Amy E. Sutherland

Maternal melatonin administration mitigates coronary stiffness and endothelial dysfunction, and improves heart resilience to insult in growth restricted lambs

Failure of the placenta to develop and perform gives rise to intrauterine growth restriction (IUGR) in the fetus. IUGR is associated with impaired heart function in childhood and can even persist long term. Oxidative stress is increased in IUGR and we asked if an antioxidant could reduce this. Melatonin is a well‐known and well‐studied hormone, present in all of us, and it has potent antioxidant properties. In this study we administered melatonin to pregnant ewes carrying twins, one with IUGR. Maternal melatonin improved oxygen delivery to the IUGR fetus and strengthened and protected its heart against infarct. After birth, the poor function and stiffness in the coronary arteries of IUGR lambs were entirely prevented by melatonin. Our results demonstrate that administration of melatonin to a mother carrying an IUGR fetus can markedly dampen the adverse heart and artery effects in the offspring following birth.

Detecting brain injury in neonatal hypoxic ischemic encephalopathy: Closing the gap between experimental and clinical research

Moderate to severe neonatal hypoxic ischemic encephalopathy remains an important cause of infant death and childhood disability. Early and accurate diagnosis of encephalopathy is difficult but critical for timely intervention. Thus, we have utilized a clinically relevant large animal model of asphyxia in-utero, followed by immediate lamb delivery, resuscitation and clinical care over the next 72h for assessment of potential biomarkers of brain injury. In-utero asphyxia was induced in twelve near-term lambs and outcomes compared with seven controls. Asphyxia resulted in bradycardia (97±12beats/min), hypotension (12.1±1mm Hg) and metabolic acidosis (pH6.9±0.02; base-excess -13.8±0.8mmol/l). 72h following asphyxia, cerebrospinal concentrations of malondialdehyde and S100B were elevated 2-fold and 5-fold, respectively, in asphyxic lambs compared to control lambs. Magnetic resonance spectroscopy (MRS) at 72h showed a significant decrease in n-acetyl aspartate: choline ratio in asphyxia lambs compared to that observed at 12h (0.56±0.23 vs. 0.82±0.15, respectively); lactate:choline ratio was not changed over this time. Marked neuropathology was observed in asphyxia lambs with neuronal degeneration in the hippocampus, thalamus, striatum and cortex. Astrogliosis was observed in the hippocampus and thalamus. Early blood markers of metabolic state showed limited predictive value of histological damage at 72h. MRS outcomes at 72h showed a modest but significant correlation with histological evidence of neuronal brain injury (lactate:N-acetyl aspartate ratio in the thalamus r(2)=0.2, p<0.01). MRS at 72h was best able to detect established brain injury, but a combination of biomarkers over multiple phases of injury may be able to assess the evolution of neonatal brain injury.

Antenatal glucocorticoids reduce growth in appropriately grown and growth-restricted ovine fetuses in a sex-specific manner

Antenatal glucocorticoids are administered to mature the fetal lungs before preterm birth. Glucocorticoids also have non-pulmonary effects, including reducing fetal body and brain growth. The present study examined whether glucocorticoid administration has a sex-specific effect on growth in appropriately grown (control) and intrauterine growth-restricted (IUGR) fetal sheep. IUGR was induced at 0.7 gestation in fetal sheep by single umbilical artery ligation. On Days 5 and 6 after surgery, IUGR or control fetuses were exposed to the synthetic glucocorticoid betamethasone (BM; 11.4mg) or saline via intramuscular maternal administration. On Day 7, a postmortem was conducted to determine fetal sex and weight. Compared with control fetuses, the birthweight of male and female IUGR fetuses was significantly reduced (by 18.5±4.4% (P=0.002) and 21.7±6.0% (P=0.001), respectively). Maternal administration of BM significantly reduced bodyweight in both control and IUGR fetuses (by 11.3±2.8% and 20.5±3.6% in control male and female fetuses, respectively; and by 22.9±3.1% and 38.3±3.4% in IUGR male and female fetuses, respectively; P<0.001 for all, versus control+saline) fetuses. In control and IUGR animals the degree of growth restriction was greater in females than males (P<0.05) following administration of BM. These data suggest that antenatal glucocorticoids reduce fetal growth in a sex-specific manner, with females more growth restricted than males.

Preterm white matter brain injury is prevented by early administration of umbilical cord blood cells

Infants born very preterm are at high risk for neurological deficits including cerebral palsy. In this study we assessed the neuroprotective effects of umbilical cord blood cells (UCBCs) and optimal administration timing in a fetal sheep model of preterm brain injury. 50 million allogeneic UCBCs were intravenously administered to fetal sheep (0.7 gestation) at 12h or 5d after acute hypoxia-ischemia (HI) induced by umbilical cord occlusion. The fetal brains were collected at 10d after HI. HI (n=7) was associated with reduced number of oligodendrocytes (Olig2+) and myelin density (CNPase+), and increased density of activated microglia (Iba-1+) in cerebral white matter compared to control fetuses (P<0.05). UCBCs administered at 12h, but not 5d after HI, significantly protected white matter structures and suppressed cerebral inflammation. Activated microglial density showed a correlation with decreasing oligodendrocyte number (P<0.001). HI caused cell death (TUNEL+) in the internal capsule and cell proliferation (Ki-67+) in the subventricular zone compared to control (P<0.05), while UCBCs at 12h or 5d ameliorated these effects. Additionally, UCBCs at 12h induced a significant systemic increase in interleukin-10 at 10d, and reduced oxidative stress (malondialdehyde) following HI (P<0.05). UCBC administration at 12h after HI reduces preterm white matter injury, via anti-inflammatory and antioxidant actions.

Cord blood mononuclear cells prevent neuronal apoptosis in response to perinatal asphyxia in the newborn lamb

Asphyxia at the time of birth is a significant cause of death or disability in newborns. There is very limited treatment available for these newborns. Autologous umbilical cord blood (UBC) mononuclear cells reduce clinical markers of brain damage following perinatal asphyxia. Autologous UBC mononuclear cells reduce neuroinflammation and neuronal apoptosis within the brain following perinatal asphyxia. Autologous UBC mononuclear cells administered 12 h after perinatal asphyxia are neuroprotective, and a well‐tolerated and feasible treatment for infants following hypoxic ischaemic encephalopathy.

The effects of betamethasone on allopregnanolone concentrations and brain development in preterm fetal sheep

The risk of preterm delivery often means that the fetus will be exposed to exogenous synthetic glucocorticoids to accelerate fetal lung maturation, but effects on other organs, particularly the brain, are not understood. The neurosteroid allopregnanolone (AP) is a GABAA receptor agonist that influences fetal brain development and has neuroprotective properties. In this study we determined the impact of maternal glucocorticoid (betamethasone) administration on brain development and AP synthesis in preterm fetal sheep. Pregnant ewes underwent surgery at 105 days gestation for implantation of fetal catheters. Ewes received either betamethasone (BM; 11.4 mg; n=10) or vehicle (saline; n=5) by i.m injection on days five (BM1) and six (BM2) following surgery. Five fetuses of the BM treated ewes received an infusion of alfaxalone (20 mg) over 48 h commencing 30 min prior to BM1. All animals were euthanased on day 7, and the fetal brains collected to determine AP concentrations and histopathology. BM significantly reduced AP levels in the fetal brain and placental cotyledons, and also in fetal plasma without altering progesterone concentrations. There was a significant decrease in the number of myelinating cells in subcortical white matter, but no change to total oligodendrocyte number. Co-administration of the AP analogue analog alfaxalone with BM prevented this change in MBP expression. BM, given at a dose clinically prescribed to accelerate lung maturation, adversely affects neurosteroid levels in the preterm fetal brain, and affects the maturational profile of white matter development; these effects were mitigated by the co-administration of alfaxolone.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses

Please cite this paper as: Tare M, Miller S, Wallace E, Sutherland A, Yawno T, Coleman H, Jenkin G, Parkington H. Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses. BJOG 2012;119:906–914.

Effects of Antenatal Melatonin Treatment on the Cerebral Vasculature in an Ovine Model of Fetal Growth Restriction

Chronic moderate hypoxia, such as occurs in fetal growth restriction (FGR) during gestation, compromises the blood-brain barrier (BBB) and results in structural abnormalities of the cerebral vasculature. We have previously determined the neuroprotective and antioxidant effects of maternal administration of melatonin (MLT) on growth-restricted newborn lambs. The potential of maternal MLT therapy for the treatment of cerebrovascular dysfunction-associated developmental hypoxia has also been demonstrated in newborn lambs. We assessed whether MLT had an effect on the previously reported structural and cerebral vascular abnormalities in chronically hypoxic FGR lambs. Single umbilical-artery ligation surgery was performed in fetuses at approximately 105 days of gestation (term: 147 days) to induce placental insufficiency and FGR, and treatment with either saline or an MLT infusion (0.1 mg/kg) was started 4 h after surgery. Ewes delivered naturally at term and lambs were euthanased 24 h later. We found a significant reduction in the number of laminin-positive blood vessels within the subcortical and periventricular white matter (SCWM and PVWM) and the subventricular zone (SVZ) in FGR (p < 0.0005) and FGR + MLT brains (p < 0.0005 vs. controls), with no difference found between FGR and FGR + MLT animals. This was associated with a significant decrease in VEGF immunoreactivity in FGR and FGR + MLT brains versus controls (p < 0.0005; SCWM and PVWM) and in the SVZ in FGR brains versus controls (p < 0.005) and also with significantly lower levels of proliferating blood vessels versus controls (p < 0.0005). Glucose transporter-1 immunoreactivity (vascular endothelium) was decreased in FGR versus control lambs (p < 0.0005) in SCWM, PVWM, and the SVZ; it was significantly increased in FGR + MLT lambs compared with FGR lambs in SCWM and PVWM (p < 0.005) and even more markedly in the SVZ (p < 0.0005). FGR brains showed a 72% reduction in pericyte coverage versus control lambs and 68% versus FGR + MLT in PVWM. In SCWM, we found a 77 and 73% reduction compared with control and FGR + MLT lambs, respectively, while in the SVZ, we observed a 68% reduction versus controls and a 70% reduction in FGR versus FGR + MLT lambs. Astrocyte end-feet coverage in the SCWM showed a significant 24% reduction in FGR versus control levels, a 42% decrease within the PVWM, and a 35% decrease within the SVZ versus controls. MLT normalized astrocyte attachment to blood vessels, with no difference seen between controls and FGR + MLT animals in any of the brain regions examined. We also observed a decrease in albumin extravasation and microhemorrhage in controls and FGR + MLT brains versus FGR lambs. Our results demonstrate that umbilicoplacental insufficiency is associated with FGR-produced vascular changes in the white matter and SVZ of FGR newborn brains and that maternal MLT prevented disruption of the BBB by protecting perivascular cells essential for the maintenance of vascular homeostasis and stability.

Systemic and transdermal melatonin administration prevents neuropathology in response to perinatal asphyxia in newborn lambs

Perinatal asphyxia remains a principal cause of infant mortality and long‐term neurological morbidity, particularly in low‐resource countries. No neuroprotective interventions are currently available. Melatonin (MLT), a potent antioxidant, anti‐inflammatory and antiapoptotic agent, offers promise as an intravenous (IV) or transdermal therapy to protect the brain. We aimed to determine the effect of melatonin (IV or transdermal patch) on neuropathology in a lamb model of perinatal asphyxia. Asphyxia was induced in newborn lambs via umbilical cord occlusion at birth. Animals were randomly allocated to melatonin commencing 30 minutes after birth (60 mg in 24 hours; IV or transdermal patch). Brain magnetic resonance spectroscopy (MRS) was undertaken at 12 and 72 hours. Animals (control n = 9; control+MLT n = 6; asphyxia n = 16; asphyxia+MLT [IV n = 14; patch n = 4]) were euthanised at 72 hours, and cerebrospinal fluid (CSF) and brains were collected for analysis. Asphyxia resulted in severe acidosis (pH 6.9 ± 0.0; lactate 9 ± 2 mmol/L) and altered determinants of encephalopathy. MRS lactate:N‐acetyl aspartate ratio was 2.5‐fold higher in asphyxia lambs compared with controls at 12 hours and 3‐fold higher at 72 hours (P < .05). Melatonin prevented this rise (3.5‐fold reduced vs asphyxia; P = .02). Asphyxia significantly increased brain white and grey matter apoptotic cell death (activated caspase‐3), lipid peroxidation (4HNE) and neuroinflammation (IBA‐1). These changes were significantly mitigated by both IV and patch melatonin. Systemic or transdermal neonatal melatonin administration significantly reduces the neuropathology and encephalopathy signs associated with perinatal asphyxia. A simple melatonin patch, administered soon after birth, may improve outcome in infants affected by asphyxia, especially in low‐resource settings.

Term vs. preterm cord blood cells for the prevention of preterm brain injury

BACKGROUNDWhite matter brain injury in preterm infants can induce neurodevelopmental deficits. Umbilical cord blood (UCB) cells demonstrate neuroprotective properties, but it is unknown whether cells obtained from preterm cord blood (PCB) vs. term cord blood (TCB) have similar efficacy. This study compared the ability of TCB vs. PCB cells to reduce white matter injury in preterm fetal sheep.METHODSHypoxia–ischemia (HI) was induced in fetal sheep (0.7 gestation) by 25 min umbilical cord occlusion. Allogeneic UCB cells from term or preterm sheep, or saline, were administered to the fetus at 12 h after HI. The fetal brain was collected at 10-day post HI for assessment of white matter neuropathology.RESULTSHI (n=7) induced cell death and microglial activation and reduced total oligodendrocytes and CNPase+myelin protein in the periventricular white matter and internal capsule when compared with control (n=10). Administration of TCB or PCB cells normalized white matter density and reduced cell death and microgliosis (P<0.05). PCB prevented upregulation of plasma tumor necrosis factor (TNF)-a, whereas TCB increased anti-inflammatory interleukin (IL)-10 (P<0.05). TCB, but not PCB, reduced circulating oxidative stress.CONCLUSIONSTCB and PCB cells reduced preterm HI-induced white matter injury, primarily via anti-inflammatory actions. The secondary mechanisms of neuroprotection appear different following TCB vs. PCB administration.