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.

Endotracheal epinephrine at standard versus high dose for resuscitation of asystolic newborn lambs.

INTRODUCTION: Endotracheal (ET) epinephrine administration is an option during neonatal resuscitation, if the preferred intravenous (IV) route is unavailable. OBJECTIVES: We assessed whether endotracheal epinephrine achieved return of spontaneous circulation (ROSC), and maintained physiological stability after ROSC, at standard and higher dose, in severely asphyxiated newborn lambs. METHODS: Near-term fetal lambs were asphyxiated until asystole. Resuscitation was commenced with ventilation and chest compressions. Lambs were randomly allocated to: IV Saline placebo (5 ml/kg), IV Epinephrine (20 micrograms/kg), Standard-dose ET Epinephrine (100 micrograms/kg), and High-dose ET Epinephrine (1 mg/kg). After three allocated treatment doses, rescue IV Epinephrine was administered if ROSC had not occurred. Lambs achieving ROSC were monitored for 60 minutes. Brain histology was assessed for microbleeds. RESULTS: ROSC in response to allocated treatment (without rescue IV Epinephrine) occurred in 1/6 Saline, 9/9 IV Epinephrine, 0/9 Standard-dose ET Epinephrine, and 7/9 High-dose ET Epinephrine lambs respectively. Blood pressure during CPR increased after treatment with IV Epinephrine and High-dose ET Epinephrine, but not Saline or Standard-dose ET Epinephrine. After ROSC, both ET Epinephrine groups had lower pH, higher lactate, and higher blood pressure than the IV Epinephrine group. Cortex microbleeds were more frequent in High-dose ET Epinephrine lambs (8/8 lambs examined, versus 3/8 in IV Epinephrine lambs). CONCLUSIONS: The currently recommended dose of ET Epinephrine was ineffective in achieving ROSC. Without convincing clinical or preclinical evidence of efficacy, use of ET Epinephrine at this dose may not be appropriate. High-dose ET Epinephrine requires further evaluation before clinical translation.

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.

A systematic review of immune-based interventions for perinatal neuroprotection: closing the gap between animal studies and human trials.

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.

The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. METHODS: Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5; 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7; 30x106 cells) or equivalent volumes of saline (LPS/Sal, n = 10; or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. RESULTS: Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. CONCLUSIONS: Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation.

Hyperpolarised gas filling station for medical imaging using polarised 129Xe and 3He.

We report the design, construction, and initial tests of a hyperpolariser to produce polarised 129Xe and 3He gas for medical imaging of the lung. The hyperpolariser uses the Spin-Exchange Optical Pumping method to polarise the nuclear spins of the isotopic gas. Batch mode operation was chosen for the design to produce polarised 129Xe and polarised 3He. Two-side pumping, electrical heating and a piston to transfer the polarised gas were some of the implemented techniques that are not commonly used in hyperpolariser designs. We have carried out magnetic resonance imaging experiments demonstrating that the 3He and 129Xe polarisation reached were sufficient for imaging, in particular for in vivo lung imaging using 129Xe. Further improvements to the hyperpolariser have also been discussed.

Maternal sildenafil impairs the cardiovascular adaptations to chronic hypoxaemia in fetal sheep.

KEY POINTS: Fetal growth restriction induces a haemodynamic response that aims to maintain blood flow to vital organs such as the brain, in the face of chronic hypoxaemia Maternal sildenafil treatment impairs the hypoxaemia-driven haemodynamic response and potentially compromises fetal development. ABSTRACT: Inadequate substrate delivery to a fetus results in hypoxaemia and fetal growth restriction (FGR). In response, fetal cardiovascular adaptations redirect cardiac output to essential organs to maintain oxygen delivery and sustain development. However, FGR infants remain at risk for cardiovascular and neurological sequelae. Sildenafil citrate (SC) has been examined as a clinical therapy for FGR, but also crosses the placenta and may exert direct effects on the fetus. We investigated the effects of maternal SC administration on maternal and fetal cardiovascular physiology in growth-restricted fetal sheep. Fetal sheep (0.7 gestation) underwent sterile surgery to induce growth restriction by single umbilical artery ligation (SUAL) or sham surgery (control, AG). Fetal catheters and flow probes were implanted to measure carotid and femoral arterial blood flows. Ewes containing SUAL fetuses were randomized to receive either maternal administration of saline or SC (36 mg i.v. per day) beginning 4 days after surgery, and continuing for 20 days. Physiological recordings were obtained throughout the study. Antenatal SC treatment reduced body weight by 32% and oxygenation by 18% in SUAL compared to AG. SC did not alter maternal or fetal heart rate or blood pressure. Femoral blood flow and peripheral oxygen delivery were increased by 49% and 30% respectively in SUALSC compared to SUAL, indicating impaired cardiovascular adaptation to chronic hypoxaemia. Antenatal SC directly impairs the fetal haemodynamic response to chronic hypoxaemia. Consideration of the consequences upon the fetus should be paramount when administering interventions to the mother during pregnancy.

Does Antenatal Betamethasone Alter White Matter Brain Development in Growth Restricted Fetal Sheep?

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.

Umbilical Cord Blood Cells Do Not Reduce Ventilation-Induced Lung Injury in Preterm Lambs.

BACKGROUND: Preterm 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. METHODS: We 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. RESULTS: VILI 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. CONCLUSION: Administration of UCB cells 1 h after ventilation onset did not reduce VILI in preterm lambs.