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.

Neuroprotective effects of maternal melatonin administration in early-onset placental insufficiency and fetal growth restriction.

BACKGROUND: Early-onset fetal growth restriction (FGR) is associated with adverse outcomes. We hypothesised that maternal melatonin administration will improve fetal brain structure in FGR. METHODS: Surgery was performed on twin-bearing ewes at 88 days (0.6 gestation), and FGR induced in one twin via single umbilical artery ligation. Melatonin was administered intravenously (6 mg/day) to a group of ewes commencing on day of surgery until 127 days (0.85 gestation), when the ewe/fetuses were euthanized, and fetal brains collected. RESULTS: Study groups were control (n = 5), FGR (n = 5), control+melatonin (control+MLT; n = 6) and FGR+melatonin (FGR + MLT; n = 6). Melatonin administration did not significantly alter fetal body or brain weights. Myelin (CNPase+) fibre density was reduced in FGR vs. control animals in most brain regions examined (p < 0.05) and melatonin treatment restored CNPase fibre density. Similar but less pronounced effect was seen with mature myelin (MBP+) staining. Significant differences in activated microglia (Iba-1) activity were seen between lamb groups (MLT mitigated FGR effect) in periventricular white matter, subventricular zone and external capsule (p < 0.05). Similar effects were seen in astrogliosis (GFAP) in intragyral white matter and cortex. CONCLUSIONS: Maternal melatonin administration in early onset FGR led to improved myelination of white matter brain regions, possibly mediated by decreased inflammation. IMPACT: Maternal melatonin administration might lead to neuroprotection in the growth-restricted fetus, possibly via dampening neuroinflammation and enhancing myelination. This preclinical study adds to the body of work on this topic, and informs clinical translation. Neuroprotection likely to improve long-term outcomes of this vulnerable infant group.

Ganaxolone versus Phenobarbital for Neonatal Seizure Management.

OBJECTIVE: Seizures are more common in the neonatal period than at any other stage of life. Phenobarbital is the first-line treatment for neonatal seizures and is at best effective in approximately 50% of babies, but may contribute to neuronal injury. Here, we assessed the efficacy of phenobarbital versus the synthetic neurosteroid, ganaxolone, to moderate seizure activity and neuropathology in neonatal lambs exposed to perinatal asphyxia. METHODS: Asphyxia was induced via umbilical cord occlusion in term lambs at birth. Lambs were treated with ganaxolone (5mg/kg/bolus then 5mg/kg/day for 2 days) or phenobarbital (20mg/kg/bolus then 5mg/kg/day for 2 days) at 6 hours. Abnormal brain activity was classified as stereotypic evolving (SE) seizures, epileptiform discharges (EDs), and epileptiform transients (ETs) using continuous amplitude-integrated electroencephalographic recordings. At 48 hours, lambs were euthanized for brain pathology. RESULTS: Asphyxia caused abnormal brain activity, including SE seizures that peaked at 18 to 20 hours, EDs, and ETs, and induced neuronal degeneration and neuroinflammation. Ganaxolone treatment was associated with an 86.4% reduction in the number of seizures compared to the asphyxia group. The total seizure duration in the asphyxia+ganaxolone group was less than the untreated asphyxia group. There was no difference in the number of SE seizures between the asphyxia and asphyxia+phenobarbital groups or duration of SE seizures. Ganaxolone treatment, but not phenobarbital, reduced neuronal degeneration within hippocampal CA1 and CA3 regions, and cortical neurons, and ganaxolone reduced neuroinflammation within the thalamus. INTERPRETATION: Ganaxolone provided better seizure control than phenobarbital in this perinatal asphyxia model and was neuroprotective for the newborn brain, affording a new therapeutic opportunity for treatment of neonatal seizures. ANN NEUROL 2022;92:1066-1079.

Feasibility of cord blood collection for autologous cell therapy applications in extremely preterm infants.

BACKGROUND AIMS: Umbilical cord blood (UCB)-derived cells show strong promise as a treatment for neonatal brain injury in pre-clinical models and early-phase clinical trials. Feasibility of UCB collection and autologous administration is reported for term infants, but data are limited for preterm infants. Here the authors assessed the feasibility of UCB-derived cell collection for autologous use in extremely preterm infants born at less than 28 weeks, a population with a high incidence of brain injury and subsequent neurodisability. METHODS: In a prospective study at a tertiary hospital in Melbourne, Australia, UCB was collected from infants born at less than 28 weeks and processed to obtain total nucleated cells (TNCs), CD34+ cells, mononuclear cells and cell viability via fluorescence-activated cell sorting prior to cryopreservation. Feasibility was pre-defined as volume adequate for cryopreservation (>9 mL UCB collected) and >25 × 106 TNCs/kg retrieved. RESULTS: Thirty-eight infants (21 male, 17 female) were included in the study. Twenty-four (63.1%) were delivered via cesarean section, 30 (78.9%) received delayed cord clamping before collection and 11 (28.9%) were a multiple birth. Median (interquartile range [IQR]) gestational age was 26.0 weeks (24.5-27.5) and mean (standard deviation) birth weight was 761.5 g (221.5). Median (IQR) UCB volume collected was 19.1 mL/kg (10.5-23.5), median (IQR) TNC count was 105.2 × 106/kg (57.4-174.4), median (IQR) CD34+ cell count was 1.5 × 106/kg (0.6-2.1) and median (IQR) cell viability pre-cryopreservation was 95% (92.1-96.0). Feasibility of collection volume and cell count suitable for cell cryopreservation was achieved in 27 (71%) and 28 (73.6%) infants, respectively. CONCLUSIONS: UCB-derived cell collection adequate for cryopreservation and subsequent autologous reinfusion was achieved in 70% of extremely preterm infants. Extremely preterm UCB demonstrated a higher CD34+:TNC ratio compared with published full-term values. Recruitment to demonstrate safety of UCB cell administration in extremely premature infants is ongoing in the CORD-SAFE study (trial registration no. ACTRN12619001637134).

Advances in neonatal cell therapies: Proceedings of the First Neonatal Cell Therapies Symposium (2022).

Despite considerable advances, there is a need to improve the outcomes of newborn infants, especially related to prematurity, encephalopathy and other conditions. In principle, cell therapies have the potential to protect, repair, or sometimes regenerate vital tissues; and improve or sustain organ function. In this review, we present highlights from the First Neonatal Cell Therapies Symposium (2022). Cells tested in preclinical and clinical studies include mesenchymal stromal cells from various sources, umbilical cord blood and cord tissue derived cells, and placental tissue and membrane derived cells. Overall, most preclinical studies suggest potential for benefit, but many of the cells tested were not adequately defined, and the optimal cell type, timing, frequency, cell dose or the most effective protocols for the targeted conditions is not known. There is as yet no clinical evidence for benefit, but several early phase clinical trials are now assessing safety in newborn babies. We discuss parental perspectives on their involvement in these trials, and lessons learnt from previous translational work of promising neonatal therapies. Finally, we make a call to the many research groups around the world working in this exciting yet complex field, to work together to make substantial and timely progress to address the knowledge gaps and move the field forward. IMPACT: Survival of preterm and sick newborn infants is improving, but they continue to be at high risk of many systemic and organ-specific complications. Cell therapies show promising results in preclinical models of various neonatal conditions and early phase clinical trials have been completed or underway. Progress on the potential utility of cell therapies for neonatal conditions, parental perspectives and translational aspects are discussed in this paper.

Brief hypoxia in late gestation sheep causes prolonged disruption of fetal electrographic, breathing behaviours and can result in early labour.

KEY POINTS: Brief episodes of severe fetal hypoxia can arise in late gestation as a result of interruption of normal umbilical blood flow Systemic parameters and blood chemistry indicate complete recovery within 1-2 hours, although the long-term effects on fetal brain functions are unknown Fetal sheep were subjected to umbilical cord occlusion (UCO) for 10 min at 131 days of gestation, and then monitored intensively until onset of labour or delivery (<145 days of gestation) Normal patterns of fetal behaviour, including breathing movements, episodes of high and low voltage electorcortical activity, eye movements and postural (neck) muscle activity, were disrupted for 3-10 days after the UCO Preterm labour and delivery occurred in a significant number of the pregnancies after UCO compared to the control (sham-UCO) cohort. ABSTRACT: Complications arising from antepartum events such as impaired umbilical blood flow can cause significant fetal hypoxia. These complications can be unpredictable, as well as difficult to detect, and thus we lack a detailed understanding of the (patho)physiological changes that occur between the antenatal in utero event and birth. In the present study, we assessed the consequences of brief (∼10 min) umbilical cord occlusion (UCO) in fetal sheep at ∼0.88 gestation on fetal plasma cortisol concentrations and fetal behaviour [electrocortical (EcoG), electo-oculargram (EOG), nuchal muscle electromyography (EMG) and breathing activities] in the days following UCO. UCO caused a rapid onset of fetal hypoxaemia, hypercapnia, and acidosis; however, by 6 h, all blood parameters and cardiovascular status were normalized and not different from the control (Sham-UCO) cohort. Subsequently, the incidence of fetal breathing movements decreased compared to the control group, and abnormal behavioural patterns developed over the days following UCO and leading up to the onset of labour, which included increased high voltage and sub-low voltage ECoG and EOG activities, as well as decreased nuchal EMG activity. Fetuses subjected to UCO went into labour 7.9 ± 3.6 days post-UCO (139.5 ± 3.2 days of gestation) compared to the control group fetuses at 13.6 ± 3.3 days post-sham UCO (144 ± 2.2 days of gestation; P < 0.05), despite comparable increases in fetal plasma cortisol and a similar body weight at birth. Thus, a single transient episode of complete UCO late in gestation in fetal sheep can result in prolonged effects on fetal brain activity and premature labour, suggesting persisting effects on fetal cerebral metabolism.

Melatonin augments the neuroprotective effects of hypothermia in lambs following perinatal asphyxia.

Therapeutic hypothermia (TH) is standard care in high-resource birth settings for infants with neonatal encephalopathy. TH is partially effective and adjuvant therapies are needed. Here, we examined whether the antioxidant melatonin (MLT) provides additive benefit with TH, compared to TH alone or MLT alone, to improve recovery from acute encephalopathy in newborn lambs. Immediately before cesarean section delivery, we induced asphyxia in fetal sheep via umbilical cord occlusion until mean arterial blood pressure fell from 55 ± 3 mm Hg in sham controls to 18-20 mm Hg (10.1 ± 1.5 minutes). Lambs were delivered and randomized to control, control + MLT (60 mg iv, from 30 minutes to 24 hours), asphyxia, asphyxia + TH (whole-body cooling to 35.1 ± 0.8°C vs. 38.3 ± 0.17°C in sham controls, from 4-28 hours), asphyxia + MLT, and asphyxia + TH + MLT. At 72 hours, magnetic resonance spectroscopy (MRS) was undertaken, and then brains were collected for neuropathology assessment. Asphyxia induced abnormal brain metabolism on MRS with increased Lactate:NAA (P = .003) and reduced NAA:Choline (P = .005), induced apoptotic and necrotic cell death across gray and white matter brain regions (P < .05), and increased neuroinflammation and oxidative stress (P < .05). TH and MLT were independently associated with region-specific reductions in oxidative stress, inflammation, and cell death, compared to asphyxia alone. There was an interaction between TH and MLT such that the NAA:Choline ratio was not significantly different after asphyxia + TH + MLT compared to sham controls but had a greater overall reduction in neuropathology than either treatment alone. This study demonstrates that, in newborn lambs, combined TH + MLT for neonatal encephalopathy provides significantly greater neuroprotection than either alone. These results will guide the development of further trials for neonatal encephalopathy.

Effects of umbilical cord blood cells, and subtypes, to reduce neuroinflammation following perinatal hypoxic-ischemic brain injury.

BACKGROUND: It is well understood that hypoxic-ischemic (HI) brain injury during the highly vulnerable perinatal period can lead to cerebral palsy, the most prevalent cause of chronic disability in children. Recently, human clinical trials have reported safety and some efficacy following treatment of cerebral palsy using umbilical cord blood (UCB) cells. UCB is made up of many different cell types, including endothelial progenitor cells (EPCs), T regulatory cells (Tregs), and monocyte-derived suppressor cells (MDSCs). How each cell type contributes individually towards reducing neuroinflammation and/or repairing brain injury is not known. In this study, we examined whether human (h) UCB, or specific UCB cell types, could reduce peripheral and cerebral inflammation, and promote brain repair, when given early after perinatal HI brain injury. METHODS: HI brain injury was induced in postnatal day (PND) 7 rat pups and cells were administered intraperitoneally on PND 8. Behavioral testing was performed 7 days post injury, and then, brains and spleens were collected for analysis. RESULTS: We found in vitro that all UCB cell types, except for EPCs, were immunomodulatory. Perinatal HI brain injury induced significant infiltration of CD4+ T cells into the injured cerebral hemisphere, and this was significantly reduced by all hUCB cell types tested. Compared to HI, UCB, Tregs, and EPCs were able to reduce motor deficits, reduce CD4+ T cell infiltration into the brain, and reduce microglial activation. In addition to the beneficial effects of UCB, EPCs also significantly reduced cortical cell death, returned CD4+ T cell infiltration to sham levels, and reduced the peripheral Th1-mediated pro-inflammatory shift. CONCLUSION: This study highlights that cells found in UCB is able to mediate neuroinflammation and is an effective neuroprotective therapy. Our study also shows that particular cells found in UCB, namely EPCs, may have an added advantage over using UCB alone. This work has the potential to progress towards tailored UCB therapies for the treatment of perinatal brain injury.

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.

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.

Early- versus Late-Onset Fetal Growth Restriction Differentially Affects the Development of the Fetal Sheep Brain.

Fetal growth restriction (FGR) is a common complication of pregnancy, principally caused by suboptimal placental function, and is associated with high rates of perinatal mortality and morbidity. Clinical studies suggest that the time of onset of placental insufficiency is an important contributor towards the neurodevelopmental impairments that are evident in children who had FGR. It is however currently unknown how early-onset and late-onset FGR differentially affect brain development. The aim of this study was to examine neuropathology in early-onset and late-onset FGR fetal sheep and to determine whether they differentially alter brain development. We induced placental insufficiency and FGR via single umbilical artery ligation at either 88 days (early-onset) or 105 days (late-onset) of fetal sheep gestation (term is approx. 147 days), reflecting a period of rapid white matter brain development. Fetal blood samples were collected for the first 10 days after surgery, and all fetuses were sacrificed at 125 days' gestation for brain collection and subsequent histopathology. Our results show that early-onset FGR fetuses became progressively hypoxic over the first 10 days after onset of placental insufficiency, whereas late-onset FGR fetuses were significantly hypoxic compared to controls from day 1 after onset of placental insufficiency (SaO2 46.7 ± 7.4 vs. 65.7 ± 3.9%, respectively, p = 0.03). Compared to control brains, early-onset FGR brains showed widespread white matter injury, with a reduction in both CNPase-positive and MBP-positive density of staining in the periventricular white matter (PVWM), subcortical white matter, intragyral white matter (IGWM), subventricular zone (SVZ), and external capsule (p < 0.05 for all). Total oligodendrocyte lineage cell counts (Olig-2-positive) did not differ across groups, but mature oligodendrocytes (MBP-positive) were reduced, and neuroinflammation was evident in early-onset FGR brains with reactive astrogliosis (GFAP-positive) in the IGWM and cortex (p < 0.05), together with an increased number of Iba-1-positive activated microglia in the PVWM, SVZ, and cortex (p < 0.05). Late-onset FGR was associated with a widespread reduction of CNPase-positive myelin expression (p < 0.05) and a reduced number of mature oligodendrocytes in all white matter regions examined (p < 0.05). NeuN-positive neuronal cell counts in the cortex were not different across groups; however, the morphology of neuronal cells was different in response to placental insufficiency, most notable in the early-onset FGR fetuses, but it was late-onset FGR that induced caspase-3-positive apoptosis within the cortex. This study demonstrates that early-onset FGR is associated with more widespread white matter injury and neuroinflammation; however, both early- and late-onset FGR are associated with complex patterns of white and grey matter injury. These results indicate that it is the timing of the onset of fetal compromise relative to brain development that principally mediates altered brain development associated with FGR.

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.

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

Perinatal asphyxia is a significant cause of death or long-term neurodevelopmental impairment. Hypothermia, currently the only effective treatment, leads to modest improvements, but new therapeutic strategies are required. Umbilical cord blood (UCB) mononuclear cells have potent anti-inflammatory properties and may reduce neuropathology. This study examined whether autologous UCB mononuclear cells were neuroprotective when administered to newborn lambs at 12 h after birth asphyxia. At caesarean section, birth asphyxia was induced by clamping the umbilical cord until mean arterial blood pressure decreased to 18-20 mmHg. Asphyxia (n = 20) or control (n = 11) lambs were resuscitated and maintained, with magnetic resonance spectroscropy (MRS) performed at 12 and 72 h, and were then killed at 72 h. Cord blood was collected once the cord was clamped, and mononuclear cells were isolated and labelled fluorescently and administered to control (n = 3) or asphyxia (n = 8) lambs. Asphyxia induced a significant increase in cellular apoptosis (caspase-3 immunopositive) within all brain regions examined, including cortex, hippocampus, thalamus, striatum and subcortical white matter (P < 0.01 vs. control). Additionally, asphyxia induced significant and widespread astrogliosis and increased inflammatory cells (activated microglia and macrophages). The administration of UCB mononuclear cells (asphyxia+UCB) significantly decreased neuronal apoptosis, astrogliosis and inflammation (P < 0.05 vs. asphyxia alone). Asphyxia+UCB lambs also demonstrated decreased brain metabolites lactate:choline (P = 0.01) and lactate:N-acetylaspartate (P < 0.01) from 12 to 72 h, detected using MRS. Autologous UCB mononuclear cell treatment restores normal brain metabolism following perinatal asphyxia, and reduces brain inflammation, astrogliosis and neuronal apoptosis, supporting its use as a neuroprotective therapy following asphyxia.

Allopregnanolone and social stress: regulation of the stress response in early pregnancy in pigs.

This experiment investigated whether allopregnanolone, a neurosteroid metabolite from progesterone, modulates the stress response during early pregnancy. Twenty-five nulliparous sows (Sus scrofa) were allocated to one of three treatments: pregnant, ovariectomized or ovariectomized administered daily intravenously with alfaxalone as a synthetic allopregnanolone analog. On days 5, 12 and 19 of pregnancy, all sows were subjected to social stress by submitting them individually to a resident-intruder test, acting as the intruder. Blood samples were collected to analyze plasma progesterone, allopregnanolone, cortisol and adrenocorticotropic hormone (ACTH) concentrations. On day 26, 10 sows across the three treatments were subjected to a dexamethasone suppression test followed by a corticotrophin-releasing hormone administration to test the functionality of their hypothalamo-pituitary-adrenal (HPA) axis through cortisol release. Pregnant sows returned more rapidly to baseline cortisol concentrations following the resident-intruder test (p = 0.006). However, there were no other differences in cortisol or ACTH concentrations according to treatment or day, or to the HPA responsivity test on day 26. Allopregnanolone concentration in pregnant sows was higher than in ovariectomized sows (p < 0.001), but stable during the first third of pregnancy. Allopregnanolone concentration was correlated with longer resident-intruder test duration (pregnant: r = 0.66, p = 0.0003; ovariectomized: r = 0.47, p = 0.03), reflecting lower aggressiveness, and with progesterone concentration (r = 0.25, p = 0.03). Alfaxalone administration raised plasma allopregnanolone concentration in alfaxalone-administered sows but resulted in little behavioral and physiological effects. These findings did not support the hypothesis that the stress response of the female pig changes in the first third of pregnancy. Allopregnanolone was associated with lower aggression in social encounters.

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

Intrauterine growth restriction (IUGR) is associated with impaired cardiac function in childhood and is linked to short- and long-term morbidities. Placental dysfunction underlies most IUGR, and causes fetal oxidative stress which may impact on cardiac development. Accordingly, we investigated whether antenatal melatonin treatment, which possesses antioxidant properties, may afford cardiovascular protection in these vulnerable fetuses. IUGR was induced in sheep fetuses using single umbilical artery ligation on day 105-110 of pregnancy (term 147). Study 1: melatonin (2 mg h(-1)) was administered i.v. to ewes on days 5 and 6 after surgery. On day 7 fetal heart function was assessed using a Langendorff apparatus. Study 2: a lower dose of melatonin (0.25 mg h(-1)) was administered continuously following IUGR induction and the ewes gave birth normally at term. Lambs were killed when 24 h old and coronary vessels studied. Melatonin significantly improved fetal oxygenation in vivo. Contractile function in the right ventricle and coronary flow were enhanced by melatonin. Ischaemia-reperfusion-induced infarct area was 3-fold greater in IUGR hearts than in controls and this increase was prevented by melatonin. In isolated neonatal coronary arteries, endothelium-dependent nitric oxide (NO) bioavailability was reduced in IUGR, and was rescued by modest melatonin treatment. Melatonin exposure also induced the emergence of an indomethacin-sensitive vasodilation. IUGR caused marked stiffening of the coronary artery and this was prevented by melatonin. Maternal melatonin treatment reduces fetal hypoxaemia, improves heart function and coronary blood flow and rescues cardio-coronary deficit induced by IUGR.

Injury of the developing cerebellum: a brief review of the effects of endotoxin and asphyxial challenges in the late gestation sheep fetus.

The vulnerability of the fetal and newborn brain to events in utero or at birth that cause damage arising from perturbations of cerebral blood flow and metabolism, such as the accumulation of free radicals and excitatory transmitters to neurotoxic levels, has received considerable attention over the last few decades. Attention has usually been on the damage to cerebral structures, particularly, periventricular white matter. The rapid growth of the cerebellum in the latter half of fetal life in species with long gestations, such as the human and sheep, suggests that this may be a particularly important time for the development of cerebellar structure and function. In this short review, we summarize data from recent studies with fetal sheep showing that the developing cerebellum is particularly sensitive to infectious processes, chronic hypoxia and asphyxia. The data demonstrates that the cerebellum should be further studied in insults of this nature as it responds differently to the remainder of the brain. Damage to this region of the brain has implications not only for the development of motor control and posture, but also for higher cognitive processes and the subsequent development of complex behaviours, such as learning, memory and attention.

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction.

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long-term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.

Therapeutic potential of extracellular vesicles derived from human amniotic epithelial cells for perinatal cerebral and pulmonary injury.

Lung and brain injury that occurs during the perinatal period leads to lifelong disability and is often driven and/or exacerbated by inflammation. Human amniotic epithelial cells (hAEC), which demonstrate immunomodulatory, anti-fibrotic, and regenerative capabilities, are being explored as a therapeutic candidate for perinatal injury. However, limitations regarding scalable manufacturing, storage, transport, and dose-related toxicity have impeded clinical translation. Isolated therapeutic extracellular vesicles (EVs) from stem and stem-like cells are thought to be key paracrine mediators of therapeutic efficacy. The unique characteristics of EVs suggest that they potentially circumvent the limitations of traditional cell-based therapies. However, given the novelty of EVs as a therapeutic, recommendations around ideal methods of production, isolation, storage, and delivery have not yet been created by regulatory agencies. In this concise review, we discuss the pertinence and limitations of cell-based therapeutics in perinatal medicine. We also review the preclinical evidence supporting the use of therapeutic EVs for perinatal therapy. Further, we summarize the arising considerations regarding adequate cell source, biodistribution, isolation and storage methods, and regulatory roadblocks for the development of therapeutic EVs.

Phenotype of early-onset fetal growth restriction in sheep.

Introduction: Fetal 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. Methods: FGR 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). Results: Body weight of FGR fetuses/lambs was significantly reduced compared to controls (p<0.0001) from 127 days of gestation (term is 148 days), with increased brain:body weight ratio (p<0.0001) indicative of brain sparing. All biometric measures of body size were reduced in the FGR group with the exception of biparietal (head) diameter. The trajectory of body growth in the last trimester of sheep pregnancy was significantly reduced in the FGR group compared to controls, and stillbirth rate increased with longer gestation. Discussion: This work provides a well characterised FGR animal model that mimics the known physiological adaptations in human pregnancy and can be used to determine the efficacy of potential interventions.

Human amnion epithelial cells reduce fetal brain injury in response to intrauterine inflammation.

Intrauterine infection, such as occurs in chorioamnionitis, is a principal cause of preterm birth and is a strong risk factor for neurological morbidity and cerebral palsy. This study aims to examine whether human amnion epithelial cells (hAECs) can be used as a potential therapeutic agent to reduce brain injury induced by intra-amniotic administration of lipopolysaccharide (LPS) in preterm fetal sheep. Pregnant ewes underwent surgery at approximately 110 days of gestation (term is approx. 147 days) for implantation of catheters into the amniotic cavity, fetal trachea, carotid artery and jugular vein. LPS was administered at 117 days; hAECs were labeled with carboxyfluorescein succinimidyl ester and administered at 0, 6 and 12 h, relative to LPS administration, into the fetal jugular vein, trachea or both. Control fetuses received an equivalent volume of saline. Brains were collected 7 days later for histological assessment of brain injury. Microglia (Iba-1-positive cells) were present in the brain of all fetuses and were significantly increased in the cortex, subcortical and periventricular white matter in fetuses that received LPS, indicative of inflammation. Inflammation was reduced in fetuses that received hAECs. In LPS fetuses, the number of TUNEL-positive cells was significantly elevated in the cortex, periventricular white matter, subcortical white matter and hippocampus compared with controls, and reduced in fetuses that received hAECs in the cortex and periventricular white matter. Within the fetal brains studied there was a significant positive correlation between the number of Iba-1-immunoreactive cells and the number of TUNEL-positive cells (R(2) = 0.19, p < 0.001). The administration of hAECs protects the developing brain when administered concurrently with the initiation of intrauterine inflammation.