Extended period of ventilation before delayed cord clamping augments left-to-right shunting and decreases systemic perfusion at birth in preterm lambs.

Previous studies have suggested that an extended period of ventilation before delayed cord clamping (DCC) augments birth-related rises in pulmonary arterial (PA) blood flow. However, it is unknown whether this greater rise in PA flow is accompanied by increases in left ventricular (LV) output and systemic arterial perfusion or whether it reflects enhanced left-to-right shunting across the ductus arteriosus and/or foramen ovale (FO), with decreased systemic arterial perfusion. Using an established preterm lamb birth transition model, this study compared the effect of a short (∼40 s, n = 11), moderate (∼2 min, n = 11) or extended (∼5 min, n = 12) period of initial mechanical lung ventilation before DCC on flow probe-derived perinatal changes in PA flow, LV output, total systemic arterial blood flow, ductal shunting and FO shunting. The LV output was relatively stable during initial ventilation but increased after DCC, with similar responses in all groups. Systemic arterial flow patterns displayed only minor differences during brief and moderate periods of initial ventilation and were similar after DCC. However, an increase in PA flow was augmented with an extended initial ventilation (P < 0.001), owing to an earlier onset of left-to-right ductal and FO shunting (P < 0.001), and was accompanied by a pronounced reduction in total systemic arterial flow (P = 0.005) that persisted for 4 min after DCC (P ≤ 0.039). These findings suggest that, owing to increased left-to-right shunting and a greater reduction in systemic arterial perfusion, an extended period of ventilation before DCC does not result in greater perinatal circulatory benefits than shorter periods of initial ventilation in the birth transition. KEY POINTS: Previous studies suggest that an extended period of initial ventilation before delayed cord clamping (DCC) augments birth-related rises in pulmonary arterial (PA) blood flow. It is unknown whether this greater rise in PA flow is accompanied by an increased left ventricular output and systemic arterial perfusion or whether it reflects enhanced left-to-right shunting across the ductus arteriosus and/or foramen ovale, with decreased systemic arterial perfusion. Anaesthetized preterm fetal lambs instrumented with central arterial flow probes underwent a brief (∼40 s), moderate (∼2 min) or extended (∼5 min) period of ventilation before DCC. Perinatal changes in left ventricular output were similar in all groups, but extended initial ventilation augmented both perinatal increases in PA flow, owing to earlier onset and greater left-to-right ductal and foramen ovale shunting, and perinatal reductions in total systemic arterial perfusion. Extended ventilation before DCC does not confer a greater perinatal circulatory benefit than shorter periods of initial ventilation.

Lung ultrasound detects regional aeration inhomogeneity in ventilated preterm lambs.

BACKGROUND: Inhomogeneous lung aeration is a significant contributor to preterm lung injury. EIT detects inhomogeneous aeration in the research setting. Whether LUS detects inhomogeneous aeration is unknown. The aim was to determine whether LUS detects regional inhomogeneity identified by EIT in preterm lambs. METHODS: LUS and EIT were simultaneously performed on mechanically ventilated preterm lambs. LUS images from non-dependent and dependent regions were acquired and reported using a validated scoring system and computer-assisted quantitative LUS greyscale analysis (Q-LUSMGV). Regional inhomogeneity was calculated by observed over predicted aeration ratio from the EIT reconstructive model. LUS scores and Q-LUSMGV were compared with EIT aeration ratios using one-way ANOVA. RESULTS: LUS was performed in 32 lambs (~125d gestation, 128 images). LUS scores were greater in upper anterior (non-dependent) compared to lower lateral (dependent) regions of the left (3.4 vs 2.9, p = 0.1) and right (3.4 vs 2.7, p < 0.0087). The left and right upper regions also had greater LUS scores compared to right lower (3.4 vs 2.7, p < 0.0087) and left lower (3.7 vs 2.9, p = 0.1). Q-LUSMGV yielded similar results. All LUS findings corresponded with EIT regional differences. CONCLUSION: LUS may have potential in measuring regional aeration, which should be further explored in human studies. IMPACT: Inhomogeneous lung aeration is an important contributor to preterm lung injury, however, tools detecting inhomogeneous aeration at the bedside are limited. Currently, the only tool clinically available to detect this is electrical impedance tomography (EIT), however, its use is largely limited to research. Lung ultrasound (LUS) may play a role in monitoring lung aeration in preterm infants, however, whether it detects inhomogeneous lung aeration is unknown. Visual LUS scores and mean greyscale image analysis using computer assisted quantitative LUS (Q-LUSMGV) detects regional lung aeration differences when compared to EIT. This suggests LUS reliably detects aeration inhomogeneity warranting further investigation in human trials.

Mechanical power made simple: validating a simplified calculation of mechanical power in preterm lungs.

BACKGROUND: The incidence of chronic lung disease is increasing, suggesting a need to explore novel ways to understand ventilator induced lung injury (VILI) in preterm infants. Mechanical power (MP) is a unifying measure of energy transferred to the respiratory system and a proposed determinant of VILI. The gold-standard method for calculating MP (geometric method) is not feasible in the clinical setting. This has prompted the derivation of simplified equations for calculating MP. OBJECTIVE: To validate the agreement between a simplified calculation of MP (MPSimple) and the true MP calculated using the geometric method (MPRef). METHODS: MPSimple and MPRef was calculated in mechanically ventilated preterm lambs (n = 71) and the agreement between both measures was determined using intraclass correlation coefficients (ICC), linear regression, and Bland-Altman analysis. RESULTS: A strong linear relationship (adjusted R2 = 0.98), and excellent agreement (ICC = 0.99, 95% CI = 0.98-0.99) between MPSimple and MPRef was demonstrated. Bland-Altman analysis demonstrated a negligible positive bias (mean difference = 0.131 J/min·kg). The 95% limits of agreement were -0.06 to 0.32 J/min·kg. CONCLUSIONS: In a controlled setting, there was excellent agreement between MPSimple and gold-standard calculations. MPSimple should be validated and explored in preterm neonates to assess the cause-effect relationship with VILI and neonatal outcomes. IMPACT STATEMENT: Mechanical power (MP) unifies the individual components of ventilator induced lung injury (VILI) and provides an estimate of total energy transferred to the respiratory system during mechanical ventilation. As gold-standard calculations of mechanical power at the bedside are not feasible, alternative simplified equations have been proposed. In this study, MP calculated using a simplified equation had excellent agreement with true MP in mechanically ventilated preterm lambs. These results lay foundations to explore the role of MP in neonatal VILI and determine its relationship with short and long term respiratory outcomes.

Inflating Pressure and Not Expiratory Pressure Initiates Lung Injury at Birth in Preterm Lambs.

Rationale: Inflation is essential for aeration at birth, but current inflating pressure settings are without an evidence base. Objectives: To determine the role of inflating pressure (ΔP), and its relationship with positive end-expiratory pressure (PEEP), in initiating early lung injury pathways in the preterm lamb lung. Methods: Preterm (124 to 127 d) steroid-exposed lambs (n = 45) were randomly allocated (8-10 per group) to 15 minutes of respiratory support with placental circulation and 20 or 30 cm H2O ΔP, with an initial high PEEP (maximum, 20 cm H2O) recruitment maneuver known to facilitate aeration (dynamic PEEP), and compared with dynamic PEEP with no ΔP or 30 cm H2O ΔP and low (4 cm H2O) PEEP. Lung mechanics and aeration were measured throughout. After an additional 30 minutes of apneic placental support, lung tissue and bronchoalveolar fluid were analyzed for regional lung injury, including proteomics. Measurements and Main Results: The 30 cm H2O ΔP and dynamic PEEP strategies resulted in quicker aeration and better compliance but higher tidal volumes (often >8 ml/kg, all P < 0.0001; mixed effects) and injury. ΔP 20 cm H2O with dynamic PEEP resulted in the same lung mechanics and aeration, but less energy transmission (tidal mechanical power), as ΔP 30 cm H2O with low PEEP. Dynamic PEEP without any tidal inflations resulted in the least lung injury. Use of any tidal inflating pressures altered metabolic, coagulation and complement protein pathways within the lung. Conclusions: Inflating pressure is essential for the preterm lung at birth, but it is also the primary mediator of lung injury. Greater focus is needed on strategies that identify the safest application of pressure in the delivery room.

Impact of tidal volume strategy at birth on initiating lung injury in preterm lambs.

Tidal ventilation is essential in supporting the transition to air-breathing at birth, but excessive tidal volume (VT) is an important factor in preterm lung injury. Few studies have assessed the impact of specific VT levels on injury development. Here, we used a lamb model of preterm birth to investigate the role of different levels of VT during positive pressure ventilation (PPV) in promoting aeration and initiating early lung injury pathways. VT was delivered as 1) 7 mL/kg throughout (VTstatic), 2) begun at 3 mL/kg and increased to a final VT of 7 mL/kg over 3 min (VTinc), or 3) commenced at 7 mL/kg, decreased to 3 mL/kg, and then returned to 7 mL/kg (VTalt). VT, inflating pressure, lung compliance, and aeration were similar in all groups from 4 min, as was postmortem histology and lung lavage protein concentration. However, transient decrease in VT in the VTalt group caused increased ventilation heterogeneity. Following TMT-based quantitative mass spectrometry proteomics, 1,610 proteins were identified in the lung. Threefold more proteins were significantly altered with VTalt compared with VTstatic or VTinc strategies. Gene set enrichment analysis identified VTalt specific enrichment of immune and angiogenesis pathways and VTstatic enrichment of metabolic processes. Our finding of comparable lung physiology and volutrauma across VT groups challenges the paradigm that there is a need to rapidly aerate the preterm lung at birth. Increased lung injury and ventilation heterogeneity were identified when initial VT was suddenly decreased during respiratory support at birth, further supporting the benefit of a gentle VT approach.NEW & NOTEWORTHY There is little evidence to guide the best tidal volume (VT) strategy at birth. In this study, comparable aeration, lung mechanics, and lung morphology were observed using static, incremental, and alternating VT strategies. However, transient reduction in VT was associated with ventilation heterogeneity and inflammation. Our results suggest that rapidly aerating the preterm lung may not be as clinically critical as previously thought, providing clinicians with reassurance that gently supporting the preterm lung maybe permissible at birth.

Aeration strategy at birth does not impact carotid haemodynamics in preterm lambs.

BACKGROUND: The impact of different respiratory strategies at birth on the preterm lung is well understood; however, concerns have been raised that lung recruitment may impede cerebral haemodynamics. This study aims to examine the effect of three different ventilation strategies on carotid blood flow, carotid artery oxygen content and carotid oxygen delivery. METHODS: 124-127-day gestation apnoeic intubated preterm lambs studied as part of a larger programme primarily assessing lung injury were randomised to positive pressure ventilation with positive end-expiratory pressure (PEEP) 8 cmH2O (No-RM; n = 12), sustained inflation (SI; n = 15) or dynamic PEEP strategy (DynPEEP; maximum PEEP 14 or 20 cmH2O, n = 41) at birth, followed by 90 min of standardised ventilation. Haemodynamic data were continuously recorded, with intermittent arterial blood gas analysis. RESULTS: Overall carotid blood flow measures were comparable between strategies. Except for mean carotid blood flow that was significantly lower for the SI group compared to the No-RM and DynPEEP groups over the first 3 min (p < 0.0001, mixed effects model). Carotid oxygen content and oxygen delivery were similar between strategies. Maximum PEEP level did not alter cerebral haemodynamic measures. CONCLUSIONS: Although there were some short-term variations in cerebral haemodynamics between different PEEP strategies and SI, these were not sustained. IMPACT: Different pressure strategies to facilitate lung aeration at birth in preterm infants have been proposed. There is minimal information on the effect of lung recruitment on cerebral haemodynamics. This is the first study that compares the effect of sustained lung inflation and dynamic and static positive end-expiratory pressure on cerebral haemodynamics. We found that the different ventilation strategies did not alter carotid blood flow, carotid oxygen content or carotid oxygen delivery. This preclinical study provides some reassurance that respiratory strategies designed to focus on lung aeration at birth may not impact cerebral haemodynamics in preterm neonates.

Quantitative lung ultrasound detects dynamic changes in lung recruitment in the preterm lamb.

BACKGROUND: Lung ultrasound (LUS) may not detect small, dynamic changes in lung volume. Mean greyscale measurement using computer-assisted image analysis (Q-LUSMGV) may improve the precision of these measurements. METHODS: Preterm lambs (n = 40) underwent LUS of the dependent or non-dependent lung during static pressure-volume curve mapping. Total and regional lung volumes were determined using the super-syringe technique and electrical impedance tomography. Q-LUSMGV and gold standard measurements of lung volume were compared in 520 images. RESULTS: Dependent Q-LUSMGV moderately correlated with total lung volume (rho = 0.60, 95% CI 0.51-0.67) and fairly with right whole (rho = 0.39, 0.27-0.49), central (rho = 0.38, 0.27-0.48), ventral (rho = 0.41, 0.31-0.51) and dorsal regional lung volumes (rho = 0.32, 0.21-0.43). Non-dependent Q-LUSMGV moderately correlated with total lung volume (rho = 0.57, 0.48-0.65) and fairly with right whole (rho = 0.43, 0.32-0.52), central (rho = 0.46, 0.35-0.55), ventral (rho = 0.36, 0.25-0.47) and dorsal lung volumes (rho = 0.36, 0.25-0.47). All correlation coefficients were statistically significant. Distinct inflation and deflation limbs, and sonographic pulmonary hysteresis occurred in 95% of lambs. The greatest changes in Q-LUSMGV occurred at the opening and closing pressures. CONCLUSION: Q-LUSMGV detected changes in total and regional lung volume and offers objective quantification of LUS images, and may improve bedside discrimination of real-time changes in lung volume. IMPACT: Lung ultrasound (LUS) offers continuous, radiation-free imaging that may play a role in assessing lung recruitment but may not detect small changes in lung volume. Mean greyscale image analysis using computer-assisted quantitative LUS (Q-LUSMGV) moderately correlated with changes in total and regional lung volume. Q-LUSMGV identified opening and closing pressure and pulmonary hysteresis in 95% of lambs. Computer-assisted image analysis may enhance LUS estimation of lung recruitment at the bedside. Future research should focus on improving precision prior to clinical translation.

Divergent effects of initial ventilation with delayed cord clamping on systemic and pulmonary arterial flows in the birth transition of preterm lambs.

A current view that delayed cord clamping (DCC) results in greater haemodynamic stability at birth than immediate cord clamping (ICC) is based on comparison of DCC vs. ICC followed by an asphyxial (∼2 min) cord clamp-to-ventilation (CC-V) interval. More recent data suggest that relatively minor perinatal differences in heart rate and blood pressure fluctuations exist between DCC and ICC with a non-asphyxial (<45 s) CC-V interval, but it is unknown how ventricular output and central arterial blood flow effects of DCC compare with those of non-asphyxial ICC. Anaesthetized preterm fetal lambs instrumented with flow probes on major central arteries were ventilated for 97 (7) s (mean (SD)) before DCC at birth (n = 10), or underwent ICC 40 (6) s before ventilation (n = 10). Compared to ICC, initial ventilation and DCC was accompanied by (1) redistribution of a similar level of ascending aortic flow away from cephalic arteries and towards the aortic isthmus after ventilation; (2) a lower right ventricular output after cord clamping that was redistributed towards the lungs, thereby maintaining the absolute contribution of this output to a similar increase in pulmonary arterial flow after birth; and (3) a lower descending thoracic aortic flow after birth, related to a more rapid decline in phasic right-to-left ductal flow only partially offset by increased aortic isthmus flow. However, systemic arterial flows were similar between DCC and non-asphyxial ICC within 5 min after birth. These findings suggest that compared to non-asphyxial ICC, initial ventilation with DCC transiently redistributed central arterial flows, resulting in lower perinatal systemic arterial, but not pulmonary arterial, flows. KEY POINTS: A current view that delayed cord clamping (DCC) results in greater haemodynamic stability at birth than immediate cord clamping (ICC) is based on comparison of DCC vs. ICC with an asphyxial (∼2 min) cord clamp-to-ventilation (CC-V) interval. Recent data suggest that relatively minor perinatal differences in heart rate and blood pressure fluctuations exist between DCC and ICC with a non-asphyxial (<45 s) CC-V interval, but how central arterial blood flow effects of DCC compare with those of non-asphyxial ICC is unknown. Anaesthetized preterm fetal lambs instrumented with central arterial flow probes underwent initial ventilation for ∼90 s before DCC at birth, or ICC for ∼40 s before ventilation. Compared to non-asphyxial ICC, initial ventilation with DCC redistributed central blood flows, resulting in lower systemic, but not pulmonary, arterial flows during this period of transition. This flow redistribution was transitory, however, with systemic arterial flows similar between DCC and non-asphyxial ICC within minutes after birth.

Characteristics and physiological basis of falls in ventricular outputs after immediate cord clamping at delivery in preterm fetal lambs.

KEY POINTS: Controversy exists about the physiological mechanism(s) underlying decreases in cardiac output after immediate clamping of the umbilical cord at birth. To define these mechanisms, the four major determinants of ventricular output (afterload, preload, heart rate and contractility) were measured concurrently in fetal lambs at 15 s intervals over a 2 min period after cord clamping and before ventilation following delivery. After cord clamping, right (but not left) ventricular output fell by 20% in the initial 30 s, due to increased afterload associated with higher arterial blood pressures, but both outputs then halved over 45 s, due to a falling heart rate and deteriorating ventricular contractility accompanying rapid declines in arterial oxygenation to asphyxial levels. Ventricular outputs subsequently plateaued from 75 to 120 s, associated with rebound rises in ventricular contractility accompanying asphyxia-induced surges in circulating catecholamines. These findings provide a physiological basis for the clinical recommendation that effective ventilation should occur within 60 s after immediate cord clamping. ABSTRACT: Controversy exists about the physiological mechanism(s) underlying large decreases in cardiac output after immediate clamping of the umbilical cord at birth. To define these mechanisms, anaesthetized preterm fetal lambs (127(1)d, n = 12) were instrumented with flow probes and catheters in major central arteries, and a left ventricular (LV) micromanometer-conductance catheter. Following immediate cord clamping at delivery, haemodynamics, LV and right ventricular (RV) outputs, and LV contractility were measured at 15 s intervals during a 2 min non-ventilatory period, with aortic blood gases and circulating catecholamine (noradrenaline and adrenaline) concentrations measured at 30 s intervals. After cord clamping, (1) RV (but not LV) output fell by 20% in the initial 30 s, due to a reduced stroke volume associated with increased arterial blood pressures, (2) both outputs then halved over the next 45 s, associated with falls in heart rate, arterial blood pressures and ventricular contractility accompanying a rapid decline in arterial oxygenation to asphyxial levels, (3) reduced outputs subsequently plateaued from 75 to 120 s, associated with rebound rises in blood pressures and ventricular contractility accompanying exponential surges in circulating catecholamines. These findings are consistent with a time-dependent decline of ventricular outputs after immediate cord clamping, which comprised (1) an initial, minor fall in RV output related to altered loading conditions, (2) ensuing large decreases in both LV and RV outputs related to the combination of bradycardia and ventricular dysfunction during emergence of an asphyxial state, and (3) subsequent stabilization of reduced LV and RV outputs during ongoing asphyxia, supported by cardiovascular stimulatory effects of marked sympathoadrenal activation.

Antenatal betamethasone augments early rise in pulmonary perfusion at birth in preterm lambs: role of ductal shunting and right ventricular outflow distribution.

The glucocorticosteroid betamethasone is routinely administered via maternal intramuscular injection to enhance fetal lung maturation before anticipated preterm birth. Although antenatal betamethasone increases fetal pulmonary arterial (PA) blood flow, whether this agent alters the contribution of 1) right ventricular (RV) output or 2) left-to-right shunting across the ductus arteriosus to rises in PA blood flow after preterm birth is unknown. To address this question, anesthetized control (n = 7) and betamethasone-treated (n = 7) preterm fetal lambs (gestation 127 ± 1 days, means ± SD) were instrumented with aortic, pulmonary, and left atrial catheters as well as ductus arteriosus and left PA flow probes to calculate RV output, with hemodynamics measured for 30 min after cord clamping and mechanical ventilation. Mean PA blood flow was higher in betamethasone-treated than in control lambs over the initial 10 min after birth (P < 0.05). This higher PA flow was accompanied by 1) a greater pulmonary vascular conductance (P ≤ 0.025), 2) a larger proportion of RV output passing to lungs (P ≤ 0.01), despite a fall in this output, and 3) earlier reversal and a greater magnitude (P ≤ 0.025) of net ductal shunting, due to the combination of higher left-to-right (P ≤ 0.025) and lesser right-to-left phasic shunting (P ≤ 0.025). These results suggest that antenatal betamethasone augments the initial rise in PA blood flow after birth in preterm lambs, with this augmented rise supported by the combination of 1) a greater redistribution of RV output toward the lungs and 2) a faster and larger reversal in net ductal shunting underpinned not only by greater left-to-right, but also by lesser right-to-left phasic shunting.

Blunted sympathoadrenal activation accompanies hemodynamic stability after early ventilation and delayed cord clamping at birth in preterm lambs.

BACKGROUND: As surges in circulating norepinephrine and epinephrine have chronotropic, pressor, and inotropic effects, we tested the hypothesis that blunted rises in these catecholamines during preterm birth accompanied hemodynamic stability observed after early ventilation and delayed cord clamping (DCC), with findings compared to immediate cord clamping (ICC) and a non-asphyxial cord clamp-to-ventilation interval. METHODS: Anesthetized preterm fetal lambs were instrumented with arterial micromanometers to obtain pressure and the maximal rate of pressure rise (dP/dtmax) as a surrogate of ventricular contractility and an aortic catheter to obtain blood samples for catecholamine assay. Fetuses were delivered and mechanically ventilated before cord clamping ∼1.5 min later (DCC, n = 9) or subjected to ICC with ventilation started ∼40 s later (n = 8). RESULTS: Perinatal hemodynamics were stable after DCC, with greater fluctuations evident following birth after ICC (P ≤ 0.05). With DCC, circulating norepinephrine and epinephrine were unchanged after early ventilation but rose following cord clamping (P ≤ 0.01), with concentrations below the threshold for hemodynamic effects. Norepinephrine was higher in the ICC group after cord clamping and immediately after ventilation (P < 0.025), but catecholamine levels were otherwise similar between groups. CONCLUSION: Hemodynamic stability at birth after DCC is accompanied by sub-threshold rises in circulating norepinephrine and epinephrine and thus blunted sympathoadrenal activation.

Greater sympathoadrenal activation with longer preventilation intervals after immediate cord clamping increases hemodynamic lability at birth in preterm lambs.

This study tested the hypothesis that varying degrees of hemodynamic fluctuations seen after birth following immediate cord clamping were related to development of asphyxia with longer cord clamp-to-ventilation intervals, resulting in higher perinatal circulating levels of the catecholamines norepinephrine (NE) and epinephrine (Epi), and thus increased heart rate, blood pressures, and cardiac contractility after birth. Anesthetized preterm fetal lambs were instrumented with 1) aortic (AoT) and pulmonary trunk (PT) micromanometers to obtain pressures and the maximal rate of pressure rise (dP/dtmax) as a surrogate measure of ventricular contractility, and 2) an AoT catheter to obtain samples for blood gas and catecholamine analyses. After delivery, immediate cord clamping was followed by ventilation ∼40 s (n = 7), ∼60 s (n = 8), ∼90 s (n = 9), or ∼120 s later (n = 8), with frequent blood sampling performed before and after ventilation. AoT O2 content fell rapidly after immediate cord clamping (P < 0.001), with an asphyxial state evident at ≥60 s. Plasma NE and Epi levels increased progressively with longer cord clamp-to-ventilation intervals, with an exponential relation between falling AoT O2 content and rising catecholamines (R2 = 0.64-0.67). Elevated circulating catecholamines persisted for some minutes after ventilation onset, with postbirth surges in heart rate, AoT and PT pressures, and AoT and PT dP/dtmax linearly related to loge of catecholamine levels (R2 = 0.41-0.54, all P < 0.001). These findings suggest that 1) a greater degree of asphyxia-induced sympathoadrenal activation (reflected in elevated circulating catecholamine levels) occurs with longer intervals between immediate cord clamping and subsequent ventilation, and 2) this activation is a major determinant of hemodynamic fluctuations evident with birth.

Maternal alcohol consumption in pregnancy enhances arterial stiffness and alters vasodilator function that varies between vascular beds in fetal sheep.

While the impact of alcohol consumption by pregnant women on fetal neurodevelopment has received much attention, the effects on the cardiovascular system are not well understood. We hypothesised that repeated exposure to alcohol (ethanol) in utero would alter fetal arterial reactivity and wall stiffness, key mechanisms leading to cardiovascular disease in adulthood. Ethanol (0.75 g (kg body weight)(-1)) was infused intravenously into ewes over 1 h daily for 39 days in late pregnancy (days 95-133 of pregnancy, term ∼147 days). Maternal and fetal plasma ethanol concentrations at the end of the hour were ∼115 mg dl(-1), and then declined to apparent zero over 8 h. At necropsy (day 134), fetal body weight and fetal brain-body weight ratio were not affected by alcohol infusion. Small arteries (250-300 µm outside diameter) from coronary, renal, mesenteric, femoral (psoas) and cerebral beds were isolated. Endothelium-dependent vasodilatation sensitivity was reduced 10-fold in coronary resistance arteries, associated with a reduction in endothelial nitric oxide synthase mRNA (P = 0.008). Conversely, vasodilatation sensitivity was enhanced 10-fold in mesenteric and renal resistance arteries. Arterial stiffness was markedly increased (P = 0.0001) in all five vascular beds associated with an increase in elastic modulus and, in cerebral vessels, with an increase in collagen Iα mRNA. Thus, we show for the first time that fetal arteries undergo marked and regionally variable adaptations as a consequence of repeated alcohol exposure. These alcohol-induced vascular effects occurred in the apparent absence of fetal physical abnormalities or fetal growth restriction.

Neonatal hyperoxia: effects on nephrogenesis and long-term glomerular structure.

Preterm neonates are born while nephrogenesis is ongoing and are commonly exposed to factors in the extrauterine environment that may impair renal development. Supplemental oxygen therapy exposes the preterm infant to a hyperoxic environment that may induce oxidative stress. Our aim was to determine the immediate and long-term effects of exposure to hyperoxia, during the period of postnatal nephrogenesis, on renal development. Newborn mice (C57BL/6J) were kept in a normoxic (room air, 21% oxygen) or a controlled hyperoxic (65% oxygen) environment from birth to postnatal day 7 (P7d). From P7d, animals were maintained in room air until early adulthood at postnatal day 56 (P56d) or middle age (10 mo; P10mo). Pups were assessed for glomerular maturity and renal corpuscle cross-sectional area at P7d (control n = 14; hyperoxic n = 14). Nephron number and renal corpuscle size were determined stereologically at P56d (control n = 14; hyperoxic n = 14) and P10mo (control n = 10; hyperoxic n = 10). At P7d, there was no effect of hyperoxia on glomerular size or maturity. In early adulthood (P56d), body weights, relative kidney weights and volumes, and nephron number were not different between groups, but the renal corpuscles were significantly enlarged. This was no longer evident at P10mo, with relative kidney weights and volumes, nephron number, and renal corpuscle size not different between groups. Furthermore, hyperoxia exposure did not significantly accelerate glomerulosclerosis in middle age. Hence, our findings show no overt long-term deleterious effects of early life hyperoxia on glomerular structure.

Alcohol exposure during late ovine gestation alters fetal liver iron homeostasis without apparent dysmorphology.

High levels of alcohol (ethanol) exposure during fetal life can affect liver development and can increase susceptibility to infection after birth. Our aim was to determine the effects of a moderate level of ethanol exposure in late gestation on the morphology, iron status, and inflammatory status of the ovine fetal liver. Pregnant ewes were chronically catheterized at 91 days of gestation (DG; term ~145 DG) for daily intravenous infusion of ethanol (0.75 g/kg maternal body wt; n = 8) or saline (n = 7) over 1 h from 95 to 133 DG. At necropsy (134 DG), fetal livers were collected for analysis. Liver weight, general liver morphology, hepatic cell proliferation and apoptosis, perivascular collagen deposition, and interleukin (IL)-1ß, IL-6, or IL-8 mRNA levels were not different between groups. However, ethanol exposure led to significant decreases in hepatic content of ferric iron and gene expression of the iron-regulating hormone hepcidin and tumor necrosis factor (TNF)-α (all P < 0.05). In the placenta, there was no difference in transferrin receptor, divalent metal transporter 1, and ferritin mRNA levels; however, ferroportin mRNA levels were increased in ethanol-exposed animals (P < 0.05), and ferroportin protein tended to be increased (P = 0.054). Plasma iron concentration was not different between control and ethanol-exposed groups; control fetuses had significantly higher iron concentrations than their mothers, whereas maternal and fetal iron concentrations were similar in ethanol-exposed animals. We conclude that daily ethanol exposure during the third-trimester-equivalent in sheep does not alter fetal liver morphology; however, decreased fetal liver ferric iron content and altered hepcidin and ferroportin gene expression indicate that iron homeostasis is altered.

Lung ultrasound of the dependent lung detects real-time changes in lung volume in the preterm lamb.

BACKGROUND: Effective lung protective ventilation requires reliable, real-time estimation of lung volume at the bedside. Neonatal clinicians lack a readily available imaging tool for this purpose. OBJECTIVE: To determine the ability of lung ultrasound (LUS) of the dependent region to detect real-time changes in lung volume, identify opening and closing pressures of the lung, and detect pulmonary hysteresis. METHODS: LUS was performed on preterm lambs (n=20) during in vivo mapping of the pressure-volume relationship of the respiratory system using the super-syringe method. Electrical impedance tomography was used to derive regional lung volumes. Images were blindly graded using an expanded scoring system. The scores were compared with total and regional lung volumes, and differences in LUS scores between pressure increments were calculated. RESULTS: Changes in LUS scores correlated moderately with changes in total lung volume (r=0.56, 95% CI 0.47-0.64, p<0.0001) and fairly with right whole (r=0.41, CI 0.30-0.51, p<0.0001), ventral (r=0.39, CI 0.28-0.49, p<0.0001), central (r=0.41, CI 0.31-0.52, p<0.0001) and dorsal (r=0.38, CI 0.27-0.49, p<0.0001) regional lung volumes. The pressure-volume relationship of the lung exhibited hysteresis in all lambs. LUS was able to detect hysteresis in 17 (85%) lambs. The greatest changes in LUS scores occurred at the opening and closing pressures. CONCLUSION: LUS was able to detect large changes in total and regional lung volume in real time and correctly identified opening and closing pressures but lacked the precision to detect small changes in lung volume. Further work is needed to improve precision prior to translation to clinical practice.

S-Nitrosoglutathione Limits Apoptosis and Reduces Pulmonary Vascular Dysfunction After Bypass.

BACKGROUND: During hypoxia or acidosis, S-nitrosoglutathione (GSNO) has been shown to protect the cardiomyocyte from ischemia-reperfusion injury. In a randomized double-blinded control study of a porcine model of paediatric cardiopulmonary bypass (CPB), we aimed to evaluate the effects of 2 different doses (low and high) of GSNO. METHODS: Pigs weighing 15-20 kg were exposed to CPB with 1 hour of aortic cross-clamp. Prior to and during CPB, animals were randomized to receive low-dose (up to 20 nmol/kg/min) GSNO (n = 8), high-dose (up to 60 nmol/kg/min) GSNO (n = 6), or normal saline (n = 7). Standard cardiac intensive care management was continued for 4 hours post-bypass. RESULTS: There was a reduction in myocyte apoptosis after administration of GSNO (P = .04) with no difference between low- and high-dose GSNO. The low-dose GSNO group had lower pulmonary vascular resistance post-CPB (P = .007). Mitochondrial complex I activity normalized to citrate synthase activity was higher after GSNO compared with control (P = .02), with no difference between low- and high-dose GSNO. CONCLUSIONS: In a porcine model of CPB, intravenous administration of GSNO limits myocardial apoptosis through preservation of mitochondrial complex I activity, and improves pulmonary vascular resistance. There appears to be a dose-dependent effect to this protection.

Effects of prenatal ethanol exposure on the lungs of postnatal lambs.

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.

Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function.

Prenatal exposure to high levels of ethanol is associated with cardiac malformations, but the effects of lower levels of exposure on the heart are unclear. Our aim was to investigate the effects of daily exposure to ethanol during late gestation, when cardiomyocytes are undergoing maturation, on the developing myocardium. Pregnant ewes were infused with either ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95 to 133 (term ∼145 days); tissues were collected at 134 days. In sheep, cardiomyocytes mature during late gestation as in humans. Within the left ventricle (LV), cardiomyocyte number was determined using unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantified using image analysis. Genes relating to cardiomyocyte proliferation and apoptosis were examined using quantitative real-time PCR. Fetal plasma ethanol concentration reached 0.11 g/dL after EtOH infusions. Ethanol exposure induced significant increases in relative heart weight, relative LV wall volume, and cardiomyocyte cross-sectional area. Ethanol exposure advanced LV maturation in that the proportion of binucleated cardiomyocytes increased by 12%, and the number of mononucleated cardiomyocytes was decreased by a similar amount. Apoptotic gene expression increased in the ethanol-exposed hearts, although there were no significant differences between groups in total cardiomyocyte number or interstitial collagen. Daily exposure to a moderate dose of ethanol in late gestation accelerates the maturation of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may program for long-term cardiac vulnerability.

Daily ethanol exposure during late ovine pregnancy: physiological effects in the mother and fetus in the apparent absence of overt fetal cerebral dysmorphology.

High levels of ethanol (EtOH) consumption during pregnancy adversely affect fetal development; however, the effects of lower levels of exposure are less clear. Our objectives were to assess the effects of daily EtOH exposure (3.8 USA standard drinks) on fetal-maternal physiological variables and the fetal brain, particularly white matter. Pregnant ewes received daily intravenous infusions of EtOH (0.75 g/kg maternal body wt over 1 h, 8 fetuses) or saline (8 fetuses) from 95 to 133 days of gestational age (DGA; term ∼145 DGA). Maternal and fetal arterial blood was sampled at 131-133 DGA. At necropsy (134 DGA) fetal brains were collected for analysis. Maternal and fetal plasma EtOH concentrations reached similar maximal concentration (∼0.11 g/dl) and declined at the same rate. EtOH infusions produced mild reductions in fetal arterial oxygenation but there were no changes in maternal oxygenation, maternal and fetal Pa(CO(2)), or in fetal mean arterial pressure or heart rate. Following EtOH infusions, plasma lactate levels were elevated in ewes and fetuses, but arterial pH fell only in ewes. Fetal body and brain weights were similar between groups. In three of eight EtOH-exposed fetuses there were small subarachnoid hemorrhages in the cerebrum and cerebellum associated with focal cortical neuronal death and gliosis. Overall, there was no evidence of cystic lesions, inflammation, increased apoptosis, or white matter injury. We conclude that daily EtOH exposure during the third trimester-equivalent of ovine pregnancy has modest physiological effects on the fetus and no gross effects on fetal white matter development.