Postnatal steroids as lung protective and anti-inflammatory in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization precede bronchopulmonary dysplasia (BPD). Glucocorticoids are anti-inflammatory and reduce ventilator requirements in preterm infants. However, high-dose glucocorticoids inhibit alveolarization. The effect of glucocorticoids on lung function and structure in preterm newborns exposed to antenatal inflammation is unknown. We hypothesise that postnatal low-dose dexamethasone reduces ventilator requirements, prevents inflammation and BPD-like lung pathology, following antenatal inflammation. METHODS: Pregnant ewes received intra-amniotic LPS (E.coli, 4 mg/mL) or saline at 126 days gestation; preterm lambs were delivered 48 h later. Lambs were randomised to receive either tapered intravenous dexamethasone (LPS/Dex, n = 9) or saline (LPS/Sal, n = 10; Sal/Sal, n = 9) commencing <3 h after birth. Respiratory support was gradually de-escalated, using a standardised protocol aimed at weaning from ventilation towards unassisted respiration. Tissues were collected at day 7. RESULTS: Lung morphology and mRNA levels for inflammatory mediators were measured. Respiratory support requirements were not different between groups. Histological analyses revealed higher tissue content and unchanged alveolarization in LPS/Sal compared to other groups. LPS/Dex lambs exhibited decreased markers of pulmonary inflammation compared to LPS/Sal. CONCLUSION: Tapered low-dose dexamethasone reduces the impact of antenatal LPS on ventilation requirements throughout the first week of life and reduces inflammation and pathological thickening of the preterm lung IMPACT: We are the first to investigate the combination of antenatal inflammation and postnatal dexamethasone therapy in a pragmatic study design, akin to contemporary neonatal care. We show that antenatal inflammation with postnatal dexamethasone therapy does not reduce ventilator requirements, but has beneficial maturational impacts on the lungs of preterm lambs at 7 days of life. Appropriate tapered postnatal dexamethasone dosing should be explored for extuabtion of oxygen-dependant neonates.

Interleukin-1 blockade attenuates white matter inflammation and oligodendrocyte loss after progressive systemic lipopolysaccharide exposure in near-term fetal sheep.

BACKGROUND: Increased systemic and tissue levels of interleukin (IL)-1ß are associated with greater risk of impaired neurodevelopment after birth. In this study, we tested the hypothesis that systemic IL-1 receptor antagonist (Ra) administration would attenuate brain inflammation and injury in near-term fetal sheep exposed to lipopolysaccharide (LPS). METHODS: Chronically instrumented near-term fetal sheep at 0.85 of gestation were randomly assigned to saline infusion (control, n = 9), repeated LPS infusions (0 h = 300 ng, 24 h = 600 ng, 48 h = 1200 ng, n = 8) or repeated LPS plus IL-1Ra infusions (13 mg/kg infused over 4 h) started 1 h after each LPS infusion (n = 9). Sheep were euthanized 4 days after starting infusions for histology. RESULTS: LPS infusions increased circulating cytokines and were associated with electroencephalogram (EEG) suppression with transiently reduced mean arterial blood pressure, and increased carotid artery perfusion and fetal heart rate (P < 0.05 vs. control for all). In the periventricular and intragyral white matter, LPS-exposure increased IL-1ß immunoreactivity, numbers of caspase 3+ cells and microglia, reduced astrocyte and olig-2+ oligodendrocyte survival but did not change numbers of mature CC1+ oligodendrocytes, myelin expression or numbers of neurons in the cortex and subcortical regions. IL-1Ra infusions reduced circulating cytokines and improved recovery of EEG activity and carotid artery perfusion. Histologically, IL-1Ra reduced microgliosis, IL-1ß expression and caspase-3+ cells, and improved olig-2+ oligodendrocyte survival. CONCLUSION: IL-1Ra improved EEG activity and markedly attenuated systemic inflammation, microgliosis and oligodendrocyte loss following LPS exposure in near-term fetal sheep. Further studies examining the long-term effects on brain maturation are now needed.

Maternal allergic asthma during pregnancy alters fetal lung and immune development in sheep: potential mechanisms for programming asthma and allergy.

KEY POINTS: Experimental maternal allergic asthma in sheep provides an experimental model in which to test impacts on progeny. Fetuses from allergic asthmatic ewes had fewer surfactant-producing cells in lungs. A greater proportion of lymphocytes from thymus were CD44 positive in fetuses from allergic asthmatic ewes than in controls. These changes to fetal development might contribute to poor neonatal lung function and increased risk of allergy seen in offspring of pregnancies complicated by asthma. ABSTRACT: Asthma is prevalent in pregnancy and increases the risk of disease in offspring, including neonatal respiratory distress and childhood asthma and allergy, but the mechanisms are not understood. We hypothesized that fetal lung structure and immune phenotype in late gestation fetal sheep would be impaired in our sheep model of maternal allergic asthma during pregnancy. Singleton-bearing ewes were either sensitized before pregnancy to house dust mite (HDM, allergic, n = 7) or were non-allergic (control, n = 5). The ewes were subsequently subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Tissues were collected at 140 ± 1 days gestational age (term, ∼147 days). The density of type II alveolar epithelial cells (surfactant protein C-immunostained) in the lungs was 30% lower in fetuses from allergic ewes than in controls (P < 0.001), but tissue-to-air space ratio and numbers of leucocytes and macrophages were not different between groups. The proportion of CD44+ lymphocytes in the fetal thymus was 3.5-fold higher in fetuses from allergic ewes than in control ewes (P = 0.043). Fewer surfactant-producing type II alveolar epithelial cells may contribute to the increased risk of neonatal respiratory distress in infants of asthmatic mothers, suggesting that interventions to promote lung maturation could improve their neonatal outcomes. If the elevated lymphocyte expression of CD44 persists postnatally, this would confer greater susceptibility to allergic diseases in progeny of asthmatic mothers, consistent with observations in humans. Further experiments are needed to evaluate postnatal phenotypes of progeny and investigate potential interventions.

Human Umbilical Cord Blood Therapy Protects Cerebral White Matter from Systemic LPS Exposure in Preterm Fetal Sheep.

BACKGROUND: Infants born preterm following exposure to in utero inflammation/chorioamnionitis are at high risk of brain injury and life-long neurological deficits. In this study, we assessed the efficacy of early intervention umbilical cord blood (UCB) cell therapy in a large animal model of preterm brain inflammation and injury. We hypothesised that UCB treatment would be neuroprotective for the preterm brain following subclinical fetal inflammation. METHODS: Chronically instrumented fetal sheep at 0.65 gestation were administered lipopolysaccharide (LPS, 150 ng, 055:B5) intravenously over 3 consecutive days, followed by 100 million human UCB mononuclear cells 6 h after the final LPS dose. Controls were administered saline instead of LPS and cells. Ten days after the first LPS dose, the fetal brain and cerebrospinal fluid were collected for analysis of subcortical and periventricular white matter injury and inflammation. RESULTS: LPS administration increased microglial aggregate size, neutrophil recruitment, astrogliosis and cell death compared with controls. LPS also reduced total oligodendrocyte count and decreased mature myelinating oligodendrocytes. UCB cell therapy attenuated cell death and inflammation, and recovered total and mature oligodendrocytes, compared with LPS. CONCLUSIONS: UCB cell treatment following inflammation reduces preterm white matter brain injury, likely mediated via anti-inflammatory actions.

Inflammation-induced preterm lung maturation: lessons from animal experimentation.

Intrauterine inflammation, or chorioamnionitis, is a major contributor to preterm birth. Prematurity per se is associated with considerable morbidity and mortality resulting from lung immaturity but exposure to chorioamnionitis reduces the risk of neonatal respiratory distress syndrome (RDS) in preterm infants. Animal experiments have identified that an increase in pulmonary surfactant production by the preterm lungs likely underlies this decreased risk of RDS in infants exposed to chorioamnionitis. Further animal experimentation has shown that infectious or inflammatory agents in amniotic fluid exert their effects on lung development by direct effects within the developing respiratory tract, and probably not by systemic pathways. Differences in the effects of intrauterine inflammation and glucocorticoids demonstrate that canonical glucocorticoid-mediated lung maturation is not responsible for inflammation-induced changes in lung development. Animal experimentation is identifying alternative lung maturational pathways, and transgenic animals and cell culture techniques will allow identification of novel mechanisms of lung maturation that may lead to new treatments for the prevention of RDS.

Differential short-term regional effects of early high dose erythropoietin on white matter in preterm lambs after mechanical ventilation.

Inadvertently injurious ventilation of preterm neonates in the delivery room can cause cerebral white matter (WM) inflammation and injury. We investigated the impact of an early high dose of recombinant human erythropoietin (EPO) on ventilation-induced WM changes in preterm lambs. Injurious ventilation, targeting a V(T) of 15 ml kg(-1) with no positive end-expiratory pressure, was initiated for 15 min in preterm lambs (0.85 gestation). Conventional ventilation was continued for a further 105 min. Lambs received either 5000 IU kg(-1) of EPO (EPREX®; Vent+EPO; n = 6) or vehicle (Vent; n = 8) via an umbilical vein at 4 ± 2 min. Markers of WM injury and inflammation were assessed using quantitative real-time PCR (qPCR) and immunohistochemistry and compared to a group of unventilated controls (UVC; n = 4). In Vent+EPO lambs compared to Vent lambs: (i) interleukin (IL)-1ß and IL-6 mRNA levels in the periventricular WM and IL-8 mRNA levels in the subcortical WM were higher (P < 0.05 for all); (ii) the density of microglia within the aggregations was not different in the periventricular WM and was lower in the subcortical WM (P = 0.001); (iii) the density of astrocytes was lower in the subcortical WM (P = 0.002); (iv) occludin and claudin-1 mRNA levels were higher in the periventricular WM (P < 0.02 for all) and (vi) the number of blood vessels with protein extravasation was lower (P < 0.05). Recombinant human EPO had variable regional effects within the WM when administered during injurious ventilation. The adverse short-term outcomes discourage the use of early high dose EPO administration in preterm ventilated babies.

Development of an experimental model of maternal allergic asthma during pregnancy.

Maternal asthma during pregnancy adversely affects pregnancy outcomes but identification of the cause/s, and the ability to evaluate interventions, is limited by the lack of an appropriate animal model. We therefore aimed to characterise maternal lung and cardiovascular responses and fetal-placental growth and lung surfactant levels in a sheep model of allergic asthma. Immune and airway functions were studied in singleton-bearing ewes, either sensitised before pregnancy to house dust mite (HDM, allergic, n = 7) or non-allergic (control, n = 5), and subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Maternal lung, fetal and placental phenotypes were characterised at 140 ± 1 days gestational age (term, ∼147 days). The eosinophil influx into lungs was greater after HDM challenge in allergic ewes than after saline challenge in control ewes before mating and in late gestation. Airway resistance increased throughout pregnancy in allergic but not control ewes, consistent with increased airway smooth muscle in allergic ewes. Maternal allergic asthma decreased relative fetal weight (-12%) and altered placental phenotype to a more mature form. Expression of surfactant protein B mRNA was 48% lower in fetuses from allergic ewes than controls, with a similar trend for surfactant protein D. Thus, allergic asthma in pregnant sheep modifies placental phenotype, and inhibits fetal growth and lung development consistent with observations from human pregnancies. Preconceptional allergen sensitisation and repeated airway challenges in pregnant sheep therefore provides an animal model to identify mechanisms of altered fetal development and adverse pregnancy outcomes caused by maternal asthma in pregnancy.

Human Amnion Epithelial Cells Modulate Ventilation-Induced White Matter Pathology in Preterm Lambs.

BACKGROUND: Preterm infants can be inadvertently exposed to high tidal volumes (VT) during resuscitation in the delivery room due to limitations of available equipment. High VT ventilation of preterm lambs produces cerebral white matter (WM) pathology similar to that observed in preterm infants who develop cerebral palsy. We hypothesized that human amnion epithelial cells (hAECs), which have anti-inflammatory and regenerative properties, would reduce ventilation-induced WM pathology in neonatal late preterm lamb brains. METHODS: Two groups of lambs (0.85 gestation) were used, as follows: (1) ventilated lambs (Vent; n = 8) were ventilated using a protocol that induces injury (VT targeting 15 ml/kg for 15 min, with no positive end-expiratory pressure) and were then maintained for another 105 min, and (2) ventilated + hAECs lambs (Vent+hAECs; n = 7) were similarly ventilated but received intravenous and intratracheal administration of 9 × 10(7) hAECs (18 × 10(7) hAECs total) at birth. Oxygenation and ventilation parameters were monitored in real time; cerebral oxygenation was measured using near-infrared spectroscopy. qPCR (quantitative real-time PCR) and immunohistochemistry were used to assess inflammation, vascular leakage and astrogliosis in both the periventricular and subcortical WM of the frontal and parietal lobes. An unventilated control group (UVC; n = 5) was also used for qPCR analysis of gene expression. Two-way repeated measures ANOVA was used to compare physiological data. Student's t test and one-way ANOVA were used for immunohistological and qPCR data comparisons, respectively. RESULTS: Respiratory parameters were not different between groups. Interleukin (IL)-6 mRNA levels in subcortical WM were lower in the Vent+hAECs group than the Vent group (p = 0.028). IL-1ß and IL-6 mRNA levels in periventricular WM were higher in the Vent+hAECs group than the Vent group (p = 0.007 and p = 0.001, respectively). The density of Iba-1-positive microglia was lower in the subcortical WM of the parietal lobes (p = 0.010) in the Vent+hAECs group but not in the periventricular WM. The number of vessels in the WM of the parietal lobe exhibiting protein extravasation was lower (p = 0.046) in the Vent+hAECs group. Claudin-1 mRNA levels were higher in the periventricular WM (p = 0.005). The density of GFAP-positive astrocytes was not different between groups. CONCLUSIONS: Administration of hAECs at the time of birth alters the effects of injurious ventilation on the preterm neonatal brain. Further studies are required to understand the regional differences in the effects of hAECs on ventilation-induced WM pathology and their net effect on the developing brain.

Prophylactic erythropoietin exacerbates ventilation-induced lung inflammation and injury in preterm lambs.

Ventilation-induced lung injury (VILI) of preterm neonates probably contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Erythropoietin (EPO) has been suggested as a therapy for BPD. The aim of this study was to determine whether prophylactic administration of EPO reduces VILI in preterm newborn lambs. Lambs at 126 days of gestation (term is 147 days) were delivered and ventilated with a high tidal volume strategy for 15 min to cause lung injury, then received gentle ventilation until 2 h of age. Lambs were randomized to receive intravenous EPO (5000 IU kg(-1): Vent+EPO; n = 6) or phosphate-buffered saline (Vent; n = 7) soon after birth: unventilated controls (UVC; n = 8) did not receive ventilation or any treatment. Physiological parameters were recorded throughout the experimental procedure. Samples of lung were collected for histological and molecular assessment of inflammation and injury. Samples of liver were collected to assess the systemic acute phase response. Vent+EPO lambs received higher F IO 2, P aO 2 and oxygenation during the first 10 min than Vent lambs. There were no differences in physiological indices beyond this time. Total lung injury score, airway wall thickness, inflammation and haemorrhage were higher in Vent+EPO lambs than in Vent lambs. Lung inflammation and early markers of lung and systemic injury were elevated in ventilated lambs relative to unventilated lambs; EPO administration further increased lung inflammation and markers of lung and systemic injury. Prophylactic EPO exacerbates VILI, which may increase the incidence and severity of long-term respiratory disease. More studies are required before EPO can be used for lung protection in preterm infants.

Exposure to intrauterine inflammation leads to impaired function and altered structure in the preterm heart of fetal sheep.

Intrauterine inflammation is a major contributor to preterm birth and has adverse effects on preterm neonatal cardiovascular physiology. Cardiomyocyte maturation occurs in late gestation in species such as humans and sheep. We tested the hypothesis that intrauterine inflammation has deleterious effects on cardiac function in preterm sheep which might be explained by altered cardiomyocyte proliferation and maturation. Pregnant ewes received an ultrasound-guided intra-amniotic injection of lipopolysaccharide (LPS) or saline 7 days prior to delivery at day 127 of pregnancy (term 147 days). Cardiac contractility was recorded in spontaneously beating hearts of the offspring, perfused in a Langendorff apparatus. Saline-filled latex balloons were inserted into the left ventricle (LV) and right ventricle (RV). Responsiveness to isoprenaline and stop-flow/reperfusion was assessed. In other experiments, hearts were perfusion-fixed, and cardiomyocyte nuclearity, volume and number were determined. ß-Adrenoceptor mRNA levels were determined in unfixed tissue. In hearts of LPS-exposed fetuses, contractility in the LV and RV was suppressed by ~40% and cardiomyocyte numbers were reduced by ~25%. Immature mono-nucleated cardiomyocytes had lower volumes (~18%), whereas mature bi-nucleated cardiomyocyte volume was ~77% greater. Although basal coronary flow was significantly increased by 21±7% in LPS-exposed hearts, following ischaemia/reperfusion (IR), end-diastolic pressure was increased 2.4±0.3-fold and infarct area was increased 3.2±0.6-fold compared with those in controls. Maximum responsiveness to isoprenaline was enhanced by LPS, without an increase in ß-adrenoceptor mRNA, suggesting altered second messenger signalling. Intrauterine inflammation altered cardiac growth, suppressed contractile function and enhanced responsiveness to stress. Although these effects may ensure immediate survival, they probably contribute to the increased vulnerability of organ perfusion in preterm neonates.

In utero LPS exposure impairs preterm diaphragm contractility.

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Intrauterine inflammation alters fetal cardiopulmonary and cerebral haemodynamics in sheep.

Intrauterine inflammation impairs fetal pulmonary vascular development and increases cerebral metabolic rate in fetal sheep. We hypothesized that these structural and metabolic effects of intrauterine inflammation would be accompanied by reduced fetal pulmonary blood flow and increased cerebral perfusion. Fetal sheep were instrumented at 112 days of gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days ewes were randomly assigned to receive intra-amniotic lipopolysaccharide (LPS, 20 mg from Escherichia coli; n = 7) or saline (control, 4 ml; n = 6). Fetal haemodynamic data were recorded continually from 1 h before intra-amniotic LPS or saline, until 144 h after. Fetal arterial blood was sampled before, and periodically after, intra-amniotic LPS or saline. End-diastolic and mean pulmonary blood flows were significantly lower than control from 48 and 96 h after LPS exposure, respectively, until the end of the experiment. Carotid blood flow was transiently increased at 96 and 120 h after LPS exposure. Carotid arterial oxygen content was lower than control from 48 h after intra-amniotic LPS. Fetal arterial lactate concentration was higher than control between 4 and 12 h after intra-amniotic LPS. Experimental intrauterine inflammation reduces pulmonary blood flow in fetal sheep, over a time course consistent with impaired pulmonary vascular development. Increased carotid blood flow after LPS administration may reflect an inflammation-induced increase in cerebral metabolic demand.

Intrauterine inflammation alters cardiopulmonary and cerebral haemodynamics at birth in preterm lambs.

Intrauterine inflammation is associated with preterm birth and poor long-term cardiopulmonary outcomes. We aimed to determine the effect of intrauterine inflammation on the cardiopulmonary and cerebral haemodynamic transition at birth, and the response to subsequent haemodynamic challenge. Fetal instrumentation was performed at ∼112 days gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days, inflammation was induced by intra-amniotic administration of lipopolysaccharide (LPS; n = 7); controls (n = 5) received intra-amniotic saline. At 125 days lambs were delivered and mechanically ventilated. Arterial blood gases, pulmonary and systemic arterial blood pressures and flows were measured during the perinatal period. At 10 min a haemodynamic challenge was administered by increasing positive end-expiratory pressure. During the first 10 min after birth, LPS-exposed lambs had higher pulmonary vascular resistance and lower pulmonary blood flow and left ventricular output than controls. Carotid arterial blood flow was higher in LPS-exposed lambs than controls between 3 and 7 min after delivery, and cerebral oxygen delivery was higher at 5 min. During the haemodynamic challenge, pulmonary blood flow and left ventricular output were reduced in controls but not in LPS-exposed lambs; a transient reduction in brachiocephalic arterial pressure occurred in LPS-exposed lambs but not in controls. Intrauterine inflammation altered the cardiopulmonary and cerebral haemodynamic transition at birth and reduced the cardiopulmonary response to a haemodynamic challenge after birth. The transient reduction in brachiocephalic arterial pressure suggests intrauterine inflammation may alter cerebrovascular control following an increase in positive end-expiratory pressure.

Human amnion epithelial cells reduce fetal brain injury in response to intrauterine inflammation.

Intrauterine infection, such as occurs in chorioamnionitis, is a principal cause of preterm birth and is a strong risk factor for neurological morbidity and cerebral palsy. This study aims to examine whether human amnion epithelial cells (hAECs) can be used as a potential therapeutic agent to reduce brain injury induced by intra-amniotic administration of lipopolysaccharide (LPS) in preterm fetal sheep. Pregnant ewes underwent surgery at approximately 110 days of gestation (term is approx. 147 days) for implantation of catheters into the amniotic cavity, fetal trachea, carotid artery and jugular vein. LPS was administered at 117 days; hAECs were labeled with carboxyfluorescein succinimidyl ester and administered at 0, 6 and 12 h, relative to LPS administration, into the fetal jugular vein, trachea or both. Control fetuses received an equivalent volume of saline. Brains were collected 7 days later for histological assessment of brain injury. Microglia (Iba-1-positive cells) were present in the brain of all fetuses and were significantly increased in the cortex, subcortical and periventricular white matter in fetuses that received LPS, indicative of inflammation. Inflammation was reduced in fetuses that received hAECs. In LPS fetuses, the number of TUNEL-positive cells was significantly elevated in the cortex, periventricular white matter, subcortical white matter and hippocampus compared with controls, and reduced in fetuses that received hAECs in the cortex and periventricular white matter. Within the fetal brains studied there was a significant positive correlation between the number of Iba-1-immunoreactive cells and the number of TUNEL-positive cells (R(2) = 0.19, p < 0.001). The administration of hAECs protects the developing brain when administered concurrently with the initiation of intrauterine inflammation.

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice.

BACKGROUND AIMS: Human amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury. METHODS: Newborn mouse pups were randomized to either normoxia (inspired O2 content (FiO2) = 0.21, n = 60) or hyperoxia (FiO2 = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14. RESULTS: Hyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-ß and platelet-derived growth factor-ß). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia. CONCLUSIONS: Intraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.

Intrauterine inflammation alters cardiopulmonary but not cerebral hemodynamics during open endotracheal tube suction in preterm lambs.

BACKGROUND: Intrauterine inflammation adversely affects cardiopulmonary, systemic, and cerebral hemodynamics in preterm neonates, but its impact on responses to endotracheal tube (ETT) suction, known to affect hemodynamics, is unknown. We hypothesized that intrauterine inflammation would alter the cardiopulmonary and cerebral hemodynamic response to open ETT suction in preterm lambs. METHODS: Chronically instrumented fetuses received intra-amniotic lipopolysaccharide (LPS; to induce intrauterine inflammation) or saline at 118 d of gestation (term ~147 d). At 125 d of gestation, lambs were delivered and mechanically ventilated. Open ETT suction was performed 30 min after delivery. Pulmonary and cerebral arterial pressures and flows were recorded continuously. RESULTS: Intrauterine inflammation reduced pulmonary blood flow (PBF) and increased pulmonary vascular resistance (PVR) after preterm birth. PBF and left-ventricular output (LVO) increased during and immediately after ETT suction in both groups, but the values were higher in LPS-exposed lambs. Preductal oxygenation significantly decreased during ETT suction but to a greater extent in LPS-exposed lambs. Cerebral blood flow and systemic arterial pressure were increased by open ETT suction similarly in the two groups. CONCLUSION: Intrauterine inflammation exacerbates the neonatal hemodynamic response to open ETT suction.

Effects of tail docking and castration on stress responses in lambs and the influence of prenatal glucocorticoid treatment.

It is common practice in Australian agriculture to remove the tails of lambs to prevent infection and to castrate males to prevent behavioural problems and unwanted reproduction. We have studied the pain and stress responses to these interventions by measuring changes in the hypothalamic-pituitary-adrenal (HPA) axis and ß-endorphin levels. Further, we have evaluated the effects of prenatal exposure to dexamethasone, which is known to affect the developing HPA axis. In control animals that had received prenatal saline treatment, plasma cortisol and adrenocorticotrophin (ACTH) levels increased after the interventions in both females and males. Plasma ß-endorphin levels also increased after the interventions, but the responses were less consistent. Prenatal dexamethasone exposure early in pregnancy (dexamethasone 0.14 mg kg(-1) ewe weight injection commenced on day 40 of pregnancy for four consecutive intramuscular injections at 12-hourly intervals) blunted the cortisol response to tail docking in female offspring, but not to combined tail docking and castration in males. It had no effect on ACTH or ß-endorphin responses in either sex. These findings describe the stress responses to these common agricultural interventions and suggest that long-term development of the HPA axis in females is altered by prenatal exposure to dexamethasone.

Prostaglandins mediate the fetal pulmonary response to intrauterine inflammation.

Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1ß and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.

Effects of caffeine on renal and pulmonary function in preterm newborn lambs.

INTRODUCTION: Caffeine administration is associated with a reduction in bronchopulmonary dysplasia, assisted ventilation, patent ductus arteriosus (DA) and cerebral palsy in preterm infants, but the mechanisms are unknown. Our aim was to determine the effects of acute caffeine administration on renal and pulmonary function in preterm lambs. METHODS: Lambs were delivered by caesarean section at ~126 days of gestation and ventilated with a tidal volume of 5 ml/kg, 60 breaths/min and 5 cmH(2)O positive end-expiratory pressure. After 30 minutes, lambs received 40 mg/kg caffeine i.v (n=7) or saline (controls; n=6) over 30 minutes and were ventilated for 2 hours. RESULTS: Arterial caffeine concentrations reached 35.9 ± 7.8 mg/l. Urine output was significantly higher after caffeine treatment than in controls (5.86 ± 1.95 vs 0.76 ± 0.94 ml/kg, area under curve p=0.041). Mean heart rate was significantly higher after caffeine treatment than in controls (211 ± 8 vs 169 ± 15 beats per minute, p<0.05) and remained higher for the experimental period. DISCUSSION: Caffeine did not affect pulmonary artery or DA blood flows or other renal, respiratory or cardiovascular parameters examined. Neonatal caffeine administration increased heart rate and urine output but had little effect on pulmonary function in ventilated preterm lambs.

The cardiopulmonary haemodynamic transition at birth is not different between male and female preterm lambs.

Males born preterm are at greater risk of illness and death than females, principally due to respiratory disease. Much of the excess morbidity occurs within the first few hours of life. Therefore, the aim of the present study was to investigate whether or not differences in the cardiopulmonary transition soon after birth underlie the increased morbidity in males after preterm birth. Nine female and thirteen male lambs (128±2 days gestation) underwent surgery immediately before delivery for implantation of a pulmonary arterial flow-probe and catheters into the main pulmonary artery and a carotid artery. After birth lambs were ventilated for 30 min (tidal volume 7 mL kg(-1)) while anaesthetised. Arterial pressures and flows were recorded in real time and left-ventricular output measured using Doppler echocardiography. Before birth, fetal cardiopulmonary haemodynamics, arterial blood gases, pH, glucose and lactate did not differ between sexes. Similarly, in the neonatal period there were no significant differences in arterial blood gas status, ventilation parameters, respiratory indices or cardiopulmonary haemodynamics between the sexes. Our data show that the cardiopulmonary transition at birth in ventilated, anaesthetised preterm lambs is not influenced by sex. Thus, the neonatal 'male disadvantage' is not explained by an impaired cardiovascular transition at birth.