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Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-based cord clamping; PBCC) maintains cardiac output and oxygenation in preterm lambs at birth, however, its efficacy after intrauterine inflammation is not known. Given the high incidence of chorioamnionitis in preterm infants, we investigated whether PBCC conferred any benefits compared to immediate cord clamping (ICC) in preterm lambs exposed antenatally to 7 days of intrauterine inflammation. Methods: Ultrasound guided intraamniotic injection of 20 mg Lipopolysaccharide (from E. coli:055:B5) was administered to pregnant ewes at 0.8 gestation. Seven days later, ewes were anesthetized, preterm fetuses exteriorised via cesarean section, and instrumented for continuous measurement of pulmonary, systemic and cerebral pressures and flows, and systemic, and cerebral oxygenation. Lambs were then randomized to either PBCC, whereupon ventilation was initiated and maintained for 3 min prior to umbilical cord clamping, or ICC where the umbilical cord was cut and ventilation initiated 30 s later. Ventilation was maintained for 30 min. Results: ICC caused a rapid fall in systemic (by 25%) and cerebral (by 11%) oxygen saturation in ICC lambs, concurrent with a rapid increase in carotid arterial pressure and heart rate. The overshoot in carotid arterial pressure was sustained in ICC lambs for the first 20 min of the study. PBCC maintained cardiac output and prevented the fall in cerebral oxygen delivery at birth. PBCC lambs had lower respiratory compliance and higher respiratory requirements throughout the study. Conclusion: PBCC mitigated the adverse effects of ICC on oxygenation and cardiac output, and therefore could be more beneficial in preterm babies exposed to antenatal inflammation as it maintains cardiac output and oxygen delivery. The increased respiratory requirements require further investigation in this sub-group of preterm infants.

Fetal growth restriction (FGR) and prematurity are associated with high risk of brain injury and long-term neurological deficits. FGR infants born preterm are commonly exposed to mechanical ventilation, but it is not known whether ventilation differentially induces brain pathology in FGR infants compared with appropriate for gestational age (AGA) infants. We investigated markers of neuropathology in moderate- to late-preterm FGR lambs, compared with AGA lambs, delivered by caesarean birth and ventilated under standard neonatal conditions for 24 h. FGR was induced by single umbilical artery ligation in fetal sheep at 88-day gestation (term, 150 days). At 125-day gestation, FGR and AGA lambs were delivered, dried, intubated, and commenced on noninjurious ventilation, with surfactant administration at 10 min. A group of unventilated FGR and AGA lambs at the same gestation was also examined. Over 24 h, circulating pH, Po2, and lactate levels were similar between groups. Ventilated FGR lambs had lower cerebral blood flow compared with AGA lambs ( P = 0.01). The brain of ventilated FGR lambs showed neuropathology compared with unventilated FGR, and unventilated and ventilated AGA lambs, with increased apoptosis (caspase-3), blood-brain barrier dysfunction (albumin extravasation), activated microglia (Iba-1), and increased expression of cellular oxidative stress (4-hydroxynonenal). The neuropathologies seen in the ventilated FGR brain were most pronounced in the periventricular and subcortical white matter but also evident in the subventricular zone, cortical gray matter, and hippocampus. Ventilation of preterm FGR lambs increased brain injury compared with AGA preterm lambs and unventilated FGR lambs, mediated via increased vascular permeability, neuroinflammation and oxidative stress.

Animal models have made and continue to make important contributions to neonatal medicine. For example, studies in fetal sheep have taught us much about the physiology of the fetal-to-neonatal transition. However, whereas animal models allow multiple factors to be investigated in a logical and systematic manner, no animal model is perfect for humans and so we need to understand the fundamental differences in physiology between the species in question and humans. Although most physiological systems are well conserved between species, some small differences exist and so wherever possible the knowledge generated from preclinical studies in animals should be tested in clinical trials. However, with the rise of evidence-based medicine the distinction between scientific knowledge generation and evidence gathering has been confused and the two have been lumped together. This misunderstands the contribution that scientific knowledge can provide. Science should be used to guide the gathering of evidence by informing the design of clinical trials, thereby increasing their likelihood of success. While scientific knowledge is not evidence, in the absence of evidence it is likely to be the best option for guiding clinical practice.

Antenatal administration of betamethasone (BM) is a common antecedent of preterm birth, but there is limited information about its impact on the acute evolution of preterm neonatal brain injury. We aimed to compare the effects of maternal BM in combination with mechanical ventilation on the white matter (WM) of late preterm sheep. At 0.85 of gestation, pregnant ewes were randomly assigned to receive intra-muscular (i.m.) saline (n = 9) or i.m. BM (n = 13). Lambs were delivered and unventilated controls (UVCSal, n = 4; UVCBM, n = 6) were humanely killed without intervention; ventilated lambs (VentSal, n = 5; VentBM, n = 7) were injuriously ventilated for 15 min, followed by conventional ventilation for 75 min. Cardiovascular and cerebral haemodynamics and oxygenation were measured continuously. The cerebral WM underwent assessment of inflammation and injury, and oxidative stress was measured in the cerebrospinal fluid (CSF). In the periventricular and subcortical WM tracts, the proportion of amoeboid (activated) microglia, the density of astrocytes, and the number of blood vessels with protein extravasation were higher in UVCBM than in UVCSal (p < 0.05 for all). During ventilation, tidal volume, mean arterial pressure, carotid blood flow, and oxygen delivery were higher in ­VentBM lambs (p < 0.05 vs. VentSal). In the subcortical WM, microglial infiltration was increased in the VentSal group compared to UVCSal. The proportion of activated microglia and protein extravasation was higher in the VentBM group compared to VentSal within the periventricular and subcortical WM tracts (p < 0.05). CSF oxidative stress was increased in the VentBM group compared to UVCSal, UVCBM, and VentSal groups (p < 0.05). Antenatal BM was associated with inflammation and vascular permeability in the WM of late preterm fetal sheep. During the immediate neonatal period, the increased carotid perfusion and oxygen delivery in BM-treated lambs was associated with increased oxidative stress, microglial activation and microvascular injury.

AIMS Approximately 6-9% pregnancies are affected by fetal growth restriction (FGR). Placental alterations related to utero-placental insufficiency in FGR may induce placental vascular remodelling to the detriment of the fetus. The objective of this article was to study histopathological features of placentae in a cohort of preterm growth-restricted infants in comparison to a cohort of preterm appropriately grown infants. METHODS In a cohort of 40 preterm infants of 28-32 weeks' gestation, placental histopathology was evaluated by a histopathologist, who was blinded to the identity of the grouping. Twenty infants had FGR, while 20 were appropriate for gestational age (AGA). Predefined histopathological characteristics were assessed based on the Amsterdam Placental Workshop Group Consensus Statement. RESULTS The gestational age and birthweight of the FGR and AGA cohorts were 29.8 ± 1.3 versus 30 ± 0.9 weeks, P = 0.78 and 923 ± 168 versus 1403 ± 237 g

PURPOSE OF REVIEW As the infant's physiology changes dramatically after birth, modern neonatal resuscitation approaches should detect and be modified in response to these changes. This review describes the changes in respiratory physiology at birth and highlights approaches that can assist these changes. RECENT FINDINGS To better target assistance given to infants at birth, the changes in lung physiology have been classified into three phases. The first phase involves lung aeration. As little or no gas exchange can occur, assistance should focus on airway liquid clearance. During the second phase, as airway liquid resides in lung tissue, assistance should focus on minimizing the complications associated with lung edema. The third phase occurs whenever the liquid is cleared from the tissue and respiratory mechanics stabilize. Although more traditional approaches are most effective during this phase, this is not the case for the first two phases. Furthermore, the glottis actively adducts during apnea in newborns and so noninvasive respiratory support requires the infant to be breathing so that the glottis will open. SUMMARY The respiratory support provided to infants at birth should match the infant's changing physiology during transition, which requires a more sophisticated approach and equipment than current recommendations.

Introduction Over five percent of infants born worldwide will need help breathing after birth. Delayed cord clamping (DCC) has become the standard of care for vigorous infants. DCC in non-vigorous infants is uncommon because of logistical difficulties in providing effective resuscitation during DCC. In Baby-Directed Umbilical Cord Clamping (Baby-DUCC), the umbilical cord remains patent until the infant’s lungs are exchanging gases. We conducted a feasibility study of the Baby-DUCC technique. Methods We obtained antenatal consent from pregnant women to enroll infants born at ≥32 weeks. Vigorous infants received ≥2 min of DCC. If the infant received respiratory support, the umbilical cord was clamped ≥60 s after the colorimetric carbon dioxide detector turned yellow. Maternal uterotonic medication was administered after umbilical cord clamping. A paediatrician and researcher entered the sterile field to provide respiratory support during a cesarean birth. Maternal and infant outcomes in the delivery room and prior to hospital discharge were analysed. Results Forty-four infants were enrolled, 23 delivered via cesarean section (8 unplanned) and 15 delivered vaginally (6 via instrumentation). Twelve infants were non-vigorous. ECG was the preferred method for recording HR. Two infants had a HR 100 BPM by 80 s after birth. Median time to umbilical cord clamping was 150 and 138 s in vigorous and non-vigorous infants, respectively. Median maternal blood loss was 300 ml. Conclusions It is feasible to provide resuscitation to term and near-term infants during DCC, after both vaginal and cesarean births, clamping the umbilical cord only when the infant is physiologically ready.

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)-1β 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.

Lung aeration stimulates the increase in pulmonary blood flow (PBF) at birth, but the spatial relationships between PBF and lung aeration and the role of increased oxygenation remain unclear. Using simultaneous phase-contrast X-ray imaging and angiography, we have investigated the separate roles of lung aeration and increased oxygenation in PBF changes at birth using near-term (30 days of gestation) rabbit kits (n = 18). Rabbits were imaged before ventilation, then the right lung was ventilated with 100% nitrogen (N2), air or 100% O2 (oxygen), before all kits were switched to ventilation in air, followed by ventilation of both lungs using air. Unilateral ventilation of the right lung with 100% N2 significantly increased heart rate (from 69.4 ± 4.9 to 93.0 ± 15.0 bpm), the diameters of both left and right pulmonary axial arteries, number of visible vessels in both left and right lungs, relative PBF index in both pulmonary arteries, and reduced bolus transit time for both left and right axial arteries (from 1.34 ± 0.39 and 1.81 ± 0.43 s to 0.52 ± 0.17 and 0.89 ± 0.21 s in the left and right axial arteries, respectively). Similar changes were observed with 100% oxygen, but increases in visible vessel number and vessel diameter of the axial arteries were greater in the ventilated right lung during unilateral ventilation. These findings confirm that PBF increase at birth is not spatially related to lung aeration and that the increase in PBF to unventilated regions is unrelated to oxygenation, although oxygen can potentiate this increase.

Chest compressions (CC) and adrenaline administration are recommended in asphyxiated newborns with persistent bradycardia despite effective ventilation. The effects of CC on cerebral blood flow in newborns at birth are unknown. Our aim was to determine the effects of CC, with or without adrenaline administration, on the return of spontaneous circulation, carotid blood flow (CBF), and carotid arterial pressure (CAP) in asphyxiated near-term lambs.Asphyxia was induced in near-term lambs by clamping the umbilical cord and delaying ventilation onset until spontaneous circulation ceased. Lambs were then resuscitated by positive pressure ventilation along with CC followed by adrenaline administration. CAP and CBF were continuously recorded.Mean CAP did not increase significantly during CC and only increased following adrenaline administration. CC did not increase mean CBF but increased CBF amplitude due to increased peak flow and the onset of retrograde flow during diastole. Adrenaline increased mean CBF from 1 ± 2 to 15 ± 5 ml/kg/min and abolished retrograde diastolic CBF, leading to the return in spontaneous circulation.We conclude that CC with adrenaline administration was required to increase CBF and restore spontaneous circulation in asphyxiated lambs. Low CBF and retrograde diastolic CBF during CC indicate hypoperfusion to the brain.

Aim Intra-uterine growth restriction (IUGR) is an important cause for prematurity as well as a significant risk factor for neurodevelopmental deficits. In this study, we aimed to examine the association between IUGR and early brain injury on neonatal cranial ultrasound in preterm infants. Methods This retrospective cohort study examined the relationship between IUGR and neonatal cranial ultrasound findings in preterm infants

Objective At birth, an initial sustained inflation (SI) uniformly aerates the lungs, increases arterial oxygenation and rapidly improves circulatory recovery in asphyxiated newborns. We hypothesised that lung aeration, in the absence of an increase in arterial oxygenation, can increase heart rate (HR) in asphyxiated near-term lambs. Interventions Lambs were delivered and instrumented at 139±2 days of gestation. Asphyxia was induced by umbilical cord clamping and then delaying the onset of ventilation until mean carotid arterial pressures (CAPs) had decreased 2 ; n=6), 5% oxygen (O 2 ; n=6), 21% O 2 (n=6) or 100% O 2 (n=6) followed by ventilation in air for 30 min. Main outcome measures HR, CAP and pulmonary blood flow (PBF) were continuously recorded. Results HR and PBF increased more quickly in lambs resuscitated with 100% and 21% O 2 than with 5% O 2 or N 2 . HR and PBF recovery in the 5% O 2 group was delayed relative to all other oxygen SI groups. HR in 5%, 21% and 100% O 2 groups reached 100 bpm before the SI was complete. HR and PBF in the N 2 group did not increase until 10 s after the SI was completed and ventilation was initiated with air. CAP tended to increase quicker in all O 2 groups than in N 2 group. Conclusions Oxygen content during an SI is important for circulatory recovery in asphyxiated lambs. This increase in HR is likely driven by the increase in PBF and venous return to the heart.

Introduction: Ventilation causes cerebral white matter inflammation and injury, which is exacerbated by intrauterine inflammation. However, the effects on cortical gray matter are not well-known. Our aim was to examine the effect of ventilation on the cerebral cortex of near-term lambs exposed to intrauterine inflammation. Method:Pregnant ewes at 119 ± 1 days gestation received an intra-amniotic injection of saline or lipopolysaccharide (LPS; 10 mg). Seven days later, lambs were randomized to either a high tidal volume injurious ventilation strategy (INJSAL N = 6, INJLPS N = 5) or a protective ventilation strategy (PROTSAL N = 5, PROTLPS N = 6). Respiratory parameters, heart rate and blood gases were monitored during the neonatal period. At post-mortem, the brain was collected and processed for immunohistochemical assessment. Neuronal density (NeuN), apoptotic cell death (caspase 8 and TUNEL), microglial density (Iba-1), astrocytic density (GFAP), and vascular protein extravasation (sheep serum) were assessed within the frontal, parietal, temporal and occipital lobes of the cerebral cortex. Results:A significant reduction in the number of neurons in all cortical layers except 4 was observed in LPS-exposed lambs compared to controls (layer #1: p = 0.041; layers #2 + 3: p = 0.023; layers #5 + 6: p = 0.016). LPS treatment caused a significant increase in gray matter area, indicative of edema. LPS+ventilation did not cause apoptotic cell death in the gray matter. Astrogliosis was not observed following PROT or INJ ventilation, with or without LPS exposure. LPS exposure was associated with vascular protein extravasation. Conclusion:Ventilation had little effect on gray matter inflammation and injury. Intrauterine inflammation reduced neuronal cell density, caused edema of the cortical gray matter, and blood vessel extravasation in the brain of near-term lambs.

BackgroundCaffeine is routinely given to preterm infants hours after birth to treat apnea of prematurity. In view of it's success, earlier administration in the delivery room is being considered, but little is known about how caffeine may effect the cardiovascular changes during the fetal to neonatal transition. Our aim was to determine the effect of prenatal caffeine administration on haemodynamic parameters in ventilated preterm lambs immediately after birth.MethodsCatheters (carotid artery and jugular vein) and ultrasonic flow probes (pulmonary artery and carotid artery) were implanted in preterm lambs (~126 ±2 days of gestation; term is 147 days), immediately before delivery by caesarean section. Before the cord was clamped, lambs were intubated and a caffeine (10mg/kg caffeine-base; n = 9) or saline (n = 5) infusion was given intravenously to the ewe and lamb over a 15-minute period. Two minutes after clamping the cord, ventilation commenced with a sustained inflation (35 cm H2O for 30 seconds) followed by ventilation for 30 minutes (target tidal volume of 6-8ml/kg).ResultsBlood gas parameters and rectal body temperature were not different between the two groups. Changes in pulmonary blood flow (PBF) and carotid blood flow (CBF) did not differ significantly between groups. PBF increased significantly after ventilation onset in both groups (caffeine p = 0.022, saline p ConclusionPrenatal caffeine infusion had no significant effect on acute haemodynamic parameters in ventilated preterm lambs during the cardiorespiratory transition.

Background For infants born moderately/late preterm (32–37 weeks of gestation), immaturity of the intestine has the potential to impact both short- and long-term gastrointestinal function. The aim of this study conducted in sheep was to compare the morphology and smooth muscle contractility of the ileum in term and late preterm lambs. Materials and methods Lambs delivered preterm (132 days gestation; n = 7) or term (147 days gestation; n = 9) were milk-fed after birth and euthanased at 2 days of age. A segment of distal ileum was collected for analysis of the length and cellular composition of the villi and crypts, smooth muscle width and contractility, and mRNA expression of the cell markers Ki67, lysozyme, mucin 2, synaptophysin, chromogranin A, olfactomedin 4, axis inhibition protein 2, and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5). Results There was no difference in the proportion of inflammatory, proliferating, apoptotic, enterocyte, or goblet cells between groups, but preterm lambs exhibited a significant upregulation of the stem cell marker LGR5 (p = 0.01). Absolute villus height (term: 1,032 ± 147 µm, preterm: 651 ± 52 µm; p

Purpose: Synchronised intermittent mandatory ventilation (SIMV) and high-frequency jet ventilation (HFJV) are accepted ventilatory strategies for treatment of respiratory distress syndrome (RDS) in preterm babies. We hypothesised that SIMV and HFJV both facilitate adequate oxygenation and ventilation but that HFJV is associated with less lung injury. Results: There were no differences in arterial oxygenation or partial pressure of carbon dioxide despite lower mean airway pressure during SIMV for most of the study. There were no consistent significant differences in end systolic and end diastolic PBF, lung injury data and static lung compliance. Methods: Preterm lambs of anaesthetised ewes were instrumented, intubated and delivered by caesarean section after intratracheal suction and instillation of surfactant. Each lamb was managed for 3 hr according to a predetermined algorithm for ventilatory support consistent with open lung ventilation. Pulmonary blood flow (PBF) was measured continuously and pulsatility index was calculated. Ventilatory parameters were recorded and arterial blood gases were measured at intervals. At postmortem, in situ pressure-volume deflation curves were recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess inflammation. Conclusions: SIMV and HFJV have comparable clinical efficacy and ventilator pressure requirements when applied with a targeted lung volume recruitment strategy.

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 × 107 hAECs (18 × 107 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-1β 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.

Fetal growth restriction (FGR) and preterm birth are frequent co-morbidities, both are independent risks for brain injury. However, few studies have examined the mechanisms by which preterm FGR increases the risk of adverse neurological outcomes. We aimed to determine the effects of prematurity and mechanical ventilation (VENT) on the brain of FGR and appropriately grown (AG, control) lambs. We hypothesized that FGR preterm lambs are more vulnerable to ventilation-induced acute brain injury. FGR was surgically induced in fetal sheep (0.7 gestation) by ligation of a single umbilical artery. After 4 weeks, preterm lambs were euthanized at delivery or delivered and ventilated for 2 h before euthanasia. Brains and cerebrospinal fluid (CSF) were collected for analysis of molecular and structural indices of early brain injury. FGRVENT lambs had increased oxidative cell damage and brain injury marker S100B levels compared with all other groups. Mechanical ventilation increased inflammatory marker IL-8 within the brain of FGRVENT and AGVENT lambs. Abnormalities in the neurovascular unit and increased blood–brain barrier permeability were observed in FGRVENT lambs, as well as an altered density of vascular tight junctions markers. FGR and AG preterm lambs have different responses to acute injurious mechanical ventilation, changes which appear to have been developmentally programmed in utero.

PURPOSE Injurious mechanical ventilation causes white matter (WM) injury in preterm infants through inflammatory and haemodynamic pathways. The relative contribution of each of these pathways is not known. We hypothesised that in vivo magnetic resonance imaging (MRI) can detect WM brain injury resulting from mechanical ventilation 24 h after preterm delivery. Further we hypothesised that the combination of inflammatory and haemodynamic pathways, induced by umbilical cord occlusion (UCO) increases brain injury at 24 h. METHODS Fetuses at 124±2 days gestation were exposed, instrumented and either ventilated for 15 min using a high tidal-volume (VT) injurious strategy with the umbilical cord intact (INJ; inflammatory pathway only), or occluded (INJ+UCO; inflammatory and haemodynamic pathway). The ventilation groups were compared to lambs that underwent surgery but were not ventilated (Sham), and lambs that did not undergo surgery (unoperated control; Cont). Fetuses were placed back in utero after the 15 min intervention and ewes recovered. Twenty-four hours later, lambs were delivered, placed on a protective ventilation strategy, and underwent MRI of the brain using structural, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) techniques. RESULTS Absolute MRS concentrations of creatine and choline were significantly decreased in INJ+UCO compared to Cont lambs (P = 0.03, P = 0.009, respectively); no significant differences were detected between the INJ or Sham groups and the Cont group. Axial diffusivities in the internal capsule and frontal WM were lower in INJ and INJ+UCO compared to Cont lambs (P = 0.05, P = 0.04, respectively). Lambs in the INJ and INJ+UCO groups had lower mean diffusivities in the frontal WM compared to Cont group (P = 0.04). DTI colour mapping revealed lower diffusivity in specific WM regions in the Sham, INJ, and INJ+UCO groups compared to the Cont group, but the differences did not reach significance. INJ+UCO lambs more likely to exhibit lower WM diffusivity than INJ lambs. CONCLUSIONS Twenty-four hours after injurious ventilation, DTI and MRS showed increased brain injury in the injuriously ventilated lambs compared to controls. DTI colour mapping threshold approach provides evidence that the haemodynamic and inflammatory pathways have additive effects on the progression of brain injury compared to the inflammatory pathway alone.

What is the central question of this study? Late preterm infants are often assumed to escape long-term morbidities known to impact earlier preterm offspring. Is this true for the cardiovascular system? What is the main finding and its importance? We show that late preterm birth is a risk factor for cardiovascular dysfunction in early adulthood and is influenced by sex. Early signs of cardiovascular dysfunction might predispose to heart disease in adulthood. Very preterm infants have an increased risk of cardiovascular disease; however, the effects of a late preterm birth on future cardiovascular function are not known. We hypothesized that after a late preterm birth, the well-described impairments in heart rate variability and baroreflex sensitivity would persist into adulthood. To test this hypothesis, sheep born preterm (0.9 gestation; nine male and seven female) or term (11 male and six female) underwent surgery at 14 months of age for insertion of femoral arterial and venous catheters and a femoral flow probe. After recovery, heart rate variability was assessed, followed by a baroreflex challenge (using the vasoactive agents phenylephrine and sodium nitroprusside) in conscious adult lambs. Our data demonstrate decreased low-frequency normalised units (LFnu) and low-frequency/high-frequency ratio in female but not male ex-preterm sheep at rest. When challenged, mature male ex-preterm sheep have an increased blood pressure response but dampened heart rate baroreflex response. We show that even a late preterm birth leads to cardiovascular dysfunction in adulthood. These early signs of cardiovascular dysfunction might underpin the later hypertension and increased risk of heart disease observed in adults born preterm. These findings are particularly important because late preterm infants are often assumed to escape the long-term morbidities known to impact on very preterm and extremely preterm offspring.

Fetal growth restriction (FGR) is associated with increased perinatal morbidity, mortality and long-term neurodevelopmental sequelae. The objective of this study was to examine whether information about early neurodevelopmental deficits was evident using three-dimensional head ultrasound and developmental assessments in preterm infants with FGR, compared with appropriate for gestational age (AGA) infants in the early post-natal period.Twenty preterm FGR infants weighing10th centile and born between 28 and 32 weeks were compared with age-matched AGA infants. In the second post-natal week after birth, we used three-dimensional ultrasound to assess cerebral ventricular volumes. Prechtl General Movement Assessments were performed at 4-6 weeks after birth. Test of Infant Motor Performance (TIMP) to measure functional motor behaviour was performed at 4-6 and 12-14 weeks corrected age.There was no statistically significant difference in the combined cerebral ventricular volume between the two groups (FGR, 0.81 ± 0.42 vs. AGA 0.72 ± 0.38 cmUltrasound in the early weeks may be useful to detect the neuropathology which could then mediate functional consequences.

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. β-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 β-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.

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 (Hi) 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.

Key points Erythropoietin (EPO) has been suggested as a potential treatment for bronchopulmonary dysplasia (BPD) in preterm infants. Ventilation-induced lung injury (VILI) is a major cause of BPD in preterm neonates. We investigated whether early high-dose EPO (i.v. 5000 IU kg−1) administration can reduce lung inflammation and injury resultant from VILI in ventilated preterm lambs. Early high-dose EPO administration increased mRNA expression of early markers of lung inflammation and injury and systemic injury controls. Early high-dose EPO worsened histological assessment of inflammation, airway wall thickness, haemorrhage and total injury compared to controls. Early high-dose EPO may increase the incidence and severity of respiratory disease in ventilated, preterm neonates. Abstract 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 , 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.

The transition to newborn life in preterm infants is complicated by immature cardiovascular and respiratory systems. Consequently, preterm infants often require respiratory support immediately after birth. Although aeration of the lung underpins the circulatory transition at birth, positive pressure ventilation can adversely affect cardiorespiratory function during this vulnerable period, reducing pulmonary blood flow and left ventricular output. Furthermore, pulmonary volutrauma is known to initiate pulmonary inflammatory responses, resulting in remote systemic involvement. This review focuses on the downstream consequences of positive pressure ventilation, in particular, interactions between cardiovascular output and the initiation of a systemic inflammatory cascade, on the immature brain. Recent studies have highlighted that positive pressure ventilation strategies are precursors of cerebral injury, probably mediated through cerebral blood flow instability. The presence of, or initiation of, an inflammatory cascade accentuates adverse cerebral blood flow, in addition to being a direct source of brain injury. Importantly, the degree of brain injury is dependent on the nature of the initial ventilation strategy used.

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.

To assess arterial morphology and mechanics in preterm infants with fetal growth restriction (FGR) compared with those appropriate for gestational age (AGA) in the early neonatal period. This observational study involved 20 preterm FGR infants (28 to 32 weeks) of gestational age (GA) and birth weight (BW)

Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1β and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease.

Key points • Intrauterine inflammation is associated with preterm birth and poor long-term cardiopulmonary and neurological outcomes. The effect of intrauterine inflammation on the cardiopulmonary and cerebral haemodynamic transition after preterm birth is poorly understood. • We demonstrated that intrauterine inflammation increased pulmonary vascular resistance, reduced pulmonary blood flow and left ventricular output and increased cerebral blood flow and cerebral oxygen delivery after preterm birth. • Increasing positive end-expiratory pressure, which causes a haemodynamic challenge, reduced pulmonary blood flow and left ventricular output in controls but not in lipopolysaccharide-exposed lambs. A transient reduction in brachiocephalic arterial pressure was observed in lipopolysaccharide-exposed lambs. • Intrauterine inflammation altered the cardiopulmonary and cerebral haemodynamic transition at birth and reduced the responsiveness of the pulmonary circulation to an increase in positive end-expiratory pressure. Abstract 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.

Delayed cord clamping improves circulatory stability in preterm infants at birth, but the underlying physiology is unclear. We investigated the effects of umbilical cord clamping, before and after ventilation onset, on cardiovascular function at birth. Prenatal surgery was performed on lambs (123 days) to implant catheters into the pulmonary and carotid arteries and probes to measure pulmonary (PBF), carotid (CaBF) and ductus arteriosus blood flows. Lambs were delivered at 126 ± 1 days and: (1) the umbilical cord was clamped at delivery and ventilation was delayed for about 2 min (Clamp 1st; n = 6), and (2) umbilical cord clamping was delayed for 3-4 min, until after ventilation was established (Vent 1st; n = 6). All lambs were subsequently ventilated for 30 min. In Clamp 1st lambs, cord clamping rapidly (within four heartbeats), but transiently, increased pulmonary and carotid arterial pressures (by ∼30%) and CaBF (from 30.2 ± 5.6 to 40.1 ± 4.6 ml min(-1) kg(-1)), which then decreased again within 30-60 s. Following ventilation onset, these parameters rapidly increased again. In Clamp 1st lambs, cord clamping reduced heart rate (by ∼40%) and right ventricular output (RVO; from 114.6 ± 14.4 to 38.8 ± 9.7 ml min(-1) kg(-1)), which were restored by ventilation. In Vent 1st lambs, cord clamping reduced RVO from 153.5 ± 3.8 to 119.2 ± 10.6 ml min(-1) kg(-1), did not affect heart rates and resulted in stable blood flows and pressures during transition. Delaying cord clamping for 3-4 min until after ventilation is established improves cardiovascular function by increasing pulmonary blood flow before the cord is clamped. As a result, cardiac output remains stable, leading to a smoother cardiovascular transition throughout the early newborn period.

Antenatal corticosteroids are used to augment fetal lung maturity in human pregnancy. Dexamethasone (DEX) is also used to treat congenital adrenal hyperplasia of the fetus in early pregnancy. We previously reported effects of synthetic corticosteroids given to sheep in early or late gestation on pregnancy length and fetal cortisol levels and glucocorticoids alter plasma insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) concentrations in late pregnancy and reduce fetal weight. The effects of administering DEX in early pregnancy on fetal organ weights and betamethasone (BET) given in late gestation on weights of fetal brain regions or organ development have not been reported. We hypothesized that BET or DEX administration at either stage of pregnancy would have deleterious effects on fetal development and associated hormones. In early pregnancy, DEX was administered as four injections at 12-hourly intervals over 48 h commencing at 40–42 days of gestation (dG). There was no consistent effect on fetal weight, or individual fetal organ weights, except in females at 7 months postnatal age. When BET was administered at 104, 111 and 118 dG, the previously reported reduction in total fetal weight was associated with significant reductions in weights of fetal brain, cerebellum, heart, kidney and liver. Fetal plasma insulin, leptin and triiodothyronine were also reduced at different times in fetal and postnatal life. We conclude that at the amounts given, the sheep fetus is sensitive to maternal administration of synthetic glucocorticoid in late gestation, with effects on growth and metabolic hormones that may persist into postnatal life.

Fetal growth restriction (FGR) is a common complication of pregnancy and, in severe cases, is associated with elevated rates of perinatal mortality, neonatal morbidity, and poor neurodevelopmental outcomes. The leading cause of FGR is placental insufficiency, with the placenta failing to adequately meet the increasing oxygen and nutritional needs of the growing fetus with advancing gestation. The resultant chronic fetal hypoxia induces a decrease in fetal growth, and a redistribution of blood flow preferentially to the brain. However, this adaptation does not ensure normal brain development. Early detection of brain injury in FGR, allowing for the prediction of short- and long-term neurodevelopmental consequences, remains a significant challenge. Furthermore, in FGR infants the detection and diagnosis of neuropathology is complicated by preterm birth, the etiological heterogeneity of FGR, timing of onset of growth restriction, its severity, and coexisting complications. In this review, we examine existing and emerging diagnostic tools from human and preclinical studies for the detection and assessment of brain injury in FGR fetuses and neonates. Increased detection rates, and early detection of brain injury associated with FGR, will offer opportunities for developing and assessing interventions to improve long-term outcomes.

Oxidative stress arising from suboptimal placental function contributes to a multitude of pathologies in infants compromised by fetal growth restriction (FGR). FGR infants are at high risk for respiratory dysfunction after birth and poor long-term lung function. Our objective was to investigate the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. Placental insufficiency and FGR was surgically induced in 13 fetal sheep at ∼105 days of gestation by ligation of a single umbilical artery. Maternal intravenous melatonin infusion was commenced in seven of the ewes 4 h after surgery and continued until birth. Lambs delivered normally at term and lungs were collected 24 h after birth for histological assessment of lung structure and injury and compared with appropriately grown control lambs ( n = 8). FGR fetuses were hypoxic and had lower glucose during gestation compared with controls. Melatonin administration prevented chronic hypoxia. Within the lung, FGR caused reduced secondary septal crest density and altered elastin deposition compared with controls. Melatonin administration had no effect on the changes to lung structure induced by FGR. We conclude that chronic FGR disrupts septation of the developing alveoli, which is not altered by melatonin administration. These findings suggest that oxidative stress is not the mechanism driving altered lung structure in FGR neonates. Melatonin administration did not prevent disrupted airway development but also had no apparent adverse effects on fetal lung development.NEW & NOTEWORTHY Fetal growth restriction (FGR) results in poor respiratory outcomes, which may be caused by oxidation in utero. We investigated the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. FGR disrupted septation of the developing alveoli, which is not altered by melatonin administration. Oxidative stress may not be the mechanism driving altered lung structure in FGR neonates.

Although beneficial in clinical practice, the INtubate-SURfactant-Extubate (IN-SUR-E) method is not successful in all preterm neonates with respiratory distress syndrome, with a reported failure rate ranging from 19 to 69 %. One of the possible mechanisms responsible for the unsuccessful IN-SUR-E method, requiring subsequent re-intubation and mechanical ventilation, is the inability of the preterm lung to achieve and maintain an “optimal” functional residual capacity. The importance of lung recruitment before surfactant administration has been demonstrated in animal studies showing that recruitment leads to a more homogeneous surfactant distribution within the lungs. Therefore, the aim of this study is to compare the application of a recruitment maneuver using the high-frequency oscillatory ventilation (HFOV) modality just before the surfactant administration followed by rapid extubation (INtubate-RECruit-SURfactant-Extubate: IN-REC-SUR-E) with IN-SUR-E alone in spontaneously breathing preterm infants requiring nasal continuous positive airway pressure (nCPAP) as initial respiratory support and reaching pre-defined CPAP failure criteria. In this study, 206 spontaneously breathing infants born at 24+0–27+6 weeks’ gestation and failing nCPAP during the first 24 h of life, will be randomized to receive an HFOV recruitment maneuver (IN-REC-SUR-E) or no recruitment maneuver (IN-SUR-E) just prior to surfactant administration followed by prompt extubation. The primary outcome is the need for mechanical ventilation within the first 3 days of life. Infants in both groups will be considered to have reached the primary outcome when they are not extubated within 30 min after surfactant administration or when they meet the nCPAP failure criteria after extubation. From all available data no definitive evidence exists about a positive effect of recruitment before surfactant instillation, but a rationale exists for testing the following hypothesis: a lung recruitment maneuver performed with a step-by-step Continuous Distending Pressure increase during High-Frequency Oscillatory Ventilation (and not with a sustained inflation) could have a positive effects in terms of improved surfactant distribution and consequent its major efficacy in preterm newborns with respiratory distress syndrome. This represents our challenge. ClinicalTrials.gov identifier: NCT02482766 . Registered on 1 June 2015.

We determined the effects of prenatal dexamethasone administration in early gestation on development of the hypothalamic-pituitary-adrenal (HPA) axis up to 7 months of postnatal age with measurements of hormone levels and gene expression. Plasma adrenocorticotropic hormone and cortisol levels after corticotropin-releasing hormone (CRH)/arginine vasopressin challenge were lower in treatment females than in control females and treatment males. Calculation of cortisol to adrenocorticotropic hormone ratios indicated however that the adrenals of treatment females were more responsive to adrenocorticotropic hormone than control females or treatment males. Effects of treatment and sex dependence at 7 months of age were observed in levels of hypothalamic CRH messenger RNA (mRNA), hypothalamic arginine vasopressin mRNA, pituitary proopiomelanocortin mRNA, pituitary prohormone convertase 1 and prohormone convertase 2, glucocorticoid receptor and mineralocorticoid receptor in the hypothalamus and hippocampus, adrenal adrenocorticotropic hormone receptor, steroidogenic acute regulatory, 3β hydroxysteroid dehydrogenase, and 11β hydroxysteroid dehydrogenase type 2 mRNA. The results indicate that exposure to glucocorticoids in early pregnancy produces persisting and sex-dependent effects on the hypothalamic-pituitary-adrenal axis at 7 months of age.

We tested the hypothesis that interleukin 1 (IL-1) mediates intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis in pretermfetal sheep. Time-mated Merino ewes with singleton fetuses received IL-1 alpha, LPS, or saline (control) by intra-amniotic injection 1 to 2 days before operative delivery at 124 +/- 1 days gestational age (N = 5-9/group; term =d 150 days). Recombinant human IL-1 receptor antagonist (rhIL-1ra) was given into the amniotic fluid 3 hours before intra-amniotic LPS or saline to block IL-1 signaling. Inflammation in the chorioamnion was determined by histology, cytokine messenger RNA (mRNA), protein expression, and by quantitation of activated inflammatory cells. Intra-amniotic IL-1 and LPS both induced chorioamnionitis. However, IL-1 blockade with IL-1ra did not decrease intra-amniotic LPS-induced increases in pro-inflammatory cytokine mRNAs, numbers of inflammatory cells, myeloperoxidase, or monocyte chemotactic protein-1-expressing cells in the chorioamnion. We conclude that IL-1 and LPS both can cause chorioamnionitis, but IL-1 is not an important mediator of LPS-induced chorioamnionitis in fetal sheep.

Perinatal inflammation is associated with adverse neurodevelopmental outcomes, which may be partly due to changes in the cerebral oxygen delivery/consumption relationship. We aimed to determine the critical oxygen delivery threshold of the brain of preterm, ventilated lambs and to determine whether the critical threshold is affected by exposure to inflammation in utero. Pregnant ewes received intra-amniotic injection of lipopolysaccharide or saline at 125 or 127 days of gestation. Pulmonary and systemic flow probes and catheters were surgically positioned in the fetus immediately before delivery at 129 days of gestation. After delivery, lambs were ventilated for 90 min using a positive end-expiratory pressure recruitment strategy. Cardio-respiratory variables and blood gases were measured regularly. Systemic and cerebral oxygen delivery, consumption (Fick), and extraction were calculated, and the relationship between cerebral delivery and consumption analyzed. Linear regression was used to define the transition or “critical” oxygen threshold as the point at which the slope of the oxygen delivery/consumption curve changed to be >10°. Four subgroups were defined according to the calculated critical threshold. A total of 150 measurements were recorded in 18 lambs. Fetal cerebral oxygen consumption was increased by antenatal lipopolysaccharide ( P < 0.05). The postnatal critical oxygen threshold was 3.6 ml·kg−1·min−1, corresponding to cerebral oxygen consumption of 0.73 ml·kg−1·min−1. High oxygen delivery and consumption were associated with increased pulmonary and carotid blood flow and systemic extraction compared with low oxygen delivery and consumption. No postnatal effect of antenatal inflammation was observed. Inflammation in utero increases fetal, but not postnatal, cerebral oxygen consumption. Adverse alterations to pulmonary blood flow can result in reduced cerebral blood flow, oxygen delivery, and consumption. Regardless of exposure to inflammation, there is a consistent postnatal relationship between cerebral oxygen delivery and consumption.

Increasing positive end-expiratory pressure (PEEP) is advocated to recruit alveoli during high-frequency jet ventilation (HFJV), but its effect on cardiopulmonary physiology and lung injury is poorly documented. We hypothesized that high PEEP would recruit alveoli and reduce lung injury but compromise pulmonary blood flow (PBF). Preterm lambs of anesthetized ewes were instrumented, intubated, and delivered by cesarean section after instillation of surfactant. HFJV was commenced with a PEEP of 5 cm H2O. Lambs were allocated randomly at delivery to remain on constant PEEP (PEEPconst, n = 6) or to recruitment via stepwise adjustments in PEEP (PEEPadj, n = 6) to 12 cm H2O then back to 8 cm H2O over the initial 60 min. PBF was measured continuously while ventilatory parameters and arterial blood gases were measured at intervals. At postmortem, in situ pressure-volume deflation curves were recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess inflammation. PEEPadj lambs had lower pressure amplitude, fractional inspired oxygen concentration, oxygenation index, and PBF and more compliant lungs. Inflammatory markers were lower in the PEEPadj group. Adjusted PEEP during HFJV improves oxygenation and lung compliance and reduces ventilator requirements despite reducing pulmonary perfusion.

Summary 1. Early postnatal events might play a critical role in the development of cardiorespiratory diseases of prematurity. Although the exact mechanism is unknown, capillary leakage resulting in increased interstitial fluid volume has been postulated to play a critical role. We investigated the effects of capillary leakage, induced by a volume load, on cardiopulmonary and systemic haemodynamics immediately after preterm delivery. 2. Fetal sheep were instrumented at 129 days gestation, delivered and ventilated. After 15 min, lambs in the volume load group received intravenous saline (50 mL/kg) infused over 10 min; control lambs received no infusion. At 30 min, lambs underwent a pulmonary challenge by increasing positive end-expiratory pressure (PEEP) by 2 cmH2O every 10 min to 10 cmH2O, with similar decrements back to baseline PEEP. Pulmonary blood flow (PBF) and arterial pressures were recorded in real-time and cardiovascular variables were measured by Doppler echocardiography. 3. Total protein concentration in the bronchoalveolar-lavage fluid was higher in volume load lambs compared with controls, and histological interstitial fluid retention was evident in volume load lambs, both indicative of capillary leak. PBF increased immediately after the volume load, but PBF, pulmonary and systemic arterial pressures, and oxygenation all deteriorated during the PEEP challenge compared with controls, coinciding with an increase in downstream pulmonary resistance. Three of six volume load lambs had pulmonary haemorrhage, which was not observed in control lambs. 4. Capillary leakage had moderate effects, but subsequent high levels of PEEP had significant negative effects on cardiopulmonary and respiratory function in preterm lambs. Capillary leakage might contribute to postnatal cardiopulmonary failure in preterm infants.

Mechanical ventilation is a risk factor for the development of bronchopulmonary dysplasia in premature infants. Fifteen minutes of high tidal volume (Vt) ventilation induces inflammatory cytokine expression in small airways and lung parenchyma within 3 h. Our objective was to describe the temporal progression of cytokine and maturation responses to lung injury in fetal sheep exposed to a defined 15-min stretch injury. After maternal anesthesia and hysterotomy, 129-day gestation fetal lambs ( n = 7–8/group) had the head and chest exteriorized. Each fetus was intubated, and airway fluid was gently removed. While placental support was maintained, the fetus received ventilation with an escalating Vt to 15 ml/kg without positive end-expiratory pressure (PEEP) for 15 min using heated, humidified 100% nitrogen. The fetus was then returned to the uterus for 1, 6, or 24 h. Control lambs received a PEEP of 2 cmH2O for 15 min. Tissue samples from the lung and systemic organs were evaluated. Stretch injury increased the early response gene Egr-1 and increased expression of pro- and anti-inflammatory cytokines within 1 h. The injury induced granulocyte/macrophage colony-stimulating factor mRNA and matured monocytes to alveolar macrophages by 24 h. The mRNA for the surfactant proteins A, B, and C increased in the lungs by 24 h. The airway epithelium demonstrated dynamic changes in heat shock protein 70 (HSP70) over time. Serum cortisol levels did not increase, and induction of systemic inflammation was minimal. We conclude that a brief period of high Vt ventilation causes a proinflammatory cascade, a maturation of lung monocytic cells, and an induction of surfactant protein mRNA.

Collins JJ, Kallapur SG, Knox CL, Nitsos I, Polglase GR, Pillow JJ, Kuypers E, Newnham JP, Jobe AH, Kramer BW. Inflammation in fetal sheep from intra-amniotic injection of Ureaplasma parvum. Am J Physiol Lung Cell Mol Physiol 299: L852-L860, 2010. First published October 8, 2010; doi:10.1152/ajplung.00183.2010.-Bronchopulmonary dysplasia is associated with chorioamnionitis and fetal lung inflammation. Ureaplasma species are the bacteria most frequently isolated from chorioamnionitis. Very chronic ureaplasma colonization of amniotic fluid causes low-grade lung inflammation and functional lung maturation in fetal sheep. Less is known about shorter exposures of the fetal lung. Therefore, we hypothesized that ureaplasmas would cause an acute inflammatory response that would alter lung development. Singleton ovine fetuses received intra-amniotic Ureaplasma parvum serovar 3 or control media at 110, 117, or 121 days and were delivered at 124 days gestational age (term = 150 days). Inflammation was assessed by 1) cell counts in bronchoalveolar lavage fluid (BALF), and 2) cytokine mRNA measurements, immunohistochemistry, and flow cytometry for inflammatory cells and elastin and alpha-smooth muscle actin (alpha-SMA) staining in lung tissue. Neutrophils were increased in BALF 3 days after exposure to ureaplasmas (P = 0.01). Myeloperoxidase-positive cells increased after 3 days (P = 0.03), and major histocompatibility complex (MHC) class II-positive cells increased after 14 days of ureaplasma exposure (P = 0.001). PU. 1 (macrophage marker)- or CD3 (T lymphocyte marker)-positive cells were not induced by ureaplasmas. CD3-positive cells in the posterior mediastinal lymph node increased in ureaplasma-exposed animals at 3, 7, and 14 days (P = 0.002). Focal elastin depositions decreased in alveolar septa at 14 days (P = 0.002), whereas alpha-SMA increased in arteries and bronchioli. U. parvum induced a mild acute inflammatory response and changed elastin and alpha-SMA deposition in the lung, which may affect lung structure and subsequent development.

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-1α given at varying intervals prior to preterm delivery at 124d gestational age. Intra-amniotic LPS or IL-1α induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2d. LPS induced intense MCP-1 expression in sub-epithelial 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.