Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

In utero ventilation induces lung parenchymal and vascular alterations in extremely preterm fetal sheep.

Extremely preterm infants are often exposed to long durations of mechanical ventilation to facilitate gas exchange, resulting in ventilation-induced lung injury (VILI). New lung protective strategies utilizing noninvasive ventilation or low tidal volumes are now common but have not reduced rates of bronchopulmonary dysplasia. We aimed to determine the effect of 24 h of low tidal volume ventilation on the immature lung by ventilating preterm fetal sheep in utero. Preterm fetal sheep at 110 ± 1(SD) days' gestation underwent sterile surgery for instrumentation with a tracheal loop to enable in utero mechanical ventilation (IUV). At 112 ± 1 days' gestation, fetuses received either in utero mechanical ventilation (IUV, n = 10) targeting 3-5 mL/kg for 24 h, or no ventilation (CONT, n = 9). At necropsy, fetal lungs were collected to assess molecular and histological markers of lung inflammation and injury. IUV significantly increased lung mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) compared with CONT, and increased surfactant protein (SP)-A1, SP-B, and SP-C mRNA expression compared with CONT. IUV produced modest structural changes to the airways, including reduced parenchymal collagen and myofibroblast density. IUV increased pulmonary arteriole thickness compared with CONT but did not alter overall elastin or collagen content within the vasculature. In utero ventilation of an extremely preterm lung, even at low tidal volumes, induces lung inflammation and injury to the airways and vasculature. In utero ventilation may be an important model to isolate the confounding mechanisms of VILI to develop effective therapies for preterm infants requiring prolonged respiratory support.NEW & NOTEWORTHY Preterm infants often require prolonged respiratory support, but the relative contribution of ventilation to the development of lung injury is difficult to isolate. In utero mechanical ventilation allows for mechanistic investigations into ventilation-induced lung injury without confounding factors associated with sustaining extremely preterm lambs ex utero. Twenty-four hours of in utero ventilation, even at low tidal volumes, increased lung inflammation and surfactant protein expression and produced structural changes to the lung parenchyma and vasculature.

Mechanical ventilation induces brainstem inflammation in preterm fetal sheep.

Background: Preterm infants have immature respiratory drive and often require prolonged periods of mechanical ventilation. Prolonged mechanical ventilation induces systemic inflammation resulting in ventilation-induced brain injury, however its effect on brainstem respiratory centers is unknown. We aimed to determine the effects of 24 h of mechanical ventilation on inflammation and injury in brainstem respiratory centres of preterm fetal sheep. Methods: Preterm fetal sheep at 110 ± 1 days (d) gestation were instrumented to provide mechanical ventilation in utero. At 112 ± 1 d gestation, fetuses received either mechanical ventilation (VENT; n = 7; 3 ml/kg) for 24 h, or no ventilation (CONT; n = 6). At post-mortem, fetal brainstems were collected for assessment of mRNA and histological markers of inflammation and injury. Results: In utero ventilation (IUV) did not alter any blood-gas parameters. IUV significantly increased systemic IL-6 and IL-8 concentrations over the 24 h period compared to CONT. The number of ameboid microglia within the nucleus tractus solitarius and the raphe nucleus increased in VENT fetuses (p < 0.05 for both vs. control). The % area fraction of GFAP + staining was not significantly higher within the preBötzinger complex (p = 0.067) and retrotrapezoid nucleus and parafacial respiratory group (p = 0.057) in VENT fetuses compared to CONT. Numbers of caspase-3 and TUNEL-positive cells were similar between groups. Gene expression (mRNA) levels of inflammation, injury, cell death and prostaglandin synthesis within the brainstem were similar between groups. Conclusion: Mechanical ventilation induces a systemic inflammatory response with only moderate inflammatory effects within the brainstem respiratory centres of preterm fetal sheep.

Progressive inflammation reduces high-frequency EEG activity and cortical dendritic arborisation in late gestation fetal sheep.

BACKGROUND: Antenatal infection/inflammation is associated with disturbances in neuronal connectivity, impaired cortical growth and poor neurodevelopmental outcomes. The pathophysiological substrate that underpins these changes is poorly understood. We tested the hypothesis that progressive inflammation in late gestation fetal sheep would alter cortical neuronal microstructure and neural function assessed using electroencephalogram band power analysis. METHODS: Fetal sheep (0.85 of gestation) were surgically instrumented for continuous electroencephalogram (EEG) recording and randomly assigned to repeated saline (control; n = 9) or LPS (0 h = 300 ng, 24 h = 600 ng, 48 h = 1200 ng; n = 8) infusions to induce inflammation. Sheep were euthanised 4 days after the first LPS infusion for assessment of inflammatory gene expression, histopathology and neuronal dendritic morphology in the somatosensory cortex. RESULTS: LPS infusions increased delta power between 8 and 50 h, with reduced beta power from 18 to 96 h (P < 0.05 vs. control). Basal dendritic length, numbers of dendritic terminals, dendritic arborisation and numbers of dendritic spines were reduced in LPS-exposed fetuses (P < 0.05 vs. control) within the somatosensory cortex. Numbers of microglia and interleukin (IL)-1ß immunoreactivity were increased in LPS-exposed fetuses compared with controls (P < 0.05). There were no differences in total numbers of cortical NeuN + neurons or cortical area between the groups. CONCLUSIONS: Exposure to antenatal infection/inflammation was associated with impaired dendritic arborisation, spine number and loss of high-frequency EEG activity, despite normal numbers of neurons, that may contribute to disturbed cortical development and connectivity.

Investigating Pathways of Ventilation Induced Brain Injury on Cerebral White Matter Inflammation and Injury After 24 h in Preterm Lambs.

Initiation of respiratory support in the delivery room increases the risk and severity of brain injury in preterm neonates through two major pathways: an inflammatory pathway and a haemodynamic pathway. The relative contribution of each pathway on preterm brain injury is not known. We aimed to assess the role of the inflammatory and haemodynamic pathway on ventilation-induced brain injury (VIBI) in the preterm lamb. Fetal lambs (125 ± 1 day gestation) were exteriorised, instrumented and ventilated with a high tidal-volume (VT) injurious strategy for 15 min either with placental circulation intact to induce the inflammatory pathway only (INJINF; n = 7) or umbilical cord occluded to induce both the inflammatory and haemodynamic pathways (INJINF+HAE; n = 7). Sham controls were exteriorised but not ventilated (SHAM; n = 5) while unoperated controls (UNOP; n = 7) did not undergo fetal instrumentation. Fetuses were returned in utero following intervention and the ewe allowed to recover. Arterial blood gases and plasma were sampled periodically. Twenty-four hours following intervention, lambs were delivered and maintained on non-injurious ventilation for ∼40 min then brains were collected post-mortem for immunohistochemistry and RT-qPCR to assess inflammation, vascular pathology and cell death within white matter regions. Compared to INJINF lambs, INJINF+HAE lambs achieved a consistently higher VT during injurious ventilation and carotid blood flow was significantly lower than baseline by the end of ventilation. Throughout the 24 h recovery period, systemic arterial IL-6 levels of INJINF+HAE lambs were significantly higher than SHAM while there was no difference between INJINF and SHAM animals. At 24 h, mRNA expression levels of pro-inflammatory cytokines, tight junction proteins, markers of cell death, and histological injury indices of gliosis, blood vessel protein extravasation, oligodendrocyte injury and cell death were not different between groups. Injurious ventilation, irrespective of strategy, did not increase brain inflammation or injury 24 h later when compared to control animals. However, the haemodynamic pathway did influence carotid blood flow adaptations during injurious ventilation and increased systemic arterial IL-6 that may underlie long-term pathology. Future studies are required to further characterise the pathways and their long-term effects on VIBI.

Increased Prostaglandin E2 in Brainstem Respiratory Centers Is Associated With Inhibition of Breathing Movements in Fetal Sheep Exposed to Progressive Systemic Inflammation.

Background: Preterm newborns commonly experience apnoeas after birth and require respiratory stimulants and support. Antenatal inflammation is a common antecedent of preterm birth and inflammatory mediators, particularly prostaglandin E2 (PGE2), are associated with inhibition of vital brainstem respiratory centers. In this study, we tested the hypothesis that exposure to antenatal inflammation inhibits fetal breathing movements (FBMs) and increases inflammation and PGE2 levels in brainstem respiratory centers, cerebrospinal fluid (CSF) and blood plasma. Methods: Chronically instrumented late preterm fetal sheep at 0.85 of gestation were randomly assigned to receive repeated intravenous saline (n = 8) or lipopolysaccharide (LPS) infusions (experimental day 1 = 300 ng, day 2 = 600 ng, day 3 = 1200 ng, n = 8). Fetal breathing movements were recorded throughout the experimental period. Sheep were euthanized 4 days after starting infusions for assessment of brainstem respiratory center histology. Results: LPS infusions increased circulating and cerebrospinal fluid PGE2 levels, decreased arterial oxygen saturation, increased the partial pressure of carbon dioxide and lactate concentration, and decreased pH (p < 0.05 for all) compared to controls. LPS infusions caused transient reductions in the % of time fetuses spent breathing and the proportion of vigorous fetal breathing movements (P < 0.05 vs. control). LPS-exposure increased PGE2 expression in the RTN/pFRG (P < 0.05 vs. control) but not the pBÖTC (P < 0.07 vs. control) of the brainstem. No significant changes in gene expression were observed for PGE2 enzymes or caspase 3. LPS-exposure reduced the numbers of GFAP-immunoreactive astrocytes in the RTN/pFRG, NTS and XII of the brainstem (P < 0.05 vs. control for all) and increased microglial activation in the RTN/pFRG, preBÖTC, NTS, and XII brainstem respiratory centers (P < 0.05 vs. control for all). Conclusion: Chronic LPS-exposure in late preterm fetal sheep increased PGE2 levels within the brainstem, CSF and plasma, and was associated with inhibition of FBMs, astrocyte loss and microglial activation within the brainstem respiratory centers. Further studies are needed to determine whether the inflammation-induced increase in PGE2 levels plays a key role in depressing respiratory drive in the perinatal period.

Increased airway liquid volumes at birth impair cardiorespiratory function in preterm and near-term lambs.

Respiratory distress is relatively common in infants born at or near-term, particularly in infants delivered following elective cesarean section. The pathophysiology underlying respiratory distress at term has largely been explained by a failure to clear airway liquid, but recent physiological evidence has indicated that it results from elevated airway liquid at the onset of air-breathing. We have investigated the effect of elevated airway liquid volumes at birth on cardiorespiratory function in preterm and near-term lambs. Preterm (130 ± 0 days gestation, term ∼147 days gestation; n = 12) and near-term (139 ± 1 days gestation; n = 13) lambs were instrumented (to measure blood pressure, blood flow, and blood gas status) and, at delivery, airway liquid volumes were adjusted to mimic levels expected following vaginal delivery (Controls; ∼7 mL/kg) or elective cesarean section with no labor (elevated liquid (EL); 37 mL/kg). Lambs were delivered, mechanically ventilated, and monitored for blood gas status, oxygenation, ventilator requirements, blood flows (carotid artery and pulmonary artery), and blood pressure during the first few hours of life. Preterm and near-term EL lambs had poorer gas exchange and required greater ventilatory support to maintain adequate oxygenation. Pulmonary blood flow was reduced and carotid artery blood flow, mean arterial blood pressure, and heart rate were reduced in EL near-term but not preterm lambs. These data provide further evidence that greater airway liquid volumes at birth adversely affect newborn cardiorespiratory function, with the effects being greater in near-term newborns.NEW & NOTEWORTHY We provide evidence for adverse effects of elevated airway liquid volumes at birth on pulmonary blood flow and gas exchange in both preterm and near-term lambs, although the effects were greatest in near-term newborns. Our study is an important step toward understanding the fundamental physiology underlying the cardiorespiratory morbidity associated with near-term newborns with elevated airway liquid volumes leading to respiratory distress soon after birth.

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

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

Magnetic Resonance Imaging Correlates of White Matter Gliosis and Injury in Preterm Fetal Sheep Exposed to Progressive Systemic Inflammation.

Progressive fetal infection/inflammation is strongly associated with neural injury after preterm birth. We aimed to test the hypotheses that progressively developing fetal inflammation leads to neuroinflammation and impaired white matter development and that the histopathological changes can be detected using high-field diffusion tensor magnetic resonance imaging (MRI). Chronically instrumented preterm fetal sheep at 0.7 of gestation were randomly assigned to receive intravenous saline (control; n = 6) or a progressive infusion of lipopolysaccharide (LPS, 200 ng intravenous over 24 h then doubled every 24 h for 5 days to induce fetal inflammation, n = 7). Sheep were killed 10 days after starting the infusions, for histology and high-field diffusion tensor MRI. Progressive LPS infusion was associated with increased circulating interleukin (IL)-6 concentrations and moderate increases in carotid artery perfusion and the frequency of electroencephalogram (EEG) activity (p < 0.05 vs. control). In the periventricular white matter, fractional anisotropy (FA) was increased, and orientation dispersion index (ODI) was reduced (p < 0.05 vs. control for both). Histologically, in the same brain region, LPS infusion increased microglial activation and astrocyte numbers and reduced the total number of oligodendrocytes with no change in myelination or numbers of immature/mature oligodendrocytes. Numbers of astrocytes in the periventricular white matter were correlated with increased FA and reduced ODI signal intensities. Astrocyte coherence was associated with increased FA. Moderate astrogliosis, but not loss of total oligodendrocytes, after progressive fetal inflammation can be detected with high-field diffusion tensor MRI.

High-CPAP Does Not Impede Cardiovascular Changes at Birth in Preterm Sheep.

Objective: Continuous positive airway pressures (CPAP) used to assist preterm infants at birth are limited to 4-8 cmH2O due to concerns that high-CPAP may cause pulmonary overexpansion and adversely affect the cardiovascular system. We investigated the effects of high-CPAP on pulmonary (PBF) and cerebral (CBF) blood flows and jugular vein pressure (JVP) after birth in preterm lambs. Methods: Preterm lambs instrumented with flow probes and catheters were delivered at 133/146 days gestation. Lambs received low-CPAP (LCPAP: 5 cmH2O), high-CPAP (HCPAP: 15 cmH2O) or dynamic HCPAP (15 decreasing to 8 cmH2O at ~2 cmH2O/min) for up to 30 min after birth. Results: Mean PBF was lower in the LCPAP [median (Q1-Q3); 202 (48-277) mL/min, p = 0.002] compared to HCPAP [315 (221-365) mL/min] and dynamic HCPAP [327 (269-376) mL/min] lambs. CBF was similar in LCPAP [65 (37-78) mL/min], HCPAP [73 (41-106) mL/min], and dynamic HCPAP [66 (52-81) mL/min, p = 0.174] lambs. JVP was similar at CPAPs of 5 [8.0 (5.1-12.4) mmHg], 8 [9.4 (5.3-13.4) mmHg], and 15 cmH2O [8.6 (6.9-10.5) mmHg, p = 0.909]. Heart rate was lower in the LCPAP [134 (101-174) bpm; p = 0.028] compared to the HCPAP [173 (139-205)] and dynamic HCPAP [188 (161-207) bpm] groups. Ventilation or additional caffeine was required in 5/6 LCPAP, 1/6 HCPAP, and 5/7 dynamic HCPAP lambs (p = 0.082), whereas 3/6 LCPAP, but no HCPAP lambs required intubation (p = 0.041), and 1/6 LCPAP, but no HCPAP lambs developed a pneumothorax (p = 0.632). Conclusion: High-CPAP did not impede the increase in PBF at birth and supported preterm lambs without affecting CBF and JVP.

Effect of lung hypoplasia on the cardiorespiratory transition in newborn lambs.

Newborns with lung hypoplasia (LH) commonly have limited respiratory function and often require ventilatory assistance after birth. We aimed to characterize the cardiorespiratory transition and respiratory function in newborn lambs with LH. LH was induced by draining fetal lung liquid in utero [110-133 days (d), term = 147d, n = 6]. At ~133d gestation, LH and Control lambs (n = 6) were instrumented and ventilated for 3 h to monitor blood-gas status, oxygenation, ventilator requirements, and hemodynamics during the transition from fetal to newborn life. Lambs with LH had significantly reduced relative wet and dry lung weights indicating hypoplastic lungs compared with Control lambs. LH lambs experienced persistent hypercapnia and acidosis during the ventilation period, had lower lung compliance, and had higher alveolar-arterial differences in oxygen and oxygenation index compared with Control lambs. As a result, LH lambs required greater respiratory support and more supplemental oxygen. Following delivery, LH lambs experienced periods of significantly lower pulmonary artery blood flow and higher carotid artery blood flow in association with the lower oxygenation levels. The detrimental effects of LH can be attributed to a reduction in lung size and poorer gas exchange capabilities. This study has provided greater understanding of the effect of LH itself on the physiology underpinning the transition from fetal to newborn life. Advances in this area is the key to identifying improved or novel management strategies for babies with LH starting in the delivery room, to favorably alter the fetal-to-newborn transition toward improved outcomes and reduced lifelong morbidity.NEW & NOTEWORTHY Current clinical management of newborns with lung hypoplasia (LH) is largely based on expert opinion rather than scientific evidence. We have generated physiological evidence for detrimental effects of LH on hemodynamics and respiratory function in newborn lambs, which mimics the morbidity observed in LH newborns clinically. The unfavorable consequences of LH can be attributed to a reduction in lung size and poorer gas exchange capabilities.

Effects of Intrauterine Inflammation on Cortical Gray Matter of Near-Term Lambs.

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 (INJSALN = 6, INJLPSN = 5) or a protective ventilation strategy (PROTSALN = 5, PROTLPSN = 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.

Exacerbation of Ventilation-Induced Lung Injury and Inflammation in Preterm Lambs by High-Dose Nanoparticles.

Mechanical ventilation of preterm neonates causes lung inflammation and injury, with potential life-long consequences. Inert 50-nm polystyrene nanoparticles (PS50G) reduce allergic inflammation in the lungs of adult mice. We aimed to confirm the anti-inflammatory effects of PS50G in a sheep asthma model, and investigate the effects of prophylactic administration of PS50G on ventilation-induced lung injury (VILI) in preterm lambs. We assessed lung inflammatory cell infiltration, with and without PS50G, after airway allergen challenge in ewes sensitised to house dust mite. Preterm lambs (0.83 gestation) were delivered by caesarean section for immediate tissue collection (n = 5) or ventilation either with (n = 6) or without (n = 5) prophylactic intra-tracheal administration of PS50G nanoparticles (3% in 2 ml). Ventilation was continued for a total of 2 h before tissue collection for histological and biomolecular assessment of lung injury and inflammation. In ewes with experimental asthma, PS50G decreased eosinophilic infiltration of the lungs. Ventilated preterm lambs showed molecular and histological signs of lung injury and inflammation, which were exacerbated in lambs that received PSG50G. PS50G treatment decreased established inflammation in the lungs of asthmatic sheep. However, prophylactic administration of PSG50 exacerbated ventilation-induced lung injury and lung inflammation in preterm lambs.

Optimizing the Dose of Erythropoietin Required to Prevent Acute Ventilation-Induced Cerebral White Matter Injury in Preterm Lambs.

Erythropoietin (EPO) is being trialed in preterm neonates for neuroprotection. We have recently demonstrated that a single high bolus dose (5,000 IU/kg) of recombinant human EPO amplified preterm lung and brain ventilation-induced injury. We aimed to determine the optimal dose of EPO to reduce ventilation-induced cerebral white matter inflammation and injury in preterm lambs. Lambs (0.85 gestation) were ventilated with an injurious strategy for 15 min followed by conventional ventilation for 105 min. Lambs were randomized to no treatment (VENT; n = 8) or received a bolus dose of EPO (EPREX®): 300 IU/kg (EPO 300; n = 5), 1,000 IU/kg (EPO 1,000; n = 5), or 3,000 IU/kg (EPO 3,000; n = 5). Physiological parameters were measured throughout the study. After 2 h, brains were collected for analysis; real-time quantitative polymerase chain reaction and immunohistochemistry were used to assess inflammation, cell death, and vascular leakage in the periventricular and subcortical white matter (PVWM; SCWM). Molecular and histological inflammatory indices in the PVWM were not different between groups. EPO 300 lambs had higher IL-6 (p = 0.006) and caspase-3 (p = 0.025) mRNA expression in the SCWM than VENT lambs. Blood-brain barrier (BBB) occludin mRNA levels were higher in EPO 3,000 lambs in the PVWM and SCWM than VENT lambs. The number of blood vessels with protein extravasation in the SCWM was lower in EPO 1,000 (p = 0.010) and EPO 3,000 (p = 0.025) lambs compared to VENT controls but not different between groups in the PVWM. Early administration of EPO at lower doses neither reduced nor exacerbated cerebral white matter inflammation or injury. 3,000 IU/kg EPO may provide neuroprotection by improving BBB integrity.

Effect of body position and ventilation on umbilical artery and venous blood flows during delayed umbilical cord clamping in preterm lambs.

OBJECTIVE: While delayed umbilical cord clamping (UCC) is thought to facilitate placental to infant blood transfusion, the physiological factors regulating flow in the umbilical arteries and veins during delayed UCC is unknown. We investigated the effects of gravity, by changing fetal height relative to the placenta, and ventilation on umbilical blood flows and the cardiovascular transition during delayed UCC at birth. METHODS: Catheters and flow probes were implanted into preterm lambs (128 days) prior to delivery to measure pulmonary, carotid, umbilical artery (UaBF) and umbilical venous (UvBF) blood flows. Lambs were placed either 10 cm below or 10 cm above the ewe. Ventilation commenced 2-3 min before UCC and continued for 30 min after UCC. RESULTS: Gravity reduced umbilical and cerebral flows when lambs were placed below the midline, but the reduction in UaBF and UvBF was similar. Ventilation during delayed UCC reduced UvBF and UaBF by similar amounts, irrespective of the lamb's position, such that flows into and out of the placenta remained balanced. The effects of ventilation on umbilical flows were much greater than the effects of gravity, but no net placental to lamb blood transfusion could be detected under any condition. Cardiovascular parameters, cerebral oxygen kinetics and final blood volumes were similar in both groups 5 min after UCC. CONCLUSIONS: Gravity caused small transient effects on umbilical and cerebral flow, but given changes were similar in umbilical arteries and veins, no net placental transfusion was detected. Ventilation during delayed UCC has a markedly greater influence on cardiovascular function in the newborn.

Lung ultrasound accurately detects pneumothorax in a preterm newborn lamb model.

AIM: Pneumothorax is a common emergency affecting extremely preterm. In adult studies, lung ultrasound has performed better than chest x-ray in the diagnosis of pneumothorax. The purpose of this study was to determine the efficacy of lung ultrasound (LUS) examination to detect pneumothorax using a preterm animal model. METHODS: This was a prospective, observational study using newborn Border-Leicester lambs at gestational age = 126 days (equivalent to gestational age = 26 weeks in humans) receiving mechanical ventilation from birth to 2 h of life. At the conclusion of the experiment, LUS was performed, the lambs were then euthanised and a post-mortem exam was immediately performed. We used previously published ultrasound techniques to identify pneumothorax. Test characteristics of LUS to detect pneumothorax were calculated, using the post-mortem exam as the 'gold standard' test. RESULTS: Nine lambs (18 lungs) were examined. Four lambs had a unilateral pneumothorax, all of which were identified by LUS with no false positives. CONCLUSIONS: This was the first study to use post-mortem findings to test the efficacy of LUS to detect pneumothorax in a newborn animal model. Lung ultrasound accurately detected pneumothorax, verified by post-mortem exam, in premature, newborn lambs.

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

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

Spatiotemporal Aeration and Lung Injury Patterns Are Influenced by the First Inflation Strategy at Birth.

Ineffective aeration during the first inflations at birth creates regional aeration and ventilation defects, initiating injurious pathways. This study aimed to compare a sustained first inflation at birth or dynamic end-expiratory supported recruitment during tidal inflations against ventilation without intentional recruitment on gas exchange, lung mechanics, spatiotemporal regional aeration and tidal ventilation, and regional lung injury in preterm lambs. Lambs (127 ± 2 d gestation), instrumented at birth, were ventilated for 60 minutes from birth with either lung-protective positive pressure ventilation (control) or as per control after either an initial 30 seconds of 40 cm H2O sustained inflation (SI) or an initial stepwise end-expiratory pressure recruitment maneuver during tidal inflations (duration 180 s; open lung ventilation [OLV]). At study completion, molecular markers of lung injury were analyzed. The initial use of an OLV maneuver, but not SI, at birth resulted in improved lung compliance, oxygenation, end-expiratory lung volume, and reduced ventilatory needs compared with control, persisting throughout the study. These changes were due to more uniform inter- and intrasubject gravity-dependent spatiotemporal patterns of aeration (measured using electrical impedance tomography). Spatial distribution of tidal ventilation was more stable after either recruitment maneuver. All strategies caused regional lung injury patterns that mirrored associated regional volume states. Irrespective of strategy, spatiotemporal volume loss was consistently associated with up-regulation of early growth response-1 expression. Our results show that mechanical and molecular consequences of lung aeration at birth are not simply related to rapidity of fluid clearance; they are also related to spatiotemporal pressure-volume interactions within the lung during inflation and deflation.

Ventilation-induced lung injury is not exacerbated by growth restriction in preterm lambs.

Intrauterine growth restriction (IUGR) and preterm birth are frequent comorbidities and, combined, increase the risk of adverse respiratory outcomes compared with that in appropriately grown (AG) infants. Potential underlying reasons for this increased respiratory morbidity in IUGR infants compared with AG infants include altered fetal lung development, fetal lung inflammation, increased respiratory requirements, and/or increased ventilation-induced lung injury. IUGR was surgically induced in preterm fetal sheep (0.7 gestation) by ligation of a single umbilical artery. Four weeks later, preterm lambs were euthanized at delivery or delivered and ventilated for 2 h before euthanasia. Ventilator requirements, lung inflammation, early markers of lung injury, and morphological changes in lung parenchymal and vascular structure and surfactant composition were analyzed. IUGR preterm lambs weighed 30% less than AG preterm lambs, with increased brain-to-body weight ratio, indicating brain sparing. IUGR did not induce lung inflammation or injury or alter lung parenchymal and vascular structure compared with AG fetuses. IUGR and AG lambs had similar oxygenation and respiratory requirements after birth and had significant, but similar, increases in proinflammatory cytokine expression, lung injury markers, gene expression, and surfactant phosphatidylcholine species compared with unventilated controls. IUGR does not induce pulmonary structural changes in our model. Furthermore, IUGR and AG preterm lambs have similar ventilator requirements in the immediate postnatal period. This study suggests that increased morbidity and mortality in IUGR infants is not due to altered lung tissue or vascular structure, or to an altered response to early ventilation.

Surfactant before the first inflation at birth improves spatial distribution of ventilation and reduces lung injury in preterm lambs.

The interrelationship between the role of surfactant and a sustained inflation (SI) to aid ex utero transition of the preterm lung is unknown. We compared the effect of surfactant administered before and after an initial SI on gas exchange, lung mechanics, spatial distribution of ventilation, and lung injury in preterm lambs. Gestational-age lambs (127 days; 9 per group) received 100 mg/kg of a surfactant (Curosurf) either prior (Surf+SI) or 10 min after birth (SI+Surf). At birth, a 20-s, 35 cmH2O SI was applied, followed by 70 min of positive pressure ventilation. Oxygenation, carbon dioxide removal, respiratory system compliance, end-expiratory thoracic volume (via respiratory inductive plethysmography), and distribution of end-expiratory volume and ventilation (via electrical impedance tomography) were measured throughout. Early markers of lung injury were analyzed using quantitative RT-PCR. During the first 15 min, oxygenation, carbon dioxide removal, and compliance were better in the Surf+SI group (all P < 0.05). End-expiratory volume on completion of the sustained inflation was higher in the Surf+SI group than the SI+Surf group; 11 ± 1 ml/kg vs. 7 ± 1 ml/kg (mean ± SE) (P = 0.043; t-test), but was not different at later time points. Although neither achieved homogenous aeration, spatial ventilation was more uniform in the Surf+SI group throughout; 50.1 ± 10.9% of total ventilation in the left hemithorax at 70 min vs. 42.6 ± 11.1% in the SI+Surf group. Surf+SI resulted in lower mRNA levels of CYR61 and EGR1 compared with SI+Surf (P < 0.001, one-way ANOVA). Surfactant status of the fetal preterm lung at birth influences the mechanical and injury response to a sustained inflation and ventilation by changing surface tension of the air/fluid interface.