolar Sat PC accounted for most variability in tissue damping (88.9%) and tissue elastance (73.4%), whereas tissue fraction contributed 2 and 6.4%, respectively. The alveolar Sat PC pool size was the sole factor contributing to change in tissue hysteresivity. No changes were observed in airway resistance. Despite the complex cascade of events initiated by antenatal endotoxin exposure, variability in lung tissue mechanics is associated primarily with changes in alveolar Sat PC pool and lung morphology.

Psychrobacter pulmonis sp. nov., isolated from the lungs of lambs.

Unusual Gram-negative, catalase- and oxidase-positive, coccus-shaped bacteria isolated from the lungs of two lambs were characterized by phenotypic and molecular-genetic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown isolates were genealogically highly related to each other (99.8% sequence similarity) and represent a novel subline within the genus Psychrobacter. The unknown bacterium was phylogenetically closely related to, but distinct from, Psychrobacter phenylpyruvicus, Psychrobacter immobilis, Psychrobacter glacincola and Psychrobacter urativorans. The novel Psychrobacter isolates were readily distinguished from all other Psychrobacter species and other Gram-negative, oxidase-positive bacteria usually responsible for lung infections in sheep by physiological and biochemical tests. Based on molecular-genetic and phenotypic evidence, it is proposed that the unknown Psychrobacter isolates from lambs be classified as Psychrobacter pulmonis sp. nov. The type strain is strain S-606T (=CECT 5989T =CCUG 46240T).

Increased IP-10 and MIG Expression after Intra-amniotic Endotoxin in Preterm Lamb Lung

Subtraction hybridization was performed to explore changes in gene expression in the fetal lung after 20 mg of intra-amniotic (IA) endotoxin. Interferon-gamma-inducible 10-kd protein (IP-10) and monokine induced by interferon-gamma (MIG) constituted 20% of 102 endotoxin-induced clones identified in the preterm lamb lung. IP-10 (CXCL10) and MIG (CXCL9) are T-cell chemoattractants that have angiostatic properties. Both IP-10 and MIG mRNA were induced 30- to 40-fold in the fetal lung at 1 to 2 days after IA endotoxin. Intense IP-10 mRNA expression was detected by in situ hybridization in the bronchiolar and peribronchiolar areas and the vascular endothelium after IA endotoxin at all time points tested. MIG mRNA expression was detected initially focally in infiltrating neutrophils (15 hours after IA endotoxin) and later in the bronchiolar and peribronchiolar areas and vascular endothelium (1 day after IA endotoxin). In contrast to endotoxin, IA tumor necrosis factor-alpha or interleukin-1 alpha did not induce IP-10 or MIG mRNA in the lung. IA endotoxin also caused a modest induction of IP-10 and MIG mRNA in the jejunum, liver, and spleen. The IP-10 and MIG receptor CXCR3 was detected in the bronchiolar epithelium of preterm lambs by immunostaining. IP-10 and MIG are potent angiostatic chemokines that may contribute to lung injury and altered pulmonary vascular development in the preterm exposed to chorioamnionitis.

Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase.

Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (P<0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (P<0.05). Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67phox, a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (P<0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (P<0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth.

Dependence of intrapulmonary pressure amplitudes on respiratory mechanics during high-frequency oscillatory ventilation in preterm lambs.

In the healthy animal lung, high-frequency oscillatory ventilation (HFOV) achieves effective ventilation at tidal volumes (V(T)) less than or equal to dead space while generating very small pressure fluctuations in the alveolar spaces (deltaP(A)). We hypothesized that the respiratory mechanical parameters influence the magnitude of the intrapulmonary pressure fluctuations during HFOV. A computer model of the neonatal respiratory system was used to examine the independent effects of altering the compliance, nonlinear and linear resistance, and inertance of the respiratory system on V(T), and cyclic intrapulmonary pressures under homogeneous and heterogeneous conditions. The impact of low compliance on the transmission of pressure from the airway opening to the trachea (deltaP(tr)/deltaP(ao)) and alveolar compartment (deltaP(A)/deltaP(ao)) during HFOV was determined in a preterm lamb lung model. In the computer model, an increase in flow-dependent resistance to simulate changing the internal diameter of the tracheal tube from 4.0 mm to 2.5 mm halved the transmission of the pressure waveform to both the carina and the alveolar compartment. Increased peripheral resistance was associated with an increased deltaP(tr)/deltaP(ao) but a reduction in deltaP(A)/deltaP(ao). The deltaP(A)/deltaP(ao) also decreased with increasing alveolar compartment compliance, a finding that was verified in the preterm lamb lung. There was an exponential decrease in the magnitude of deltaP(A1) compared with deltaP(A2) as the ratio of the time constants of the two parallel compartments (tau(1)/tau(2)) increased in the heterogeneous computer lung model. The transmission of driving pressure amplitude to both the proximal airways and lung tissue during HFOV is dependent on lung mechanics and may be greater in the poorly compliant lung than that observed previously in experiments on healthy animals.

Dependence of Intrapulmonary Pressure Amplitudes on Respiratory Mechanics during High-Frequency Oscillatory Ventilation in Preterm Lambs

In the healthy animal lung, high-frequency oscillatory ventilation (HFOV) achieves effective ventilation at tidal volumes (VT) less than or equal to dead space while generating very small pressure fluctuations in the alveolar spaces (ΔPA). We hypothesized that the respiratory mechanical parameters influence the magnitude of the intrapulmonary pressure fluctuations during HFOV. A computer model of the neonatal respiratory system was used to examine the independent effects of altering the compliance, nonlinear and linear resistance, and inertance of the respiratory system on VT, and cyclic intrapulmonary pressures under homogeneous and heterogeneous conditions. The impact of low compliance on the transmission of pressure from the airway opening to the trachea (ΔPtr/ΔPao) and alveolar compartment (ΔPA/ΔPao) during HFOV was determined in a preterm lamb lung model. In the computer model, an increase in flow-dependent resistance to simulate changing the internal diameter of the tracheal tube from 4.0 mm to 2.5 mm halved the transmission of the pressure waveform to both the carina and the alveolar compartment. Increased peripheral resistance was associated with an increased ΔPtr/ΔPao but a reduction in ΔPA/ΔPao. The ΔPA/ΔPao also decreased with increasing alveolar compartment compliance, a finding that was verified in the preterm lamb lung. There was an exponential decrease in the magnitude of ΔPA1 compared with ΔPA2 as the ratio of the time constants of the two parallel compartments (τ1/τ2) increased in the heterogeneous computer lung model. The transmission of driving pressure amplitude to both the proximal airways and lung tissue during HFOV is dependent on lung mechanics and may be greater in the poorly compliant lung than that observed previously in experiments on healthy animals.

Decreased indicators of lung injury with continuous positive expiratory pressure in preterm lambs.

Continuous positive airway pressure (CPAP) is being used clinically to avoid mechanical ventilation of preterm infants as a strategy to minimize lung injury. There is little experimental information about how CPAP might minimize lung injury after preterm birth. We induced preterm labor in antenatal glucocorticoid-treated sheep carrying twins at 133 d gestation with an inhibitor of progesterone synthesis. The lambs delivered spontaneously approximately 2 d later and were randomized to three groups: no ventilation (n = 4), conventional mechanical ventilation to a target PCO(2) of 40 mm Hg (n = 5), or CPAP using a bubble CPAP device set to deliver 5 cm H(2)O pressure (n = 6). The CPAP lambs breathed without distress and maintained PCO(2) values of approximately 60 mm Hg. At 2 h of age, the lungs of the CPAP lambs held 74 +/- 4 mL/kg air at 40 cm H(2)O pressure, which was more than the 60 +/- 3 mL/kg for the ventilated lambs (p < 0.05). Lymphocyte and monocyte numbers in alveolar washes were equivalent in the unventilated, ventilated, and CPAP lambs. However, no neutrophils were found in the unventilated lambs, and the ventilated lambs had 6.6 times more neutrophils in alveolar washes than did the CPAP lambs (p < 0.05). The cells in alveolar wash from CPAP lambs contained less hydrogen peroxide than did the cells from ventilated lambs (p < 0.05). In this model in preterm lambs CPAP results in lower indicators of acute lung injury than does mechanical ventilation during the first 2 h of life.

Decreased Indicators of Lung Injury with Continuous Positive Expiratory Pressure in Preterm Lambs

Continuous positive airway pressure (CPAP) is being used clinically to avoid mechanical ventilation of preterm infants as a strategy to minimize lung injury. There is little experimental information about how CPAP might minimize lung injury after preterm birth. We induced preterm labor in antenatal glucocorticoid-treated sheep carrying twins at 133 d gestation with an inhibitor of progesterone synthesis. The lambs delivered spontaneously approximately 2 d later and were randomized to three groups: no ventilation (n = 4), conventional mechanical ventilation to a target Pco2 of 40 mm Hg (n = 5), or CPAP using a bubble CPAP device set to deliver 5 cm H2O pressure (n = 6). The CPAP lambs breathed without distress and maintained Pco2 values of approximately 60 mm Hg. At 2 h of age, the lungs of the CPAP lambs held 74 ± 4 mL/kg air at 40 cm H2O pressure, which was more than the 60 ± 3 mL/kg for the ventilated lambs (p < 0.05). Lymphocyte and monocyte numbers in alveolar washes were equivalent in the unventilated, ventilated, and CPAP lambs. However, no neutrophils were found in the unventilated lambs, and the ventilated lambs had 6.6 times more neutrophils in alveolar washes than did the CPAP lambs (p < 0.05). The cells in alveolar wash from CPAP lambs contained less hydrogen peroxide than did the cells from ventilated lambs (p < 0.05). In this model in preterm lambs CPAP results in lower indicators of acute lung injury than does mechanical ventilation during the first 2 h of life.

Effects of intra-amniotic endotoxin on lung structure and function two months after term birth in sheep

Intra-amniotic endotoxin causes chorioamnionitis and results in improved lung function after preterm delivery in sheep. Our aim was to determine the effects on lung structure and function after term birth in lambs exposed to intra-amniotic or intra-allantoic endotoxin. At 119 days' gestation, pregnant ewes bearing singleton fetuses received intra-amniotic or intra-allantoic injections of either saline (allantoic, n = 1; amniotic, n = 10) or Escherichia coli (055:B5) endotoxin (allantoic, n = 5; amniotic, n = 7). Amniotic or allantoic fluid was aspirated for white blood cell counts 24 hours after the injections. Ewes (n = 20) were allowed to deliver spontaneously. At 8 weeks' postnatal age we measured ventilation, lung volumes, and compliance in the offspring and collected their lungs for morphologic analysis. Higher amniotic or allantoic cell counts were confined to the fluid space into which endotoxin was injected. Saline injections did not increase amniotic or allantoic white blood cell counts. Gestation length, birth weight, and postnatal growth were unaffected by endotoxin treatments. Lung volumes and compliances at 8 weeks of age were not different between saline-treated and endotoxin-treated groups. Lung morphometry was not significantly altered by endotoxin, with one minor exception: interlobular septal volume was increased by intra-amniotic endotoxin, but this effect had no functional consequences. Intra-amniotic or intra-allantoic endotoxin caused a localized inflammatory response but did not cause preterm delivery or intrauterine growth restriction. The functional improvements and corresponding structural alterations in the lungs of preterm lambs, reported previously in this model, were not associated with improved or impaired lung function or marked alterations in lung structure 2 months after birth at term.

Injury, inflammation, and remodeling in fetal sheep lung after intra-amniotic endotoxin.

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intra-amniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung.

Injury Responses to Different Surfactants in Ventilated Premature Lamb Lungs

The lung of the preterm infant is easily injured and an initial indication of the injury is an inflammatory response. Surfactant treatment and gentle ventilation will minimize the initiation and progression of injury. We asked if the initial lung injury response differed when preterm ventilated lambs were treated with complete natural sheep surfactant, a lipid extract of sheep surfactant, a surfactant used to treat RDS (Survanta®), or a synthetic surfactant containing recombinant SP-C (Venticute®). We used a gentle style of ventilation and a positive end expiratory pressure of 4 cmH20 to minimize injury. The surfactants were not distinguishable based on gas exchange, compliance or lung gas volumes over the 6h ventilation period. When compared with unventilated controls the ventilated lambs had increased protein and inflammatory cells in alveolar lavages. The cells from the alveolar lavages produced more H202, expressed more surface adhesion antigens and CD-14 receptors, and expressed more mRNA for the pro-inflammatory cytokines IL-1β and IL-8 than did cells from unventilated lungs. Lung tissue expressed primarily increased IL-6 mRNA relative to unventilated controls. However, there were no consistent differences in any of the inflammatory indicators between the different surfactant treated groups. Because endotoxin free natural surfactant containing SP-A was not superior to three other surfactants containing differing amounts of the surfactant proteins, additions of these proteins to clinical surfactants may not decrease the indicators of lung inflammation that accompany the initiation of ventilation of the preterm lung.

Injury responses to different surfactants in ventilated premature lamb lungs.

The lung of the preterm infant is easily injured and an initial indication of the injury is an inflammatory response. Surfactant treatment and gentle ventilation will minimize the initiation and progression of injury. We asked if the initial lung injury response differed when preterm ventilated lambs were treated with complete natural sheep surfactant, a lipid extract of sheep surfactant, a surfactant used to treat RDS (Survanta), or a synthetic surfactant containing recombinant SP-C (Venticute). We used a gentle style of ventilation and a positive end expiratory pressure of 4 cmH(2)0 to minimize injury. The surfactants were not distinguishable based on gas exchange, compliance or lung gas volumes over the 6h ventilation period. When compared with unventilated controls the ventilated lambs had increased protein and inflammatory cells in alveolar lavages. The cells from the alveolar lavages produced more H(2)0(2), expressed more surface adhesion antigens and CD-14 receptors, and expressed more mRNA for the pro-inflammatory cytokines IL-1 beta and IL-8 than did cells from unventilated lungs. Lung tissue expressed primarily increased IL-6 mRNA relative to unventilated controls. However, there were no consistent differences in any of the inflammatory indicators between the different surfactant treated groups. Because endotoxin free natural surfactant containing SP-A was not superior to three other surfactants containing differing amounts of the surfactant proteins, additions of these proteins to clinical surfactants may not decrease the indicators of lung inflammation that accompany the initiation of ventilation of the preterm lung.