Delivery of medications to preterm neonates receiving non-invasive ventilation (NIV) represents o... more Delivery of medications to preterm neonates receiving non-invasive ventilation (NIV) represents one of the most challenging scenarios for aerosol medicine. This challenge is highlighted by the undersized anatomy and the complex (patho)physiological characteristics of the lungs in such infants. Key physiological restraints include low lung volumes, low compliance, and irregular respiratory rates, which significantly reduce lung deposition. Such factors are inherent to premature birth and thus can be regarded to as the intrinsic factors that affect lung deposition. However, there are a number of extrinsic factors that also impact lung deposition: such factors include the choice of aerosol generator and its configuration within the ventilation circuit, the drug formulation, the aerosol particle size distribution, the choice of NIV type, and the patient interface between the delivery system and the patient. Together, these extrinsic factors provide an opportunity to optimize the lung de...
Aims: Recent literature suggests hypothermia may protect against lung injury. We evaluated body t... more Aims: Recent literature suggests hypothermia may protect against lung injury. We evaluated body temperature as a variable in lung inflammation due to oxygenation and mechanical ventilation following delivery of near-term lambs. Methods: Twin fetuses were randomized prior to delivery at 140 d GA (term 150 d): unventilated controls, normothermic ventilated with room air, normothermic ventilated with 100% oxygen, low temperature ventilated (target 35 • C) with 100% oxygen, and high temperature (target 40 • C) with 100% oxygen. Lambs were intubated for gentle mechanical ventilation (tidal volume 7-8 ml/kg). Temperature targeting was with radiant warmers and plastic wrap for normothermia, with heat lamps for hyperthermia, and with ice packs for hypothermia. Lambs were euthanized after 2 h mechanical ventilation. Post-mortem, bronchoalveolar lavage fluid and lung tissue samples were evaluated for inflammatory responses by measuring inflammatory cell counts, protein, myeloperoxidase, protein carbonyl, and pro-inflammatory cytokine mRNA. Results: Target temperatures were achieved by 30 min of age and tightly maintained for the 2 h study. There were no differences in physiologic variables among groups except those directly resulting from study protocol-PaO 2 from air vs. 100% oxygen and body temperature. Indicators of inflammation increased similarly in all ventilated groups compared to unventilated controls. Conclusion: Moderate hyperthermia or hypothermia did not affect lung injury responses to the initiation of ventilation at birth in near-term lambs.
Background: Increased pro-inflammatory cytokines in tracheal aspirates correlate with the develop... more Background: Increased pro-inflammatory cytokines in tracheal aspirates correlate with the development of BPD in preterm infants. Ventilation of preterm lambs increases pro-inflammatory cytokines and causes lung inflammation. Objective: We tested the hypothesis that selective inhibitors of pro-inflammatory signaling would decrease lung inflammation induced by ventilation in preterm newborn lambs. We also examined if the variability in injury response was explained by variations in the endogenous surfactant pool size. Methods: Date-mated preterm lambs (n = 28) were operatively delivered and mechanically ventilated to cause lung injury (tidal volume escalation to 15 mL/kg by 15 min at age). The lambs then were ventilated with 8 mL/kg tidal volume for 1 h 45 min. Groups of animals randomly received specific inhibitors for IL-8, IL-1, or NF-B. Unventilated lambs (n = 7) were the controls. Bronchoalveolar lavage fluid (BALF) and lung samples were used to quantify inflammation. Saturated phosphatidylcholine (Sat PC) was measured in BALF fluid and the data were stratified based on a level of 5 μmol/kg (~8 mg/kg surfactant). Results: The inhibitors did not decrease the cytokine levels or inflammatory response. The inflammation increased as Sat PC pool size in BALF decreased. Ventilated lambs with a Sat PC level > 5 μmol/kg had significantly decreased markers of injury and lung inflammation compared with those lambs with < 5 μmol/kg. Conclusion: Lung injury caused by high tidal volumes at birth were decreased when endogenous surfactant pool sizes were larger. Attempts to decrease inflammation by blocking IL-8, IL-1 or NF-B were unsuccessful.
Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding vent... more Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding ventilation heterogeneities: whether to achieve this gradually or quickly remains unclear. We compared the effect of gradual vs. constant tidal inflations and a pressure-limited sustained inflation (SI) at birth on gas exchange, lung mechanics, gravity-dependent lung volume distribution, and lung injury in 131-day gestation preterm lambs. Lambs were resuscitated with either 1) a 20-s, 40-cmH2O pressure-limited SI (PressSI), 2) a gradual increase in tidal volume (Vt) over 5-min from 3 ml/kg to 7 ml/kg (IncrVt), or 3) 7 ml/kg Vt from birth. All lambs were subsequently ventilated for 15 min with 7 ml/kg Vt with the same end-expiratory pressure. Lung mechanics, gas exchange and spatial distribution of end-expiratory volume (EEV), and tidal ventilation (electrical impedance tomography) were recorded regularly. At 15 min, early mRNA tissue markers of lung injury were assessed. The IncrVt group res...
Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymu... more Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development. Methods: Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated. Results: Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intraamniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes. Conclusion: Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.
Background: Ventilated preterm infant lungs are vulnerable to overdistension and underinflation. ... more Background: Ventilated preterm infant lungs are vulnerable to overdistension and underinflation. The optimal ventilator-delivered tidal volume (VT) in these infants is unknown and may depend on the extent of alveolarisation at birth. Objectives: We aimed to calculate respiratory dead space (VD) from the molar mass (MM) signal of an ultrasonic flowmeter (VD,MM) in very preterm infants on volume-targeted ventilation (VT target, 4-5 ml/kg) and to study the association between gestational age (GA) and VD,MM-to-VT ratio (VD,MM/VT), alveolar tidal volume (VA) and alveolar minute volume (AMV). Methods: This was a single-centre, prospective, observational, cohort study in a neonatal intensive care unit. Tidal breathing analysis was performed in ventilated very preterm infants (GA range 23-32 weeks) on day 1 of life. Results: Valid measurements were obtained in 43/51 (87%) infants. Tidal breathing variables were analysed using multivariable linear regression. VD,MM/VT was negatively associat...
Background: Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory respon... more Background: Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum. Method: Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1a at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days). Results: 3 and 7 d after IL-1a administration, intestinal mRNA levels for IL-4, IL-10, IFN-c and TNF-a were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1. Conclusion: Intraamniotic IL-1a causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process.
The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigeni... more The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2610 4 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulentderived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p.0.05). MBA size variants (32-170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1b, IL-6 and IL-8 expression in chorioamnion tissue (p,0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.
Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilatio... more Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilation, but its role in protecting the lung from injury during the initiation of ventilation in the delivery room is not established. We aimed to evaluate whether PEEP and/or tidal volume (V T) within the first 15-min of ventilation are protective against lung injury. Operatively delivered preterm lambs (133 Ϯ 1 d gestation) were randomly assigned to unventilated controls or to one of four 15 min ventilation interventions: 1) V T 15 mL/kg, PEEP 0 cm H 2 O; 2) V T 15 mL/kg, PEEP 5 cm H 2 O; 3) V T 8 mL/kg, PEEP 0 cm H 2 O; and 4) V T 8 mL/kg, PEEP 5 cm H 2 O. Each group was subsequently ventilated with V T Ͻ10 mL/kg, PEEP 5 cm H 2 O for 1 h 45 min. Lung function was assessed and measurements of lung injury were evaluated postmortem. After the 15 min ventilation maneuver, the V T 15 groups were hypocarbic, had higher oxygenation, and required lower pressures than the V T 8 groups; no consistent effect of PEEP was found. Markers of lung injury were significantly elevated in all ventilation groups compared with unventilated controls; no effect of PEEP was found. Ventilation resulted in localization of IL-6 to the small airways. Initial ventilation of preterm lambs with PEEP and/or V T of 8 mL/kg did not prevent an inflammatory injury to the lung.
Basic Science Investigation nature publishing group Background: Intra-amniotic lipopolysaccharide... more Basic Science Investigation nature publishing group Background: Intra-amniotic lipopolysaccharide (LPS) exposure may affect neonatal outcome by altering fetal lung and immune system development. We hypothesized that intra-amniotic LPS exposure would cause persistent fetal pulmonary responses as the lungs develop in utero. Methods: Fetal lambs were exposed to intra-amniotic LPS at 118 or at 118 and 123 d of gestational age (GA) with delivery at 125, 133, or 140 d (term = 147 d). Immune responses, PU.1 expression, toll-like receptor (tLR)-1,2,4,6 mRNA levels, mast cell levels, and pulmonary elastin deposition were evaluated. results: After a single dose of LPS, pulmonary inflammatory responses were observed with increases of (i) PU.1 and tLR1 at 125 d GA and (ii) monocytes, lymphocytes, tLR2, and tLR6 at 133 d GA. Repetitive LPS exposure resulted in (i) increases of neutrophils, monocytes, PU.1, and tLR1 at 125 d GA; (ii) increases of neutrophils, PU.1, and tLR2 at 133 d GA; and (iii) decreases of mast cells, elastin foci, tLR4, and tLR6 at early gestation. At 140 d GA, only PU.1 was increased after repetitive LPS exposure. conclusion: The preterm fetal lung can respond to a single exposure or repeated exposures from intra-amniotic LPS in multiple ways, but the absence of inflammatory and structural changes in LPS-exposed fetuses delivered near term suggest that the fetus can resolve an inflammatory stimulus in utero with time.
Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia and... more Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia and persistent lung disease in childhood. We report where injury occurred within the lung after brief ventilation at birth. Preterm sheep (129 d gestation) were ventilated with an escalating tidal volume to 15 mL/kg by 15 min to injure the lungs, with the placental circulation intact (fetal) or after delivery (newborn). Fetal lambs were returned to the uterus for 2 h 45 min, whereas newborn lambs were maintained with gentle ventilatory support for the same period. The control group was not ventilated. Bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed. In both fetal and newborn lambs, ventilation caused bronchial epithelial disruption in mediumsized airways. Early growth response protein 1 (Egr-1), monocyte chemotactic protein 1 (MCP-1), IL-6, and IL-1 mRNA increased in the lung tissue from fetal and newborn lambs. Egr-1, MCP-1, and IL-6 mRNA were induced in mesenchymal cells surrounding small airways, whereas IL-1 mRNA localized to the epithelium of medium/small airways. Ventilation caused loss of heat shock protein 70 (HSP70) mRNA from the bronchial epithelium, but induced mRNA in the smooth muscle surrounding large airways. HSP70 protein decreased in the lung tissue and increased in BALF with ventilation. Initiation of ventilation induced a stress response and inflammatory cytokines in small and medium-sized airways.
Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis,... more Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis, and IL-1 contributes to the pathogenesis of chorioamnionitis-induced lung inflammation and fetal inflammatory responses. We tested the hypothesis that IL-1 mediates the systemic and pulmonary induction of MCP-1 and MCP-2 in response to lipopolysaccharide (LPS)-induced chorioamnionitis. MCP-1 mRNA, MCP-2 mRNA, and MCP-1 protein expression were measured in two models: 1) intra-amniotic LPS and 2) intra-amniotic recombinant sheep IL-1␣ given at varying intervals before preterm delivery at 124 d GA. Intra-amniotic LPS or IL-1␣ induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2 d. LPS induced intense MCP-1 expression in subepithelial mesenchymal cells and interstitial inflammatory cells in the lung. Inhibition of IL-1 signaling with recombinant human IL-1 receptor antagonist (rhIL-1ra) did not attenuate LPS induced increase in MCP-1 or MCP-2 expression. MCP-1 and MCP-2 were not induced in liver or chorioamnion, but MCP-1 increased in cord plasma. LPS or IL-1 can induce robust expression of MCP-1 or MCP-2 in the fetal lung. LPS induction of MCP-1 is not IL-1 dependent in fetal sheep. MCP-1 and MCP-2 may be significant contributors to fetal inflammation.
Basic Science Investigation nature publishing group Background: Antenatal inflammation and matern... more Basic Science Investigation nature publishing group Background: Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intraamniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids. Methods: Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age. results: IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger β-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post-LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA. conclusion: IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.
This paper is the fourth in a series of reviews that will summarize available data and critically... more This paper is the fourth in a series of reviews that will summarize available data and critically discuss the potential role of lung-function testing in infants with acute neonatal respiratory disorders and chronic lung disease of infancy. The current paper addresses information derived from tidal breathing measurements within the framework outlined in the introductory paper of this series, with particular reference to how these measurements inform on control of breathing. Infants with acute and chronic respiratory illness demonstrate differences in tidal breathing and its control that are of clinical consequence and can be measured objectively. The increased incidence of significant apnea in preterm infants and infants with chronic lung disease, together with the reportedly increased risk of sudden unexplained death within the latter group, suggests that control of breathing is affected by both maturation and disease. Clinical observations are supported by formal comparison of tidal breathing parameters and control of breathing indices in the research setting.
We report on four children who received cis-platinum simultaneously with, or in one case 10 month... more We report on four children who received cis-platinum simultaneously with, or in one case 10 months after, cranial irradiation and experienced exaggerated ototoxicity affecting all audible frequencies. The hearing loss was severe, affecting the critical areas for speech perception, and necessitated the provision of bilateral hearing aids. The audiograms of these patients are shown and compared to those of four children who had received cis-platinum as part of their treatment for neuroblastoma but without cranial irradiation. The precipitation of the exaggerated hearing loss with the administration of cis-platinum in one patient 10 months after finishing cranial irradiation suggests that care should be taken in the timing of cis-platinum administration in relation to concurrent or previous cranial irradiation.
To assess whether lung volume and ventilation inhomogeneity in preterm infants at 15-18 months co... more To assess whether lung volume and ventilation inhomogeneity in preterm infants at 15-18 months corrected age, and the change in these outcomes from the newborn period to 15-18 months corrected age, depend on gestational age (GA) at birth and the severity of neonatal lung disease. Preterm (GA range, 23-32 weeks) and term healthy control infants were studied in quiet sedated sleep at 15-18 months corrected age by multiple breath washout with 5% sulfur hexafluoride using an ultrasonic flowmeter. Valid measurements were obtained from 58 infants. Multivariate and multilevel regression was used to analyze outcomes. Functional residual capacity (FRC), lung clearance index, and first and second to zeroeth moment ratios were calculated. After accounting for body size at test, FRC at follow-up, and the increase in FRC from the newborn period to 15-18 months corrected age were positively associated with GA and negatively associated with the duration of endotracheal ventilation. Indices of ventilation inhomogeneity were unaltered by GA and the duration of endotracheal ventilation. In very preterm infants, GA and the duration of endotracheal ventilation are independently associated with reduced lung volume and lung growth during infancy, although the effect size of these findings is small.
Journal of Developmental Origins of Health and Disease, 2012
Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our pre... more Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our previous work, we demonstrated that the fetal liver is involved in the systemic inflammation associated with chorioamnionitis, leading to metabolic changes. On the basis of these findings, we hypothesized that chorioamnionitis can lead to postnatal inflammation-related liver injury and disturbed lipid metabolism. Chorioamnionitis was induced in sheep by intra-amniotic injection of lipopolysaccharide (LPS) or saline at 90, 100 and 110 days of gestation. Liver homeostasis and lipid metabolism were analyzed at term and at 7 weeks of age. At term, hepatic T-lymphocytes and apoptotic hepatocytes were increased. In addition, hepatic cholesterol and triglyceride levels were decreased in LPS-exposed animals compared with controls. At 7 weeks of age, no hepatic inflammation could be detected. However, liver triglycerides and plasma cholesterol levels were increased in LPS-exposed animals relative to controls. The changes in lipid levels at 7 weeks of age were associated with increased leptin receptor mRNA levels, increased lipid peroxidation, increased expression of cytochrome c oxidase subunit 4 as a marker for mitochondrial function and increased circulating ceramide levels. These findings demonstrate that chorioamnionitis-mediated antenatal inflammation-related liver disturbances have long-lasting postnatal effects on lipid metabolism.
Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infa... more Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infants, but the effects on cardiopulmonary hemodynamics are understood poorly. We aimed to determine the effect of increased PEEP on cardiopulmonary hemodynamics and to compare measurements from indwelling flow probes with Doppler echocardiography. Preterm lambs (129 ± 1 days) were ventilated initially with a tidal volume of 7 ml/kg and 4 cmH2O of PEEP. In ramp lambs ( n = 7), PEEP was increased by 2-cmH2O increments to 10 cmH2O and then in decrements back to 4 cmH2O. PEEP was unchanged in controls ( n = 6). Doppler echocardiographic flow measurements in the left pulmonary artery (LPA) and ductus arteriosus (DA) were correlated with flow probe measurements. Compared with controls, high PEEP reduced LPA flow from baseline (10-cmH2O PEEP: 43 ± 8% vs. control: 83 ± 21%; P = 0.029). High PEEP increased the proportion of right-to-left (R-L) shunting through the DA, with a trend to an increased o...
Delivery of medications to preterm neonates receiving non-invasive ventilation (NIV) represents o... more Delivery of medications to preterm neonates receiving non-invasive ventilation (NIV) represents one of the most challenging scenarios for aerosol medicine. This challenge is highlighted by the undersized anatomy and the complex (patho)physiological characteristics of the lungs in such infants. Key physiological restraints include low lung volumes, low compliance, and irregular respiratory rates, which significantly reduce lung deposition. Such factors are inherent to premature birth and thus can be regarded to as the intrinsic factors that affect lung deposition. However, there are a number of extrinsic factors that also impact lung deposition: such factors include the choice of aerosol generator and its configuration within the ventilation circuit, the drug formulation, the aerosol particle size distribution, the choice of NIV type, and the patient interface between the delivery system and the patient. Together, these extrinsic factors provide an opportunity to optimize the lung de...
Aims: Recent literature suggests hypothermia may protect against lung injury. We evaluated body t... more Aims: Recent literature suggests hypothermia may protect against lung injury. We evaluated body temperature as a variable in lung inflammation due to oxygenation and mechanical ventilation following delivery of near-term lambs. Methods: Twin fetuses were randomized prior to delivery at 140 d GA (term 150 d): unventilated controls, normothermic ventilated with room air, normothermic ventilated with 100% oxygen, low temperature ventilated (target 35 • C) with 100% oxygen, and high temperature (target 40 • C) with 100% oxygen. Lambs were intubated for gentle mechanical ventilation (tidal volume 7-8 ml/kg). Temperature targeting was with radiant warmers and plastic wrap for normothermia, with heat lamps for hyperthermia, and with ice packs for hypothermia. Lambs were euthanized after 2 h mechanical ventilation. Post-mortem, bronchoalveolar lavage fluid and lung tissue samples were evaluated for inflammatory responses by measuring inflammatory cell counts, protein, myeloperoxidase, protein carbonyl, and pro-inflammatory cytokine mRNA. Results: Target temperatures were achieved by 30 min of age and tightly maintained for the 2 h study. There were no differences in physiologic variables among groups except those directly resulting from study protocol-PaO 2 from air vs. 100% oxygen and body temperature. Indicators of inflammation increased similarly in all ventilated groups compared to unventilated controls. Conclusion: Moderate hyperthermia or hypothermia did not affect lung injury responses to the initiation of ventilation at birth in near-term lambs.
Background: Increased pro-inflammatory cytokines in tracheal aspirates correlate with the develop... more Background: Increased pro-inflammatory cytokines in tracheal aspirates correlate with the development of BPD in preterm infants. Ventilation of preterm lambs increases pro-inflammatory cytokines and causes lung inflammation. Objective: We tested the hypothesis that selective inhibitors of pro-inflammatory signaling would decrease lung inflammation induced by ventilation in preterm newborn lambs. We also examined if the variability in injury response was explained by variations in the endogenous surfactant pool size. Methods: Date-mated preterm lambs (n = 28) were operatively delivered and mechanically ventilated to cause lung injury (tidal volume escalation to 15 mL/kg by 15 min at age). The lambs then were ventilated with 8 mL/kg tidal volume for 1 h 45 min. Groups of animals randomly received specific inhibitors for IL-8, IL-1, or NF-B. Unventilated lambs (n = 7) were the controls. Bronchoalveolar lavage fluid (BALF) and lung samples were used to quantify inflammation. Saturated phosphatidylcholine (Sat PC) was measured in BALF fluid and the data were stratified based on a level of 5 μmol/kg (~8 mg/kg surfactant). Results: The inhibitors did not decrease the cytokine levels or inflammatory response. The inflammation increased as Sat PC pool size in BALF decreased. Ventilated lambs with a Sat PC level > 5 μmol/kg had significantly decreased markers of injury and lung inflammation compared with those lambs with < 5 μmol/kg. Conclusion: Lung injury caused by high tidal volumes at birth were decreased when endogenous surfactant pool sizes were larger. Attempts to decrease inflammation by blocking IL-8, IL-1 or NF-B were unsuccessful.
Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding vent... more Support of the mechanically complex preterm lung needs to facilitate aeration while avoiding ventilation heterogeneities: whether to achieve this gradually or quickly remains unclear. We compared the effect of gradual vs. constant tidal inflations and a pressure-limited sustained inflation (SI) at birth on gas exchange, lung mechanics, gravity-dependent lung volume distribution, and lung injury in 131-day gestation preterm lambs. Lambs were resuscitated with either 1) a 20-s, 40-cmH2O pressure-limited SI (PressSI), 2) a gradual increase in tidal volume (Vt) over 5-min from 3 ml/kg to 7 ml/kg (IncrVt), or 3) 7 ml/kg Vt from birth. All lambs were subsequently ventilated for 15 min with 7 ml/kg Vt with the same end-expiratory pressure. Lung mechanics, gas exchange and spatial distribution of end-expiratory volume (EEV), and tidal ventilation (electrical impedance tomography) were recorded regularly. At 15 min, early mRNA tissue markers of lung injury were assessed. The IncrVt group res...
Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymu... more Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development. Methods: Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated. Results: Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intraamniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes. Conclusion: Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.
Background: Ventilated preterm infant lungs are vulnerable to overdistension and underinflation. ... more Background: Ventilated preterm infant lungs are vulnerable to overdistension and underinflation. The optimal ventilator-delivered tidal volume (VT) in these infants is unknown and may depend on the extent of alveolarisation at birth. Objectives: We aimed to calculate respiratory dead space (VD) from the molar mass (MM) signal of an ultrasonic flowmeter (VD,MM) in very preterm infants on volume-targeted ventilation (VT target, 4-5 ml/kg) and to study the association between gestational age (GA) and VD,MM-to-VT ratio (VD,MM/VT), alveolar tidal volume (VA) and alveolar minute volume (AMV). Methods: This was a single-centre, prospective, observational, cohort study in a neonatal intensive care unit. Tidal breathing analysis was performed in ventilated very preterm infants (GA range 23-32 weeks) on day 1 of life. Results: Valid measurements were obtained in 43/51 (87%) infants. Tidal breathing variables were analysed using multivariable linear regression. VD,MM/VT was negatively associat...
Background: Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory respon... more Background: Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum. Method: Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1a at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days). Results: 3 and 7 d after IL-1a administration, intestinal mRNA levels for IL-4, IL-10, IFN-c and TNF-a were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1. Conclusion: Intraamniotic IL-1a causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process.
The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigeni... more The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2610 4 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulentderived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p.0.05). MBA size variants (32-170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1b, IL-6 and IL-8 expression in chorioamnion tissue (p,0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.
Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilatio... more Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilation, but its role in protecting the lung from injury during the initiation of ventilation in the delivery room is not established. We aimed to evaluate whether PEEP and/or tidal volume (V T) within the first 15-min of ventilation are protective against lung injury. Operatively delivered preterm lambs (133 Ϯ 1 d gestation) were randomly assigned to unventilated controls or to one of four 15 min ventilation interventions: 1) V T 15 mL/kg, PEEP 0 cm H 2 O; 2) V T 15 mL/kg, PEEP 5 cm H 2 O; 3) V T 8 mL/kg, PEEP 0 cm H 2 O; and 4) V T 8 mL/kg, PEEP 5 cm H 2 O. Each group was subsequently ventilated with V T Ͻ10 mL/kg, PEEP 5 cm H 2 O for 1 h 45 min. Lung function was assessed and measurements of lung injury were evaluated postmortem. After the 15 min ventilation maneuver, the V T 15 groups were hypocarbic, had higher oxygenation, and required lower pressures than the V T 8 groups; no consistent effect of PEEP was found. Markers of lung injury were significantly elevated in all ventilation groups compared with unventilated controls; no effect of PEEP was found. Ventilation resulted in localization of IL-6 to the small airways. Initial ventilation of preterm lambs with PEEP and/or V T of 8 mL/kg did not prevent an inflammatory injury to the lung.
Basic Science Investigation nature publishing group Background: Intra-amniotic lipopolysaccharide... more Basic Science Investigation nature publishing group Background: Intra-amniotic lipopolysaccharide (LPS) exposure may affect neonatal outcome by altering fetal lung and immune system development. We hypothesized that intra-amniotic LPS exposure would cause persistent fetal pulmonary responses as the lungs develop in utero. Methods: Fetal lambs were exposed to intra-amniotic LPS at 118 or at 118 and 123 d of gestational age (GA) with delivery at 125, 133, or 140 d (term = 147 d). Immune responses, PU.1 expression, toll-like receptor (tLR)-1,2,4,6 mRNA levels, mast cell levels, and pulmonary elastin deposition were evaluated. results: After a single dose of LPS, pulmonary inflammatory responses were observed with increases of (i) PU.1 and tLR1 at 125 d GA and (ii) monocytes, lymphocytes, tLR2, and tLR6 at 133 d GA. Repetitive LPS exposure resulted in (i) increases of neutrophils, monocytes, PU.1, and tLR1 at 125 d GA; (ii) increases of neutrophils, PU.1, and tLR2 at 133 d GA; and (iii) decreases of mast cells, elastin foci, tLR4, and tLR6 at early gestation. At 140 d GA, only PU.1 was increased after repetitive LPS exposure. conclusion: The preterm fetal lung can respond to a single exposure or repeated exposures from intra-amniotic LPS in multiple ways, but the absence of inflammatory and structural changes in LPS-exposed fetuses delivered near term suggest that the fetus can resolve an inflammatory stimulus in utero with time.
Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia and... more Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia and persistent lung disease in childhood. We report where injury occurred within the lung after brief ventilation at birth. Preterm sheep (129 d gestation) were ventilated with an escalating tidal volume to 15 mL/kg by 15 min to injure the lungs, with the placental circulation intact (fetal) or after delivery (newborn). Fetal lambs were returned to the uterus for 2 h 45 min, whereas newborn lambs were maintained with gentle ventilatory support for the same period. The control group was not ventilated. Bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed. In both fetal and newborn lambs, ventilation caused bronchial epithelial disruption in mediumsized airways. Early growth response protein 1 (Egr-1), monocyte chemotactic protein 1 (MCP-1), IL-6, and IL-1 mRNA increased in the lung tissue from fetal and newborn lambs. Egr-1, MCP-1, and IL-6 mRNA were induced in mesenchymal cells surrounding small airways, whereas IL-1 mRNA localized to the epithelium of medium/small airways. Ventilation caused loss of heat shock protein 70 (HSP70) mRNA from the bronchial epithelium, but induced mRNA in the smooth muscle surrounding large airways. HSP70 protein decreased in the lung tissue and increased in BALF with ventilation. Initiation of ventilation induced a stress response and inflammatory cytokines in small and medium-sized airways.
Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis,... more Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis, and IL-1 contributes to the pathogenesis of chorioamnionitis-induced lung inflammation and fetal inflammatory responses. We tested the hypothesis that IL-1 mediates the systemic and pulmonary induction of MCP-1 and MCP-2 in response to lipopolysaccharide (LPS)-induced chorioamnionitis. MCP-1 mRNA, MCP-2 mRNA, and MCP-1 protein expression were measured in two models: 1) intra-amniotic LPS and 2) intra-amniotic recombinant sheep IL-1␣ given at varying intervals before preterm delivery at 124 d GA. Intra-amniotic LPS or IL-1␣ induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2 d. LPS induced intense MCP-1 expression in subepithelial mesenchymal cells and interstitial inflammatory cells in the lung. Inhibition of IL-1 signaling with recombinant human IL-1 receptor antagonist (rhIL-1ra) did not attenuate LPS induced increase in MCP-1 or MCP-2 expression. MCP-1 and MCP-2 were not induced in liver or chorioamnion, but MCP-1 increased in cord plasma. LPS or IL-1 can induce robust expression of MCP-1 or MCP-2 in the fetal lung. LPS induction of MCP-1 is not IL-1 dependent in fetal sheep. MCP-1 and MCP-2 may be significant contributors to fetal inflammation.
Basic Science Investigation nature publishing group Background: Antenatal inflammation and matern... more Basic Science Investigation nature publishing group Background: Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intraamniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids. Methods: Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age. results: IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger β-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post-LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA. conclusion: IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.
This paper is the fourth in a series of reviews that will summarize available data and critically... more This paper is the fourth in a series of reviews that will summarize available data and critically discuss the potential role of lung-function testing in infants with acute neonatal respiratory disorders and chronic lung disease of infancy. The current paper addresses information derived from tidal breathing measurements within the framework outlined in the introductory paper of this series, with particular reference to how these measurements inform on control of breathing. Infants with acute and chronic respiratory illness demonstrate differences in tidal breathing and its control that are of clinical consequence and can be measured objectively. The increased incidence of significant apnea in preterm infants and infants with chronic lung disease, together with the reportedly increased risk of sudden unexplained death within the latter group, suggests that control of breathing is affected by both maturation and disease. Clinical observations are supported by formal comparison of tidal breathing parameters and control of breathing indices in the research setting.
We report on four children who received cis-platinum simultaneously with, or in one case 10 month... more We report on four children who received cis-platinum simultaneously with, or in one case 10 months after, cranial irradiation and experienced exaggerated ototoxicity affecting all audible frequencies. The hearing loss was severe, affecting the critical areas for speech perception, and necessitated the provision of bilateral hearing aids. The audiograms of these patients are shown and compared to those of four children who had received cis-platinum as part of their treatment for neuroblastoma but without cranial irradiation. The precipitation of the exaggerated hearing loss with the administration of cis-platinum in one patient 10 months after finishing cranial irradiation suggests that care should be taken in the timing of cis-platinum administration in relation to concurrent or previous cranial irradiation.
To assess whether lung volume and ventilation inhomogeneity in preterm infants at 15-18 months co... more To assess whether lung volume and ventilation inhomogeneity in preterm infants at 15-18 months corrected age, and the change in these outcomes from the newborn period to 15-18 months corrected age, depend on gestational age (GA) at birth and the severity of neonatal lung disease. Preterm (GA range, 23-32 weeks) and term healthy control infants were studied in quiet sedated sleep at 15-18 months corrected age by multiple breath washout with 5% sulfur hexafluoride using an ultrasonic flowmeter. Valid measurements were obtained from 58 infants. Multivariate and multilevel regression was used to analyze outcomes. Functional residual capacity (FRC), lung clearance index, and first and second to zeroeth moment ratios were calculated. After accounting for body size at test, FRC at follow-up, and the increase in FRC from the newborn period to 15-18 months corrected age were positively associated with GA and negatively associated with the duration of endotracheal ventilation. Indices of ventilation inhomogeneity were unaltered by GA and the duration of endotracheal ventilation. In very preterm infants, GA and the duration of endotracheal ventilation are independently associated with reduced lung volume and lung growth during infancy, although the effect size of these findings is small.
Journal of Developmental Origins of Health and Disease, 2012
Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our pre... more Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our previous work, we demonstrated that the fetal liver is involved in the systemic inflammation associated with chorioamnionitis, leading to metabolic changes. On the basis of these findings, we hypothesized that chorioamnionitis can lead to postnatal inflammation-related liver injury and disturbed lipid metabolism. Chorioamnionitis was induced in sheep by intra-amniotic injection of lipopolysaccharide (LPS) or saline at 90, 100 and 110 days of gestation. Liver homeostasis and lipid metabolism were analyzed at term and at 7 weeks of age. At term, hepatic T-lymphocytes and apoptotic hepatocytes were increased. In addition, hepatic cholesterol and triglyceride levels were decreased in LPS-exposed animals compared with controls. At 7 weeks of age, no hepatic inflammation could be detected. However, liver triglycerides and plasma cholesterol levels were increased in LPS-exposed animals relative to controls. The changes in lipid levels at 7 weeks of age were associated with increased leptin receptor mRNA levels, increased lipid peroxidation, increased expression of cytochrome c oxidase subunit 4 as a marker for mitochondrial function and increased circulating ceramide levels. These findings demonstrate that chorioamnionitis-mediated antenatal inflammation-related liver disturbances have long-lasting postnatal effects on lipid metabolism.
Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infa... more Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infants, but the effects on cardiopulmonary hemodynamics are understood poorly. We aimed to determine the effect of increased PEEP on cardiopulmonary hemodynamics and to compare measurements from indwelling flow probes with Doppler echocardiography. Preterm lambs (129 ± 1 days) were ventilated initially with a tidal volume of 7 ml/kg and 4 cmH2O of PEEP. In ramp lambs ( n = 7), PEEP was increased by 2-cmH2O increments to 10 cmH2O and then in decrements back to 4 cmH2O. PEEP was unchanged in controls ( n = 6). Doppler echocardiographic flow measurements in the left pulmonary artery (LPA) and ductus arteriosus (DA) were correlated with flow probe measurements. Compared with controls, high PEEP reduced LPA flow from baseline (10-cmH2O PEEP: 43 ± 8% vs. control: 83 ± 21%; P = 0.029). High PEEP increased the proportion of right-to-left (R-L) shunting through the DA, with a trend to an increased o...
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Papers by Jane Pillow