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Abstract 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.

BackgroundIntrauterine inflammation and the requirement for mechanical ventilation independently increase the risk of perinatal brain injury and adverse neurodevelopmental outcomes. We aimed to investigate the effects of mechanical ventilation for 24 h, with and without prior exposure to intrauterine inflammation, on markers of brain inflammation and injury in the preterm sheep brain.MethodsChronically instrumented fetal sheep at ~115 days of gestation were randomly allocated to receive a single intratracheal dose of 1 mg lipopolysaccharide (LPS) or isovolumetric saline, then further randomly allocated 1 h after to receive mechanical ventilation with room air or no mechanical ventilation (unventilated control + saline [UVC, n = 7]; in utero mechanical ventilation + saline [VENT, n = 8], unventilated control + intratracheal LPS [UVC + LPS, n = 7]; in utero ventilation + intratracheal LPS [VENT + LPS, n = 7]). Serial fetal blood and plasma samples were collected throughout the experimental protocol for assessment of blood biochemistry and plasma interleukin (IL)-6 levels. After 24 h of mechanical ventilation, fetal brains were collected for RT-qPCR and immunohistochemical analyses.ResultsLPS exposure increased numbers of microglia and upregulated pro-inflammatory related genes within the cortical gray matter (GM) and subcortical white matter (SCWM) (pLPS

Abstract Background Perinatal infection/inflammation is associated with a high risk for neurological injury and neurodevelopmental impairment after birth. Despite a growing preclinical evidence base, anti-inflammatory interventions have not been established in clinical practice, partly because of the range of potential targets. We therefore systematically reviewed preclinical studies of immunomodulation to improve neurological outcomes in the perinatal brain and assessed their therapeutic potential. Methods We reviewed relevant studies published from January 2012 to July 2023 using PubMed, Medline (OvidSP) and EMBASE databases. Studies were assessed for risk of bias using the SYRCLE risk of bias assessment tool (PROSPERO; registration number CRD42023395690). Results Forty preclinical publications using 12 models of perinatal neuroinflammation were identified and divided into 59 individual studies. Twenty-seven anti-inflammatory agents in 19 categories were investigated. Forty-five (76%) of 59 studies reported neuroprotection, from all 19 categories of therapeutics. Notably, 10/10 (100%) studies investigating anti-interleukin (IL)-1 therapies reported improved outcome, whereas half of the studies using corticosteroids (5/10; 50%) reported no improvement or worse outcomes with treatment. Most studies (49/59, 83%) did not control core body temperature (a known potential confounder), and 25 of 59 studies (42%) did not report the sex of subjects. Many studies did not clearly state whether they controlled for potential study bias. Conclusion Anti-inflammatory therapies are promising candidates for treatment or even prevention of perinatal brain injury. Our analysis highlights key knowledge gaps and opportunities to improve preclinical study design that must be addressed to support clinical translation.

IntroductionFetal growth restriction (FGR) is a common pregnancy complication, caused by placental insufficiency, with serious adverse consequences for development in utero and postnatal wellbeing. There are no antenatal treatments to improve growth or organ development in FGR, and animal models are essential to mimic the physiological adaptations in FGR and to assess potential interventions. This study aimed to identify the temporal nature of reduced developmental trajectory in fetuses with FGR, and to examine the effects of common factors that may mediate differential growth such as glucocorticoid treatment. We hypothesised that the trajectory of growth would be adversely impacted by FGR.MethodsFGR was induced via surgical placental insufficiency in fetal sheep (89 days gestation/0.6 gestation; n=135) and compared to age-matched controls over the last third of gestation and into neonatal life (n=153).ResultsBody weight of FGR fetuses/lambs was significantly reduced compared to controls (p

Abstract 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

BackgroundPreterm 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.MethodsPreterm 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.ResultsIn 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

Abstract Background International guidelines recommend delayed umbilical cord clamping (DCC) up to 1 min in preterm infants, unless the condition of the infant requires immediate resuscitation. However, clamping the cord prior to lung aeration may severely limit circulatory adaptation resulting in a reduction in cardiac output and hypoxia. Delaying cord clamping until lung aeration and ventilation have been established (physiological-based cord clamping, PBCC) allows for an adequately established pulmonary circulation and results in a more stable circulatory transition. The decline in cardiac output following time-based delayed cord clamping (TBCC) may thus be avoided. We hypothesise that PBCC, compared to TBCC, results in a more stable transition in very preterm infants, leading to improved clinical outcomes. The primary objective is to compare the effect of PBCC on intact survival with TBCC. Methods The Aeriation, Breathing, Clamping 3 (ABC3) trial is a multicentre randomised controlled clinical trial. In the interventional PBCC group, the umbilical cord is clamped after the infant is stabilised, defined as reaching heart rate > 100 bpm and SpO2 > 85% while using supplemental oxygen

BackgroundUmbilical cord milking (UCM) at birth causes surges in arterial blood pressure and blood flow to the brain, which may explain the high risk of intraventricular haemorrhage (IVH) in extremely preterm infants receiving UCM. This high risk of IVH has not been reported in older infants.ObjectiveWe hypothesized that lung aeration before UCM, reduces the surge in blood pressure and blood flow induced by UCM.MethodsAt 126 days' gestation, fetal lambs (N = 8) were exteriorised, intubated and instrumented to measure umbilical, pulmonary, cerebral blood flows, and arterial pressures. Prior to ventilation onset, the umbilical cord was briefly (2–3 s) occluded (8 times), which was followed by 8 consecutive UCMs when all physiological parameters had returned to baseline. Lambs were then ventilated. After diastolic pulmonary blood flow markedly increased in response to ventilation, the lambs received a further 8 consecutive UCMs. Ovine umbilical cord is shorter than the human umbilical cord, with ∼10 cm available for UCMs. Therefore, 8 UCMs/occlusions were done to match the volume reported in the human studies. Umbilical cord clamping occurred after the final milk.ResultsBoth umbilical cord occlusions and UCM caused significant increases in carotid arterial blood flow and pressure. However, the increases in systolic and mean arterial blood pressure (10 ± 3 mmHg vs. 3 ± 2 mmHg, p = 0.01 and 10 ± 4 mmHg vs. 6 ± 2 mmHg, p = 0.048, respectively) and carotid artery blood flow (17 ± 6 ml/min vs. 10 ± 6 ml/min, p = 0.02) were significantly greater when UCM occurred before ventilation onset compared with UCM after ventilation.ConclusionsUCM after ventilation onset significantly reduces the increases in carotid blood flow and blood pressure caused by UCM.

Abstract Background Increased systemic and tissue levels of interleukin (IL)-1β 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-1β 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-1β 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.

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.

Abstract Background Neonatal ventilation exacerbates brain injury in lambs with fetal growth restriction (FGR), characterized by neuroinflammation and reduced blood-brain barrier integrity, which is normally maintained by the neurovascular unit. We examined whether umbilical cord blood stem cell (UCBC) treatment stabilized the neurovascular unit and reduced brain injury in preterm ventilated FGR lambs. Methods Surgery was performed in twin-bearing pregnant ewes at 88 days’ gestation to induce FGR in one fetus. At 127 days, FGR and appropriate for gestational age (AGA) lambs were delivered, carotid artery flow probes and umbilical lines inserted, lambs intubated and commenced on gentle ventilation. Allogeneic ovine UCBCs (25 × 106 cells/kg) were administered intravenously to lambs at 1 h of life. Lambs were ventilated for 24 h and then euthanized. Results FGR (n = 6) and FGR+UCBC (n = 6) lambs were growth restricted compared to AGA (n = 6) and AGA+UCBC (n = 6) lambs (combined weight, FGR 2.3 ± 0.4 vs. AGA 3.0 ± 0.3 kg; p = 0.0002). UCBC therapy did not alter mean arterial blood pressure or carotid blood flow but decreased cerebrovascular resistance in FGR+UCBC lambs. Circulating TNF-α cytokine levels were lower in FGR+UCBC vs. FGR lambs (p

BackgroundPreterm 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.MethodsChronically 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.ResultsLPS 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).ConclusionChronic 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.

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.

Background: Physiologic-based cord clamping (PBCC) involves deferring umbilical cord clamping until after lung aeration. It is unclear if infant is at risk of becoming hypothermic during PBCC.Objectives: To test if PBCC would maintain core temperature more effectively than immediate cord clamping (ICC).Design: At 0.93 gestation, fetal lambs were surgically exteriorized and instrumented from pregnant ewes under general anesthesia. Prior to the start of the experiment, lambs were thoroughly dried, placed on hot water bottles, and core temperature was continuously monitored using a rectal thermometer. PBCC lambs (n = 21), received intermittent positive pressure ventilation (iPPV) for ≥5 min prior to umbilical cord clamping. In ICC lambs (n = 23), iPPV commenced within 60 s after umbilical cord clamping. iPPV was provided with heated/humidified gas. Lambs were moved under a radiant warmer after umbilical cord clamping. Additional warmth was provided using a plastic overlay, hairdryer, and extra water bottles, as needed. Two-way mixed and repeated measures one-way ANOVAs were used to compare temperature changes between and within a single group, respectively, over time.Results: Basal fetal parameters including core temperature were similar between groups. ICC lambs had a significant reduction in temperature compared to PBCC lambs (p < 0.001), evident by 1 min (p = 0.002). ICC lambs decreased temperature by 0.51°C (± 0.42) and 0.79°C (± 0.55) at 5 and 10 min respectively (p < 0.001). In PBCC lambs, temperature did not significantly change before or after umbilical cord clamping (p = 0.4 and p = 0.3, respectively).Conclusions: PBCC stabilized core temperature at delivery better than ICC in term lambs. Hypothermia may not be a significant risk during PBCC.

Many preterm neonates require mechanical ventilation which increases the risk of cerebral inflammation and white matter injury in the immature brain. In this review, we discuss the links between ventilation and brain injury with a focus on the immediate period after birth, incorporating respiratory support in the delivery room and subsequent mechanical ventilation in the neonatal intensive care unit. This review collates insight from large animal models in which acute injurious ventilation and prolonged periods of ventilation have been used to create clinically relevant brain injury patterns. These models are valuable resources in investigating the pathophysiology of ventilation-induced brain injury and have important translational implications. We discuss the challenges of reconciling lung and brain maturation in commonly used large animal models. A comprehensive understanding of ventilation-induced brain injury is necessary to guide the way we care for preterm neonates, with the goal to improve their neurodevelopmental outcomes.

Background: Current guidelines recommend immediate umbilical cord clamping (UCC) for newborns requiring chest compressions (CCs). Physiological-based cord clamping (PBCC), defined as delaying UCC until after lung aeration, has advantages over immediate UCC in mildly asphyxiated newborns, but its efficacy in asystolic newborns requiring CC is unknown. The aim of this study was to compare the cardiovascular response to CCs given prior to or after UCC in asystolic near-term lambs.Methods: Umbilical, carotid, pulmonary, and femoral arterial flows and pressures as well as systemic and cerebral oxygenation were measured in near-term sheep fetuses [139 ± 2 (SD) days gestation]. Fetal asphyxia was induced until asystole ensued, whereupon lambs received ventilation and CC before (PBCC; n = 16) or after (n = 12) UCC. Epinephrine was administered 1 min after ventilation onset and in 3-min intervals thereafter. The PBCC group was further separated into UCC at either 1 min (PBCC1, n = 8) or 10 min (PBCC10, n = 8) after return of spontaneous circulation (ROSC). Lambs were maintained for a further 30 min after ROSC.Results: The duration of CCs received and number of epinephrine doses required to obtain ROSC were similar between groups. After ROSC, we found no physiological benefits if UCC was delayed for 1 min compared to immediate cord clamping (ICC). However, if UCC was delayed for 10 min after ROSC, we found significant reductions in post-asphyxial rebound hypertension, cerebral blood flow, and cerebral oxygenation. The prevention of the post-asphyxial rebound hypertension in the PBCC10 group occurred due to the contribution of the placental circulation to a low peripheral resistance. As a result, left and right ventricular outputs continued to perfuse the placenta and were evidenced by reduced mean pulmonary blood flow, persistence of right-to-left shunting across the ductus arteriosus, and persistence of umbilical arterial and venous blood flows.Conclusion: It is possible to obtain ROSC after CC while the umbilical cord remains intact. There were no adverse effects of PBCC compared to ICC; however, the physiological changes observed after ROSC in the ICC and early PBCC groups may result in additional cerebral injury. Prolonging UCC after ROSC may provide significant physiological benefits that may reduce the risk of harm to the cerebral circulation.

Objectives: Neonatal resuscitation guidelines recommend administering intravenous (IV) adrenaline if bradycardia persists despite adequate ventilation and chest compressions (CC). Rapid IV access is challenging, but little evidence exists for other routes of administration. We compared IV, endotracheal (ET), and intranasal routes for adrenaline administration during resuscitation of asphyxiated newborn lambs.Study design: Near-term lambs (n = 22) were delivered by caesarean section. Severe asphyxia was induced by clamping the umbilical cord while delaying ET ventilation until blood flow in the carotid artery ceased. Following a 30 s sustained inflation and ventilation for 30 s, we commenced uncoordinated CC at 90/min. We randomized four groups receiving repeated treatment doses (Tds) every 3rd min of (i) IV-Adrenaline (50 μg), (ii) ET-Adrenaline (500 μg), (iii) Nasal-Adrenaline via an atomizer (500 μg), and (iv) IV-saline. If return of spontaneous circulation (ROSC) was not achieved after three Tds by the assigned route, up to two rescue doses (Rds) of IV adrenaline were administered. Main outcome measures were achievement of ROSC and time from start of CC to ROSC, defined as heart rate >100/min, and mean carotid arterial pressure >30 mmHg.Results: In the IV-Adrenaline group, 5/6 lambs achieved ROSC after the first Td, whereas 1 lamb required two Tds before achieving ROSC. In the ET-Adrenaline group, 1/5 lambs required one Td, 1 lamb required three Tds, 2 lambs required 2 Rds, and 1 did not achieve ROSC. In the Nasal-Adrenaline group, 1/6 lambs required one Td, 2 required two Tds, whereas 3 lambs required either one (2 lambs) or two (1 lamb) Rds of adrenaline to achieve ROSC. In the IV-saline group, no lambs achieved ROSC until adrenaline Rds; 4/5 lambs required one Rd and 1 lamb required two Rds. Time to ROSC was shorter using IV-Adrenaline (2.4 ± 0.4 min) compared with ET-Adrenaline (10.3 ± 2.4 min), Nasal-Adrenaline (9.2 ± 2.2 min), and IV-saline (11.2 ± 1.2 min).Conclusion: IV adrenaline had superior efficacy compared with nasal or ET administration. Nasal administration had a similar effect as ET administration and is an easier route for early application. Nasal high-dose adrenaline administration for neonatal resuscitation merits further investigation.

Background: Preterm infants undergoing intensive care often experience painful procedures such as heel lance for blood sampling. Knowledge of the cerebral hemodynamic response to painful stimuli contributes to understanding of cortical pain processing and the neurovascular network in the preterm brain. Previous research has demonstrated cerebral hemodynamic responses using near-infrared spectroscopy (NIRS) after noxious stimuli in infants appropriately grown for age (AGA). But this has not been studied in infants born small for gestational age after fetal growth restriction (FGR). FGR infants differ in brain development due to utero-placental insufficiency leading to the intrauterine growth restriction, and cerebral response to pain may be altered.Objectives: We aimed to compare the cerebral hemodynamic response to painful stimuli (heel lance) in FGR and AGA infants.Methods: Preterm FGR infants (n = 20) and AGA infants (n = 15) born at 28–32 weeks' gestation were studied at mean ± SD postnatal age of 11.5 ± 2.4 and 10.5 ± 2.4 days, respectively. Infants had baseline echocardiographic assessment of ductus arteriosus and stroke volume. They were monitored with NIRS for changes in tissue oxygenation index (TOI, %), and oxygenated, deoxygenated, and total hemoglobin (ΔO2Hb, ΔHHb, and ΔTHb) in contralateral and ipsilateral cerebral hemispheres, during a heel lance.Results: At baseline, FGR infants had significantly lower TOI, higher heart rate, and lower stroke volume compared to AGA infants. Most infants in both groups showed increase in each of the NIRS parameters in the contralateral hemisphere following heel lance. However, more AGA infants (6/15) showed decreased ΔTHb compared to FGR infants (1/20) (p = 0.016). The magnitude of cerebral hemodynamic response and time to response did not differ between FGR and AGA infants. FGR infants showed larger ΔO2Hb in the contralateral compared to ipsilateral cortex (p = 0.05).Conclusion: Preterm FGR infants have reduced stroke volume and lower cerebral oxygenation compared to AGA infants in the second to third week of life. FGR infants show similar cerebral hemodynamic responses to noxious stimuli compared to AGA infants. However, FGR infants are less likely to have a cerebral vasoconstrictive response, possibly due to cerebrovascular changes following placental insufficiency and brain sparing in-utero.

Fetal growth restriction (FGR) is a common complication of pregnancy often associated with neurological impairments. Currently, there is no treatment for FGR, hence it is likely these babies will be delivered prematurely, thus being exposed to antenatal glucocorticoids. While there is no doubt that antenatal glucocorticoids reduce neonatal mortality and morbidities, their effects on the fetal brain, particularly in FGR babies, are less well recognized. We investigated the effects of both short- and long-term exposure to antenatal betamethasone treatment in both FGR and appropriately grown fetal sheep brains. Surgery was performed on pregnant Border-Leicester Merino crossbred ewes at 105–110 days gestation (term ~150 days) to induce FGR by single umbilical artery ligation (SUAL) or sham surgery. Ewes were then treated with a clinical dose of betamethasone (11.4 mg intramuscularly) or saline at 113 and 114 days gestation. Animals were euthanized at 115 days (48 h following the initial betamethasone administration) or 125 days (10 days following the initial dose of betamethasone) and fetal brains collected for analysis. FGR fetuses were significantly smaller than controls (115 days: 1.68 ± 0.11 kg vs. 1.99 ± 0.11 kg, 125 days: 2.70 ± 0.15 kg vs. 3.31 ± 0.20 kg, P < 0.001) and betamethasone treatment reduced body weight in both control (115 days: 1.64 ± 0.10 kg, 125 days: 2.53 ± 0.10 kg) and FGR fetuses (115 days: 1.41 ± 0.10 kg, 125 days: 2.16 ± 0.17 kg, P < 0.001). Brain: body weight ratios were significantly increased with FGR (P < 0.001) and betamethasone treatment (P = 0.002). Within the fetal brain, FGR reduced CNPase-positive myelin staining in the subcortical white matter (SCWM; P = 0.01) and corpus callosum (CC; P = 0.01), increased GFAP staining in the SCWM (P = 0.02) and reduced the number of Olig2 cells in the periventricular white matter (PVWM; P = 0.04). Betamethasone treatment significantly increased CNPase staining in the external capsule (EC; P = 0.02), reduced GFAP staining in the CC (P = 0.03) and increased Olig2 staining in the SCWM (P = 0.04). Here we show that FGR has progressive adverse effects on the fetal brain, particularly within the white matter. Betamethasone exacerbated growth restriction in the FGR offspring, but betamethasone did not worsen white matter brain injury.

Fetal growth restriction (FGR) and prematurity are often co-morbidities, and both are risk factors for lung disease. Despite advances in early delivery combined with supportive ventilation, rates of ventilation-induced lung injury (VILI) remain high. There are currently no protective treatments or interventions available that target lung morbidities associated with FGR preterm infants. Stem cell therapy, such as umbilical cord blood (UCB) cell administration, demonstrates an ability to attenuate inflammation and injury associated with VILI in preterm appropriately grown animals. However, no studies have looked at the effects of stem cell therapy in growth restricted newborns. We aimed to determine if UCB treatment could attenuate acute inflammation in the first 24 h of ventilation, comparing effects in lambs born preterm following FGR with those born preterm but appropriately grown (AG). Placental insufficiency (FGR) was induced by single umbilical artery ligation in twin-bearing ewes at 88 days gestation, with twins used as control (appropriately grown, AG). Lambs were delivered preterm at ~126 days gestation (term is 150 days) and randomized to either immediate euthanasia (unventilated controls, AGUVC and FGRUVC) or commenced on 24 h of gentle supportive ventilation (AGV and FGRV) with additional cohorts receiving UCB treatment at 1 h (AGCELLS, FGRCELLS). Lungs were collected at post-mortem for histological and biochemical examination. Ventilation caused lung injury in AG lambs, as indicated by decreased septal crests and elastin density, as well as increased inflammation. Lung injury in AG lambs was attenuated with UCB therapy. Ventilated FGR lambs also sustained lung injury, albeit with different indices compared to AG lambs; in FGR, ventilation reduced septal crest density, reduced alpha smooth muscle actin density and reduced cell proliferation. UCB treatment in ventilated FGR lambs further decreased septal crest density and increased collagen deposition, however, it increased angiogenesis as evidenced by increased vascular endothelial growth factor (VEGF) expression and vessel density. This is the first time that a cell therapy has been investigated in the lungs of growth restricted animals. We show that the uterine environment can alter the response to both secondary stress (ventilation) and therapy (UCB). This study highlights the need for further research on the potential impact of novel therapies on a growth restricted offspring.

BackgroundPreterm infants often have immature lungs and, consequently, many require respiratory support at birth. However, respiratory support causes lung inflammation and injury, termed ventilation-induced lung injury (VILI). Umbilical cord blood (UCB) contains five cell types that have been shown to reduce inflammation and injury. The aim of this study was to determine whether UCB cells can reduce VILI in preterm lambs.MethodsWe assessed lung inflammation and injury, with and without UCB cell administration. Fetal lambs at 125 ± 1 days gestation underwent sterile surgery and were randomly allocated to one of four groups; unoperated controls (UNOP), sham controls (SHAM), injuriously ventilated lambs (VILI), and injuriously ventilated lambs that received UCB cells via the jugular vein 1 h after ventilation (VILICELLS). Ventilated lambs received an injurious ventilation strategy for 15 min, before they were returned to the uterus and the lamb and ewe recovered for 24 h. After 24 h, lambs were delivered via caesarean section and euthanized and the lungs were collected for histological and molecular assessment of inflammation and injury.ResultsVILI led to increased immune cell infiltration, increased cellular proliferation, increased tissue wall thickness, and significantly reduced alveolar septation compared to controls. Further, extracellular matrix proteins collagen and elastin had abnormal deposition following VILI compared to control groups. Administration of UCB cells did not reduce any of these indices.ConclusionAdministration of UCB cells 1 h after ventilation onset did not reduce VILI in preterm lambs.

Placental transfusion has been thought to be the main benefit of delayed umbilical cord clamping (DCC) in preterm neonates. However, the importance of cardiovascular stability provided by allowing lung aeration prior to cord clamping has recently been highlighted. We aimed to determine the influence of blood volume changes on cardiovascular stability at birth. Preterm lambs (0.85 gestation) were instrumented for measurement of pulmonary, systemic and cerebral blood pressures and flows, systemic oxygen saturation and cerebral oxygenation. Left ventricular output (LVO) was assessed by Doppler Echocardiography. Lambs underwent immediate cord clamping followed by (1) 25 ml/kg infusion of whole blood over (90 s; or 2) withdrawal of 10 ml/kg blood over 90 s. Ventilation was initiated 30 s after volume change (2 min after cord clamping) and was maintained for 30 min. Blood infusion significantly increased pulmonary blood flow (PBF) which maintained systemic cardiac output during the infusion, and increased carotid arterial pressure, flow and heart rate, which remained elevated until after ventilation onset. Upon completion of transfusion PBF rapidly returned to control levels and LVO decreased. Conversely, blood withdrawal decreased PBF and LVO. The cardiovascular changes that accompanied ventilation onset were similar between groups. Providing a blood volume transfusion immediately after umbilical cord clamping maintains PBF and cardiac output during the transfusion, which does not persist beyond the period of the transfusion. Our study implies that an apneic newborn cannot maintain cardiac output through an increase blood volume alone. Importantly, delaying umbilical cord clamping until after breathing/aeration of the lung may be a way to maintain cardiac output throughout delivery at birth.

Background: Most preterm infants fail to aerate their immature lungs at birth and need respiratory support for cardiopulmonary stabilization. Cord clamping before lung aeration compromises cardiovascular function. Delaying cord clamping until the lung has aerated may be beneficial for preterm infants by optimizing hemodynamic transition and placental transfusion. A new purpose-built resuscitation table (the Concord) has been designed making it possible to keep the cord intact after preterm birth until the lung is aerated and the infant is respiratory stable and breathing [Physiological-Based Cord Clamping (PBCC)]. The aim of this study is to test the hypothesis whether stabilizing preterm infants by PBCC is at least as effective as the standard approach using time-based Delayed Cord Clamping (DCC).Study design: This is a randomized controlled non-inferiority study including 64 preterm infants born at

Being born small lays the foundation for short-term and long-term implications for life. Intrauterine or fetal growth restriction describes the pregnancy complication of pathological reduced fetal growth, leading to significant perinatal mortality and morbidity, and subsequent long-term deficits. Placental insufficiency is the principal cause of FGR, which in turn underlies a chronic undersupply of oxygen and nutrients to the fetus. The neonatal morbidities associated with FGR depend on the timing of onset of placental dysfunction and growth restriction, its severity, and the gestation at birth of the infant. In this review, we explore the pathophysiological mechanisms involved in the development of major neonatal morbidities in FGR, and their impact on the health of the infant. Fetal cardiovascular adaptation and altered organ development during gestation are principal contributors to postnatal consequences of FGR. Clinical presentation, diagnostic tools and management strategies of neonatal morbidities are presented. We also present information on the current status of targeted therapies. A better understanding of neonatal morbidities associated with FGR will enable early neonatal detection, monitoring and management of potential adverse outcomes in the newborn period and beyond.

Background: Fetal growth restriction (FGR) is a serious pregnancy complication associated with increased risk of adverse neurodevelopment and neuromorbidity. Current imaging techniques, including conventional magnetic resonance imaging (MRI), are not sensitive enough to detect subtle structural abnormalities in the FGR brain. We examined whether advanced MRI analysis techniques have the capacity to detect brain injury (particularly white matter injury) caused by chronic hypoxia-induced fetal growth restriction in newborn preterm lambs. Methods: Surgery was undertaken in twin bearing pregnant ewes at 88–90 days gestation (term = 150 days) to induce FGR in one fetus. At 127 days gestation (~32 weeks human brain development), FGR and control (appropriate for gestational age, AGA) lambs were delivered by caesarean section, intubated and ventilated. Conventional and advanced brain imaging was conducted within the first two hours of life using a 3T MRI scanner. T1-weighted (T1w) and T2-weighted (T2w) structural imaging, magnetic resonance spectroscopy (MRS), and diffusion MRI (dMRI) data were acquired. Diffusion tensor imaging (DTI) modelling and analysis of dMRI data included the following regions of interest (ROIs): subcortical white matter, periventricular white matter, cerebellum, hippocampus, corpus callosum and thalamus. Fixel-based analysis of 3-tissue constrained spherical deconvolution (CSD) of the dMRI data was performed and compared between FGR and AGA lambs. Lambs were euthanised immediately after the scans and brain histology performed in the regions of interest to correlate with imaging. Results: FGR and AGA lamb (body weight, mean (SD): 2.2(0.5) vs. 3.3(0.3) kg, p = .002) MRI brain scans were analysed. There were no statistically significant differences observed between the groups in conventional T1w, T2w or MRS brain data. Mean, axial and radial diffusivity, and fractional anisotropy indices obtained from DTI modelling also did not show any statistically significant differences between groups in the ROIs. Fixel-based analysis of 3-tissue CSD, however, did reveal a decrease in fibre cross-section (FC, p

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.

Cerebrovascular injury is one of the major detrimental consequences of preterm birth. Recent studies have focused their attention on factors that contribute to the development of brain lesions immediately after birth. Among those factors, hypothermia and lower cerebral oxygen saturation during delivery room resuscitation and high tidal volumes delivered during respiratory support are associated with increased risk of severe neurologic injury. In preterm infants, knowledge about causes and prevention of brain injury must be applied before and at birth. Preventive and therapeutic approaches, including correct timing of cord clamping, monitoring of physiological changes during delivery room resuscitation using pulse oximetry, respiratory function monitoring, near infrared spectroscopy, and alpha EEG, may minimize brain injury, Furthermore, postnatal administration of caffeine or other potential novel treatments (e.g., proangiogenic therapies, antioxidants, hormones, or stem cells) might improve long-term neurodevelopmental outcomes in preterm infants.

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.

Preterm birth is a major cause for neonatal morbidity and mortality, and is frequently associated with adverse neurological outcomes. The transition from intrauterine to extrauterine life at birth is particularly challenging for preterm infants. The main physiological driver for extrauterine transition is the establishment of spontaneous breathing. However, preterm infants have difficulty clearing lung liquid, have insufficient surfactant levels, and underdeveloped lungs. Further, preterm infants have an underdeveloped brainstem, resulting in reduced respiratory drive. These factors facilitate the increased requirement for respiratory support. A principal cause of preterm birth is intrauterine infection/inflammation (chorioamnionitis), and infants with chorioamnionitis have an increased risk and severity of neurological damage, but also demonstrate impaired autoresuscitation capacity and prevalent apnoeic episodes. The brainstem contains vital respiratory centers which provide the neural drive for breathing, but the impact of preterm birth and/or chorioamnionitis on this brain region is not well understood. The aim of this review is to provide an overview of the role and function of the brainstem respiratory centers, and to highlight the proposed mechanisms of how preterm birth and chorioamnionitis may affect central respiratory functions.

BackgroundFor 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 methodsLambs 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).ResultsThere 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

Preterm birth is a major cause of perinatal mortality and long-term morbidity. Chorioamnionitis is a common cause of preterm birth. Clinical chorioamnionitis, characterised by maternal fever, leukocytosis, tachycardia, uterine tenderness, and preterm rupture of membranes, is less common than subclinical/histologic chorioamnionitis, which is asymptomatic and defined by inflammation of the chorion, amnion, and placenta. Chorioamnionitis is often associated with a fetal inflammatory response. The fetal inflammatory response syndrome (FIRS) is defined by increased systemic inflammatory cytokine concentrations, funisitis, and fetal vasculitis. Clinical and epidemiological studies have demonstrated that FIRS leads to poor cardiorespiratory, neurological, and renal outcomes. These observations are further supported by experimental studies that have improved our understanding of the mechanisms responsible for these outcomes. This paper outlines clinical and experimental studies that have improved our current understanding of the mechanisms responsible for chorioamnionitis-induced preterm birth and explores the cellular and physiological mechanisms underlying poor cardiorespiratory, neural, retinal, and renal outcomes observed in preterm infants exposed to chorioamnionitis.