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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.

Background: Dopamine is frequently used as inotropic agent in preterm infants. Its cardiovascular actions, as well as effects on neurovascular interactions may be neuroprotective during hypoxic-ischaemic events. Using a preterm lamb model we aimed to test the impact of intravenous dopamine on hypoxic-ischaemic brain injury. Method(s): Nine fetal lambs (91-93d gestation) were instrumented with catheters in carotid artery and jugular vein, and an umbilical cord occluder. Four days after surgery, intravenous dopamine (DA, 10 mug/kg/ min, n = 5) (or saline, n = 4) was commenced. Then a hypoxic-ischaemic insult was induced with umbilical cord occlusion for 25 min. Infusions were continued for another 72 h before euthanasia. Fetal brains were collected for immunohistopathology. Result(s): Dopamine infusion increased fetal heart rate (184 +/- 1 to 203 +/- 1 bpm, P < 0.05) while arterial pressure was unchanged. Three animals in the DA group showed tachycardic response to cord occlusion, while the other two animals showed bradycardic response similar to the saline group. In the periventricular white matter, the saline group had higher number of microglia (lectin positive) than the DA group (10 +/- 3 vs. 6 +/- 2 per 0.04 mm2, P < 0.05). The saline group tended to have shorter myelinated fibre lengths (CNPase) compared with the DA group (15.0 +/- 2.0 vs. 18.4 +/- 5.7 mum respectively, P = ns). No histological differences were evident between DA animals exhibiting a tachycardic or bradycardic response during cord occlusion. Conclusion(s): Intravenous dopamine reduces hypoxic-ischaemic white matter injury in preterm lambs, independent of the cardiovascular response during the hypoxic-ischaemia., F.Y. Wong, Ritchie Centre, MIMR, Monash University, Melbourne, VIC, Australia. E-mail: flora.wong@med.monash.edu.au, CONFERENCE ABSTRACT

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 +/- 0.8degreeC vs. 38.3 +/- 0.17degreeC 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.Copyright © 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Direct fetal creatine supplementation increased plasma and cerebral creatine concentrations but did not alter fetal body weight, basal cardiovascular output, or blood chemistry. Creatine-treated fetuses displayed changes to arterial oxygenation 24–72 h after acute global hypoxia. An increase in striatum total creatine levels following UCO was also noted and suggests that increasing creatine tissue availability may be an adaptive response against the effects of hypoxia.

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.

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. Method(s): 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 x 106 cells/kg) were administered intravenously to lambs at 1 h of life. Lambs were ventilated for 24 h and then euthanized. Result(s): 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-alpha cytokine levels were lower in FGR+UCBC vs. FGR lambs (p < 0.05). Brain histopathology showed decreased neuroinflammation and oxidative stress, increased endothelial cell proliferation, pericyte stability, and greater integrity of the neurovascular unit in FGR+UCBC vs. FGR lambs. Conclusion(s): Umbilical cord blood stem cell therapy mitigates perinatal brain injury due to FGR and ventilation, and the neuroprotective benefits may be mediated by stabilization of the neurovascular unit.Copyright © 2020 The Author(s).

Fetal growth restriction (FGR) and prematurity are associated with high risk of brain injury and long-term neurological deficits. FGR infants born preterm are commonly exposed to mechanical ventilation, but it is not known whether ventilation differentially induces brain pathology in FGR infants compared with appropriate for gestational age (AGA) infants. We investigated markers of neuropathology in moderate-to late-preterm FGR lambs, compared with AGA lambs, delivered by caesarean birth and ventilated under standard neonatal conditions for 24 h. FGR was induced by single umbilical artery ligation in fetal sheep at 88-day gestation (term, 150 days). At 125-day gestation, FGR and AGA lambs were delivered, dried, intubated, and commenced on noninjurious ventilation, with surfactant administration at 10 min. A group of unventilated FGR and AGA lambs at the same gestation was also examined. Over 24 h, circulating pH, PO2, and lactate levels were similar between groups. Ventilated FGR lambs had lower cerebral blood flow compared with AGA lambs (P = 0.01). The brain of ventilated FGR lambs showed neuropathology compared with unventilated FGR, and unventilated and ventilated AGA lambs, with increased apoptosis (caspase-3), blood-brain barrier dysfunction (albumin extravasation), activated microglia (Iba-1), and increased expression of cellular oxidative stress (4-hydroxynonenal). The neuropathologies seen in the ventilated FGR brain were most pronounced in the periventricular and subcortical white matter but also evident in the subventricular zone, cortical gray matter, and hippocampus. Ventilation of preterm FGR lambs increased brain injury compared with AGA preterm lambs and unventilated FGR lambs, mediated via increased vascular permeability, neuroinflammation and oxidative stress.Copyright © 2018 the American Physiological Society.

Background: Seizures in neonates are relatively common, and are strong predictors of long-term cognitive and developmental impairment like cerebral palsy. Current anti-seizure therapies (phenobarbitone (pheno)) can be neurotoxic. We propose that ganaxolone (ganax), a GABAA agonist, will reduce seizures, and reduce markers of brain injury following hypoxia ischemia (HI). We investigated the effects of ganax or pheno on seizure burden and neuropathology in HI lambs. Method(s): HI was induced via umbilical cord occlusion in term lambs (n = 21; sham n = 7). HI lambs were treated with either ganax (5 mg/kg/bolus followed by 5 mg/kg/d for 2 days) or pheno (20mg/kg/bolus followed by 5 mg/kg/day for 2 days) at 6 h. Seizure burden was assessed using continuous amplitudeintegrated electroencephalogram (aEEG) recording. At 48 h, lambs were euthanized for brain collection and analysis of neuropathology. Result(s): At birth, HI lambs had a pH <7.0 and blood pressure was reduced to 16.4 +/- 1.2 mmHg, indicating severe birth asphyxia. All HI lambs demonstrated electrographic seizures; mean number of seizures/h (6-12 h) was 4.8 +/- 2.2. Ganax treatment reduced the number of seizures to 1.0 +/- 0.9/h compared to pheno (3.2 +/- 1.6/ h) (P = 0.02). Brain histology revealed improved neuronal survival (NeuN+ cell number) and reduced cell death (TUNEL+) in the cortex and basal ganglia after ganax treatment, compared to pheno (P = 0.048 and P = 0.03), respectively. Conclusion(s): Ganax treatment in lambs with HI decreased seizure activity, improved neuronal survival and reduced cell death. Ganax has a high margin of safety and proved to be a better therapy.

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.Copyright © 2018 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd

Background: Impaired cerebral autoregulation in preterm infants makes circulatory management important to avoid cerebral hypoxic-ischemic injury. Dobutamine is frequently used as inotropic treatment in preterm neonates, but its effects on the brain exposed to cerebral hypoxia are unknown. We hypothesized that dobutamine would protect the immature brain from cerebral hypoxic injury. Method(s): In preterm (0.6 gestation) fetal sheep, dobutamine (Dob, 10 mug/kg/min) or saline (Sal) was infused intravenously for 74 h. Two hours after the beginning of the infusion, umbilical cord occlusion (UCO) was performed to produce fetal asphyxia (Sal+UCO: n = 9, Dob+UCO: n = 7), or sham occlusion (Sal+sham: n = 7, Dob+sham: n = 6) was performed. Brains were collected 72 h later for neuropathology. Result(s): Dobutamine did not induce cerebral changes in the sham UCO group. UCO increased apoptosis and microglia density in white matter, hippocampus, and caudate nucleus, and astrocyte density in the caudate nucleus. Dobutamine commenced before UCO reduced microglia infiltration in the white matter, and microglial and astrocyte density in the caudate. Conclusion(s): In preterm hypoxia-induced brain injury, dobutamine decreases neuroinflammation in the white matter and caudate, and reduces astrogliosis in the caudate. Early administration of dobutamine in preterm infants for cardiovascular stabilization appears safe and may be neuroprotective against unforeseeable cerebral hypoxic injury.Copyright © 2018, International Pediatric Research Foundation, Inc.

Background: Infants born preterm following exposure to in utero inflammation/chorioamnionitis are at high risk of brain injury and life-long neurological deficits. In this study, we assessed the efficacy of early intervention umbilical cord blood (UCB) cell therapy in a large animal model of preterm brain inflammation and injury. We hypothesised that UCB treatment would be neuroprotective for the preterm brain following subclinical fetal inflammation. Method(s): Chronically instrumented fetal sheep at 0.65 gestation were administered lipopolysaccharide (LPS, 150 ng, 055:B5) intravenously over 3 consecutive days, followed by 100 million human UCB mononuclear cells 6 h after the final LPS dose. Controls were administered saline instead of LPS and cells. Ten days after the first LPS dose, the fetal brain and cerebrospinal fluid were collected for analysis of subcortical and periventricular white matter injury and inflammation. Result(s): LPS administration increased microglial aggregate size, neutrophil recruitment, astrogliosis and cell death compared with controls. LPS also reduced total oligodendrocyte count and decreased mature myelinating oligodendrocytes. UCB cell therapy attenuated cell death and inflammation, and recovered total and mature oligodendrocytes, compared with LPS. Conclusion(s): UCB cell treatment following inflammation reduces preterm white matter brain injury, likely mediated via anti-inflammatory actions.Copyright © 2018 S. Karger AG, Basel.

Background: Seizures in neonates are relatively common, and are strong predictors of long-term cognitive and developmental impairment. Current anti-seizure therapies (phenobarbitone) have the potential to be neurotoxic. We propose that ganaxolone, a GABAA agonist, will reduce seizures, and reduce markers of brain injury following hypoxia ischemia. We investigated the effects of ganaxolone or phenobarbitone, given at 6h following hypoxia ischemia. Method(s): Hypoxic ischemic encephalopathy (HIE) was induced via umbilical cord occlusion in term lambs. HIE lambs were treated with either ganaxolone or phenobarbitone from 6h following HI. Seizures were assessed using continuous amplitudeintegrated electroencephalogram (aEEG) recording. At 48h, lambs were euthanized for brain collection and analysis of neuropathology. Result(s): At birth, HIE lambs had a pH <7.0, base excess >12mmol/L, and blood pressure was reduced to 16.4 +/- 1.2 mmHg, indicating severe birth asphyxia. All HIE lambs demonstrated electrographic seizures (aEEG) and the mean number of seizures per hour (6-12hrs) was 4.8 +/- 2.2. Ganaxolone treatment reduced the number of seizures to 1.0 +/- 0.9 per hour, which was a greater reduction attributable to phenobarbitone (3.2 +/- 1.6 per hour). Brain histology revealed improved neuronal survival and reduced cell death in the cortex and basal ganglia after ganaxolone treatment, compared to phenobarbitone. Conclusion(s): Ganaxolone treatment in lambs with HIE decreased seizure activity and cell death, and improved neuronal survival. Ganaxolone may be a better therapy than phenobarbitone.

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.

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.

PURPOSE: The aim of this study is to examine whether advanced magnetic resonance imaging (MRI) techniques can detect early brain injury caused by intrauterine inflammation and inappropriate initial respiratory support in preterm lambs. HYPOTHESIS: Neuropathology caused by intrauterine inflammation is exacerbated by mechanical ventilation at birth and is detectable with advanced MRI techniques. METHODS: Pregnant ewes received intra-amniotic lipopolysaccharide (LPS) 7 days prior to delivery at ~125 days of gestation (85% of gestation), whereupon lambs were delivered and randomised to receive an injurious (LPS + INJ, n = 6) or protective (LPS + PROT, n = 6) ventilation strategy. MRI of the brain was conducted 90 min after preterm delivery, using structural, diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS) techniques. A colour map threshold technique was utilised to compare distributions of low diffusivity voxels in the brains of LPS-exposed lambs with those not exposed to LPS (PROT, n = 7 PROT and INJ, n = 10). RESULTS: No overt cerebral injury was identified on structural MRI images of any lamb. However, on DTI, axial diffusivity, radial diffusivity, and mean diffusivity values were lower and significantly more heterogeneous in specific brain regions of lambs in the LPS + INJ group compared to the LPS + PROT group. Colour mapping revealed lower diffusivity in the thalamus, periventricular white matter, internal capsule, and frontal white matter in the LPS + INJ group compared to LPS + PROT group. The MRS peak area ratios of lactate, relative to those for the metabolites creatine, choline, and N-acetylaspartate, were not different between LPS-exposed groups. Lambs exposed to LPS had lower diffusivity within the white matter regions assessed than non-LPS-treated control lambs. CONCLUSION: DTI colour map threshold techniques detected early brain injury in preterm lambs exposed to intrauterine inflammation and detected differences between injuriou

PURPOSE: Injurious mechanical ventilation causes white matter (WM) injury in preterm infants through inflammatory and haemodynamic pathways. The relative contribution of each of these pathways is not known. We hypothesised that in vivo magnetic resonance imaging (MRI) can detect WM brain injury resulting from mechanical ventilation 24 h after preterm delivery. Further we hypothesised that the combination of inflammatory and haemodynamic pathways, induced by umbilical cord occlusion (UCO) increases brain injury at 24 h. METHODS: Fetuses at 124±2 days gestation were exposed, instrumented and either ventilated for 15 min using a high tidal-volume (VT) injurious strategy with the umbilical cord intact (INJ; inflammatory pathway only), or occluded (INJ+UCO; inflammatory and haemodynamic pathway). The ventilation groups were compared to lambs that underwent surgery but were not ventilated (Sham), and lambs that did not undergo surgery (unoperated control; Cont). Fetuses were placed back in utero after the 15 min intervention and ewes recovered. Twenty-four hours later, lambs were delivered, placed on a protective ventilation strategy, and underwent MRI of the brain using structural, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) techniques. RESULTS: Absolute MRS concentrations of creatine and choline were significantly decreased in INJ+UCO compared to Cont lambs (P = 0.03, P = 0.009, respectively); no significant differences were detected between the INJ or Sham groups and the Cont group. Axial diffusivities in the internal capsule and frontal WM were lower in INJ and INJ+UCO compared to Cont lambs (P = 0.05, P = 0.04, respectively). Lambs in the INJ and INJ+UCO groups had lower mean diffusivities in the frontal WM compared to Cont group (P = 0.04). DTI colour mapping revealed lower diffusivity in specific WM regions in the Sham, INJ, and INJ+UCO groups compared to the Cont group, but the differences did not reach significance. INJ+UCO lambs more

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.Copyright © 2016 The Physiological Society.

Introduction: Reduced blood flow into the fetal left ventricle (LV) may underlie development of Hypoplastic Left Heart Syndrome (HLHS), for which palliative surgery is the only treatment option. Currently there is no HLHS model in a large fetal mammalian animal for testing and clinical translation of new therapy. We have recently published a percutaneous trans-hepatic technique to catheterise the fetal heart, which could be utilised to produce an animal model of HLHS. Using a percutaneous trans-hepatic technique to deliver an occluder into the fetal heart, we aimed to create a fetal lamb model of HLHS by occluding the foramen ovale (FO). Method(s): Three fetal lambs (110-117days, term=147days) under maternal general anaesthesia were used. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured through the maternal abdomen using a 14GA IV-cannula. A coronary catheter and guidewire were inserted and advanced into the fetal IVC and right atrium. An Amplatzer Duct Occluder II (ADO II 4x4 mm, St Jude Medical) was delivered to occlude the fetal FO. In 2 fetuses, a self-expandable stent (8x12mm Superflex-DS 4Fr deliverable stent, Opti-Med Inc. Germany) was positioned across the FO first, and the ADO II was anchored within the stent. Serial ultrasounds were performed to monitor cardiac development. Euthanasia and post-mortem examination was performed 3 weeks after. A twin fetus which did not undergo the procedure was used as control for comparison. Morphometric measurements were made on digital images (Image J1.48b, NIH) of transverse sections of the heart at the level of atrioventricular valves. Result(s): All 3 fetal lambs survived well to 3 weeks without any fetal compromise. At post-mortem examination, the LV lumen was markedly reduced (Fig 1a) with lower LV/right ventricular (RV) chamber volume ratio, LV/RV lumen area ratio and increased septal thickness at level of atrioventricular valves (Table), compared with the control fetal heart (Fig 1b).

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 12 h or 5 d after acute hypoxia-ischemia (HI) induced by umbilical cord occlusion. The fetal brains were collected at 10 d 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 12 h, but not 5 d 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 12 h or 5 d ameliorated these effects. Additionally, UCBCs at 12 h induced a significant systemic increase in interleukin-10 at 10 d, and reduced oxidative stress (malondialdehyde) following HI (P < 0.05). UCBC administration at 12 h after HI reduces preterm white matter injury, via anti-inflammatory and antioxidant actions.Copyright © 2016

Dopamine is often used to treat hypotension in preterm infants; these infants are at risk of developing brain injury due to impaired autoregulation and cerebral hypoperfusion. However the effects of dopamine on the immature brain under conditions of cerebral hypoxia are not known. We hypothesized that pretreatment with dopamine would protect the immature brain from injury caused by cerebral hypoxia. Preterm fetal sheep were used to determine the effects of intravenous dopamine on hypoxia-induced brain injury.In 16 pregnant sheep at 90. days of gestation (0.6 of term, term=147. days) catheters were implanted aseptically into the fetal carotid artery and jugular vein; an inflatable occluder was placed loosely around the umbilical cord for later induction of fetal hypoxemia. At 5. days after surgery, dopamine (10mug/kg/min, n=7 fetuses) or saline (n=9 fetuses) was infused for 74h. Two hours after commencing the dopamine/saline infusion, we induced umbilical cord occlusion (UCO) for up to 25. min to produce fetal asphyxia. Fetuses were allowed to recover, and brains were collected 72. h later for assessment of neuropathology. Un-operated twin fetuses were used as age-matched non-UCO controls (n=8).In UCO+saline fetuses, microglial and apoptotic cell density in the subcortical and periventricular white matter, caudate nucleus and hippocampus was greater than that in age-matched controls; oxidative stress was elevated in the subcortical and periventricular white matter and caudate nucleus compared to that in age-matched controls. In UCO+dopamine fetuses microglial density and oxidative stress in the cerebral white matter and caudate nucleus were not different to that of age-matched controls. Apoptotic cell death was decreased in the cerebral white matter of UCO+dopamine brains, relative to UCO+saline brains.We conclude that pretreatment with dopamine does not exacerbate hypoxia-induced injury in the immature brain and may be neuroprotective because it led to decreased apo

Purpose: Synchronised intermittent mandatory ventilation (SIMV) and high-frequency jet ventilation (HFJV) are accepted ventilatory strategies for treatment of respiratory distress syndrome (RDS) in preterm babies. We hypothesised that SIMV and HFJV both facilitate adequate oxygenation and ventilation but that HFJV is associated with less lung injury. Results: There were no differences in arterial oxygenation or partial pressure of carbon dioxide despite lower mean airway pressure during SIMV for most of the study. There were no consistent significant differences in end systolic and end diastolic PBF, lung injury data and static lung compliance. Methods: Preterm lambs of anaesthetised ewes were instrumented, intubated and delivered by caesarean section after intratracheal suction and instillation of surfactant. Each lamb was managed for 3 hr according to a predetermined algorithm for ventilatory support consistent with open lung ventilation. Pulmonary blood flow (PBF) was measured continuously and pulsatility index was calculated. Ventilatory parameters were recorded and arterial blood gases were measured at intervals. At postmortem, in situ pressure-volume deflation curves were recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess inflammation. Conclusions: SIMV and HFJV have comparable clinical efficacy and ventilator pressure requirements when applied with a targeted lung volume recruitment strategy.

Objective: We aimed to assess the feasibility of using a percutaneous transhepatic cardiac catheterization technique to perform fetal pulmonary valvuloplasty and valvulotomy under ultrasound guidance at mid-gestation. Method(s): In 13 mid-gestation fetal lambs without cardiac pathology, percutaneous transhepatic cardiac catheterization was used to position a coronary angioplasty catheter within the pulmonary valve. The balloon was inflated/deflated several times, simulating pulmonary valvuloplasty. In another two fetal lambs, a guidewire tip was positioned against the pulmonary valve, and unipolar diathermy was applied to simulate perforation of an atretic valve. Result(s): Percutaneous access followed by right heart catheterization was successful in all cases. One fetus died following right ventricle perforation. Simulated pulmonary valvuloplasty was successful in nine cases using catheters with 6-mm-long balloons but unsuccessful in two cases (both survived) using 12-mm-long balloons. In one case, the catheter could not be inserted as the cannula became dislodged. Diathermy of the pulmonary valve was successful in both attempts. Conclusion(s): We successfully simulated in utero perforation and dilation of the pulmonary valve using percutaneous transhepatic access in fetal lambs. The technique has potential for clinical translation into treatment for human fetuses with critical pulmonary stenosis or pulmonary atresia with intact ventricular septum.Copyright © 2014 John Wiley & Sons, Ltd.

Background - Intact or highly restricted intra-atrial septum can be reliably diagnosed in the human fetus as early as 22 to 24 weeks of gestation. Fetal interventions targeting the atrial septum have used a direct approach through the atrial wall. Here, we report stenting of the foramen ovale with a large, open-cell stent via percutaneous access through the fetal hepatic vein in a sheep model. Methods and Results - In 5 fetal sheep of 109 to 111 days of gestation (term, 147 days), the fetal hepatic vein was punctured percutaneously under ultrasound guidance and a 13.3-cm 14-gauge intravenous catheter was inserted. After catheterization of the inferior vena cava, right atrium, foramen ovale, and left atrium with a guidewire and 1.8F to 2.6F tapered catheter, a self-expandable, 8x12-mm flexible open-cell stent was positioned in an unrestricted foramen ovale. Flow and fetal well-being were documented for 45 minutes after the procedure. Access to the left atrium was achieved in all 5 animals and all survived. In 4 animals, the stent was successfully positioned in the foramen ovale. One fetus was born at term and euthanized on day 3: postmortem examination confirmed the patency of the stent. The other 3 fetuses were well after being monitored by ultrasound for 45 minutes. In 1 animal, the stent dislodged immediately after release obstructing the mitral valve. This fetus developed ascites and was euthanized after 4 days. Conclusions - It is feasible to safely advance a large diameter, self-expandable, open-cell design stent into the fetal atrial septum via a percutaneous access route through the fetal hepatic vein.Copyright © 2015 American Heart Association, Inc.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Background: Dobutamine is often used to treat hypotension and sustain cerebral perfusion in preterm infants. Despite widespread use, little is known about the effect of dobutamine on the developing brain. Method(s): At 90 days gestation (term = 147 days) non-occlusive carotid artery and jugular vein catheters and an umbilical occluder were implanted into fetal lambs. Four days post-surgery continuous dobutamine (n = 7, 10 mug/kg/min) or saline (n = 7) infusion commenced for 74 h. Two hours after starting the infusion fetal acidemic-hypoxia was produced by umbilical cord occlusion (UCO) lasting 25 mins, or until fetal mean arterial pressure (MAP) decreased to 10 mmHg. Dobutamine infusion without UCO (n = 4), and unoperated fetuses were controls (n = 8). Brains were collected 72 h later for neuropathology analysis. Data is presented as mean +/- SE. Result(s): UCO induced bradycardia, hypotension, acidosis and hypoxia in both the saline + UCO and dobutamine + UCO groups; 3/7 saline + UCO and 0/7 dobutamine + UCO fetuses became severely hypotensive (MAP < 10 mmHg) during UCO. Dobutamine increased heart rate before, and for 6.5 h after UCO, when compared to saline + UCO fetuses. Microglia density (Iba + cells/mm2) was increased in periventricular and subcortical white matter in saline + UCO (292 +/- 48, 376 +/- 54 respectively) brains compared to unoperated controls (107 +/- 15, 161 +/- 33 respectively). In contrast, microglia density was not significantly increased after UCO in dobutamine-treated fetuses (270 +/- 55, 294 +/- 40 respectively), in comparison to unoperated and dobutamine + sham (120 +/- 24, 208 +/- 61 respectively) groups. Conclusion(s): Dobutamine decreases white matter microglia density following hypoxia-ischemia, possibly leading to decreased neuroinflammation. Cardiovascular function was improved during hypoxicischemia in preterm lambs treated with dobutamine, with all dobutamine-infused fetuses tolerating 25 mins of UCO.

Background: Reduced blood flow into the fetal left ventricle (LV) may underlie development of Hypoplastic Left Heart Syndrome (HLHS). Currently there is no animal model of HLHS for testing new therapy. We recently published a percutaneous trans-hepatic technique for catheterising the fetal heart under ultrasound guidance. We aimed to create a lamb model of HLHS, by percutaneous catheterisation and occlusion of the fetal foramen ovale (FO). Method(s): In three fetal lambs (110-117 days, term = 147 days) under maternal anaesthesia, the fetal hepatic vein was percutaneously punctured through the maternal abdomen under ultrasound guidance using a 14GA IV-cannula. A coronary catheter was advanced into the fetal IVC and right atrium. A self-expandable stent (8 x 12 mm Superflex-DS stent, Opti-Med Inc.) was positioned across the FO, followed by an Amplatzer Duct Occluder II (4 x 4 mm, St Jude Medical) being anchored within the stent to occlude the FO. Serial ultrasounds were performed to monitor cardiac development. Post-mortem examination was performed 3 weeks after. Morphometric measurements were made at the level of atrioventricular valves and compared with 6 controls. Result(s): All 3 fetal lambs survived well to 3 weeks post procedure. The LV lumens were markedly reduced, with lower LV/right ventricular (RV) chamber volume ratio (0.2-0.6) and LV/RV lumen area ratio (0.8-1.0) compared with the control fetal heart (1.0 and 1.5 respectively). Conclusion(s): Occlusion of the fetal FO using percutaneous catheterisation leads to phenotype simulating HLHS. Our results demonstrate the potential to develop the world's first large animal model of HLHS, critical for understanding the disease and devising new therapies.

Introduction: Reduced blood flow into the fetal left ventricle (LV) may lead to development of Hypoplastic Left Heart Syndrome (HLHS), for which palliative surgery is the only treatment option. Currently there is no large fetal animal model of HLHS for testing and clinical translation of new therapies. We have recently published a percutaneous transhepatic technique of cardiac catheterisation in a fetal lamb model, which could be utilised to produce an animal model of HLHS. Objective(s): To use a percutaneous transhepatic catheter technique to deliver an occluder into the foramen ovale (FO) of the fetal heart, with the aim of creating a fetal lamb model of HLHS. Method(s): Three fetal lambs (110-117days, term=147days) under maternal general anaesthesia were used. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured through the maternal abdomen using a 14GA IV-cannula. A coronary catheter and guidewire were inserted and advanced into the fetal IVC and right atrium. An Amplatzer Duct Occluder II (4x4mm, St Jude Medical) was delivered to occlude the fetal FO. In 2 fetuses, a self-expandable stent (8x12 mm Superflex-DS, 4Fr deliverable stent, OptiMed Inc., Germany) was positioned across theFO first, and theADOII was anchored within the stent. Serial ultrasounds were performed to monitor cardiac development. Euthanasia and postmortem examination was performed after 3 weeks. A twin fetus, which did not undergo the procedure, was used as control. Morphometric measurements were made on digital images (Image J1.48b, NIH) of transverse sections of the heart at the level of the atrioventricular valves. Result(s): All fetal lambs survived, without evidence of compromise, until the planned euthanasia. At postmortem examination, the LV lumen was markedly reduced (Fig 1a) with lower LV/right ventricular (RV) chamber volume ratio and LV/RV lumen area ratio, as well as increased septal thickness at the level of the atrioventricular valves (Table 1), in comp

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 r2=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. © 2014 Elsevier Inc.

OBJECTIVES: Reduced blood flow into the fetal left ventricle (LV) may lead to development of Hypoplastic Left Heart Syndrome (HLHS), for which palliative surgery is the only treatment option. Currently there is no large animal fetal HLHS model for testing and clinical translation of new therapies. We have recently published a percutaneous transhepatic technique of cardiac catheterisation in a fetal lamb model, which could be utilised to produce an animal model of HLHS. Hence, in this study our objective was to use a percutaneous transhepatic catheter technique to deliver an occluder into the foramen ovale (FO) of the fetal heart, with the aim of creating a fetal lamb model of HLHS. METHOD(S): Three fetal lambs (110-117 days, term = 147 days) under maternal general anaesthesia were used. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured through the maternal abdomen using a 14GA IV-cannula. A coronary catheter and guidewire were inserted and advanced into the fetal IVC and right atrium. An Amplatzer Duct Occluder II (4 x 4 mm, St Jude Medical) was delivered to occlude the fetal FO. In 2 fetuses, a selfexpandable stent (8 x 12 mm Superflex-DS, 4Fr deliverable stent, OptiMed Inc., Germany) was positioned across the FO first, and the ADO II was anchored within the stent. Serial ultrasounds were performed to monitor cardiac development. Euthanasia and postmortem examination was performed after 3 weeks. A twin fetus, which did not undergo the procedure, was used as control. Morphometric measurements were made on digital images of transverse sections of the heart at the level of the atrioventricular valves. RESULT(S): All fetal lambs survived, without evidence of compromise, until the planned euthanasia. At postmortem examination, the LV lumen was markedly reduced with lower LV/ RV chamber volume ratio and lower LV/RV lumen area ratio, as well as increased septal thickness at the level of the atrioventricular valves, in comparison to the control

Background: Systemic hypotension is a common complication in very preterm newborns. To maintain cerebral perfusion, inotropes such as dopamine or dobutamine are often administered, although their effects within the brain are yet to be elucidated. Aim(s): To determine the neuropathological and cardiovascular effects of intravenous dopamine or dobutamine in response to hypoxia-ischaemia, in a preterm fetal lamb model. Method(s): At 90 days gestation (term = 147 days) non-occlusive carotid artery and jugular vein catheters and an umbilical occluder were implanted into fetal lambs. Four days post-surgery continuous dopamine (n = 7, 10 mug/kg/min), dobutamine (n = 6, 10 mug/kg/min) or saline (n = 9) infusion was commenced. Two hours post-infusion, 25 mins umbilical cord occlusion (UCO) was performed. Brains were collected 72 h later for immunohistochemical analysis. Data presented mean +/- SE. Result(s): Pre-UCO, dopamine and dobutamine significantly elevated fetal heart rate (HR, 220 +/- 8 and 220 +/- 7 bpm) compared to saline infusion (192 +/- 7 bpm, p < 0.05) though did not increase mean arterial pressure (MAP). UCO was ceased early in 5/9 saline and 1/6 dopamine-infused lambs due to MAP < 10 mmHg or asystole. One dobutamine-infused lamb died after UCO. Post-UCO, HR remained elevated in dopamine and dobutamine- infused lambs. There was a significant increase in inflammatory cells (Isolectin-B4, cells/mm2) in the subcortical white matter of saline (1321 +/- 155) and dobutamine-infused (1645 +/- 322) lambs, compared to unoperated controls (263 +/- 96, p < 0.05), but no significant difference between dopamine-infused lambs (1090 +/- 293) and unoperated controls. There were no differences in inflammatory cell density in the periventricular white matter. Conclusion(s): Dopamine and dobutamine sustained MAP during UCO. Dopamine may decrease inflammatory cell infiltration in the subcortical white matter in preterm hypoxic-ischaemic injury.

Background/hypothesis: Fetal pulmonary valvuloplasty may ameliorate hypoplastic right heart syndrome and mitigate postnatal disease. Fetal heart access by direct fetal heart puncture is well-described. We have recently developed a percutaneous transhepatic fetal cardiac catheterization technique, which may be safer and offer technical advantages. We hypothesized that fetal pulmonary valvuloplasty could be performed by a percutaneous transhepatic approach at mid-gestational age. Material(s) and Method(s): Nine fetal lambs at 97-100 (term 147) days gestation (average weight: 1,215 g) under maternal general anesthesia were studied. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured using a 16GA IV cannula with needle in situ. A 2.6/1.8 Fr coronary catheter (FineCrossTM MG, Terumo) was inserted into the cannula over a 0.014 inch floppy guidewire, and the IVC, RA, RV, pulmonary artery, ductus arteriosus, and descending aorta catheterized. After removing the guiding catheter, but with the guidewire in place, a coronary percutaneous coronary angioplasty (PTCA) catheter was positioned across the pulmonary valve, and several inflations of the balloon were performed simulating a valvuloplasty. Seven fetuses were euthanized postprocedure, and two were euthanized after term-delivery, for postmortem examination. Result(s): Percutaneous cannulation of the fetal hepatic vein followed by RA and RV catheterization was successful in all cases. One fetus died during catheterization following RV perforation. In the remaining eight cases the coronary catheter was advanced to the descending aorta. Pulmonary valvuloplasty was successful in five cases using catheters with a 6-mm long balloon, and postmortem showed minimal hemorrhage without cardiac trauma. The procedure was unsuccessful in two cases (both survived) using a 12-mm long balloon which could not be turned into the pulmonary artery, but the fetuses survived and postmortem showed small RV perforations.

Introduction: Fetal blood flow and pressure remain an area of high scientific interest. Non-invasive techniques in assessing these parameters are limited in accuracy and precision. Fetal surgery to insert flow probes of catheters creates an artificial environment for the fetus that might limit the generalization of such measurements. Here, we report on the feasibility of flow and pressure measurements in the venous and arterial circulation of the fetal lamb using an ultrasound guided percutaneous transhepatic fetal cardiac catheterization technique through the maternal abdominal wall. Method(s): Eight fetal lambs at 97-100 days gestation (term 147 days, average weight: 1,252 g) under maternal general anesthesia were examined. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured using a 16GA intravenous cannula with needle in situ. A 2.6/1.8 Fr Terumo Finecross catheter (Terumo Cardiovascular, Leuven, Belgium) was inserted over a 0.014 inch floppy-guidewire, and the IVC, RA, RV, pulmonary artery, ductus arteriosus, and descending aorta catheterized. After removal of the guidewire, blood pressure was recorded in the descending aorta (DAO). A 0.014 inch Doppler guide wire was inserted and the Doppler tracing was recorded (Flowire and Combomap Volcano Cooperation, Cordova, CA) Results: In all fetuses, the catheter was successfully advanced into the abdominal aorta through IVC, RA, RV, MPA, DA and DAO. Mean BP in the DAO was 34.1 mm Hg (range 24.8-46.2 mm Hg). High quality Doppler traces could be recorded in DAO, MPA, and DA. In the DAO, systolic blood flow velocity ranged mainly from 45 to 65 cm/sec while diastolic blood flow being documented between 10 and 15 cm/sec. In the MPA, systolic blood flow velocity ranged mainly from 25 to 35 cm/sec while diastolic blood flow being documented between 10 and 15 cm/sec. Conclusion(s): The ultrasound guided percutaneous transhepatic fetal cardiac catheterization technique presented here is feasible in th

Rationale: Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure. Methods: Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles. Results: LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis. Conclusion: Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorti

The absorption of medetomidine released by continuous infusion from an osmotic pump in the abdominal cavity was studied in pregnant sheep during the 24 h postoperative period. Additionally pain and sedation was assessed. Eleven sheep were studied: six were treated with a medetomidine loaded osmotic pump delivering 10 mu L/h (3 mu g/kg/h medetomidine); and five with a saline loaded osmotic pump (control). Serial blood samples were taken and analysed to determine plasma medetomidine levels. Medetomidine was absorbed from the peritoneal cavity and a steady plasma concentration was achieved within 10 h, mean (SD) peak concentration was 2.87 (0.22) ng/mL. Sheep receiving medetomidine analgesia had significantly lower pain scores at 10 h than controls. Four control sheep required rescue analgesia, compared with 0 in the treatment group. Delivery of 3 mu g/kg/h medetomidine by an intraperitoneal osmotic pump to pregnant sheep in the 24 h postoperative period provides adequate plasma concentrations of medetomidine for analgesia without sedation.

Background: Dopamine is frequently used as inotropic agent in preterm infants. Its cardiovascular actions, as well as effects on neurovascular interactions may be neuroprotective during hypoxic-ischaemic events. Using a preterm lamb model we aimed to test the impact of intravenous dopamine on hypoxic-ischaemic brain injury. Method(s): Nine fetal lambs (91-93d gestation) were instrumented with catheters in carotid artery and jugular vein, and an umbilical cord occluder. Four days after surgery, intravenous dopamine (DA, 10 mug/kg/ min, n = 5) (or saline, n = 4) was commenced. Then a hypoxic-ischaemic insult was induced with umbilical cord occlusion for 25 min. Infusions were continued for another 72 h before euthanasia. Fetal brains were collected for immunohistopathology. Result(s): Dopamine infusion increased fetal heart rate (184 +/- 1 to 203 +/- 1 bpm, P < 0.05) while arterial pressure was unchanged. Three animals in the DA group showed tachycardic response to cord occlusion, while the other two animals showed bradycardic response similar to the saline group. In the periventricular white matter, the saline group had higher number of microglia (lectin positive) than the DA group (10 +/- 3 vs. 6 +/- 2 per 0.04 mm2, P < 0.05). The saline group tended to have shorter myelinated fibre lengths (CNPase) compared with the DA group (15.0 +/- 2.0 vs. 18.4 +/- 5.7 mum respectively, P = ns). No histological differences were evident between DA animals exhibiting a tachycardic or bradycardic response during cord occlusion. Conclusion(s): Intravenous dopamine reduces hypoxic-ischaemic white matter injury in preterm lambs, independent of the cardiovascular response during the hypoxic-ischaemia.

Background: Fetal cardiac intervention may alter the evolution of cardiac conditions and reduce severity of the postnatal disease. Fetal heart access by direct puncture of the fetal heart under ultrasound guidance has been established, but with considerable technical difficulties. Alternative routes for percutaneous access to the fetal heart have been sought. Aim(s): To investigate the feasibility of fetal cardiac catheterization using a percutaneous transhepatic approach in the mid-gestational fetal lamb, as a model for possible cardiac intervention in the midgestational human fetus. Method(s): The procedure was performed in eight fetal lambs of 95-97 days gestation (term 147 days) under maternal general anesthesia. Intraabdominal vessels and heart structures were located using ultrasound with 2-7MHz curved array transducer. Under ultrasound guidance, the fetal right hepatic vein was punctured percutaneously, through the maternal abdominal wall, using a 16GA intravenous cannula (3 Fr ID, 5.25 inch, BD Angiocath, BD Infusion Therapy Systems, Utah) with needle insitu. A coronary catheter (FineCrossTM MG, Terumo, Somerset, NJ) was inserted into the 16 GA cannula, over a 0.014 inch guidewire, and then advanced into the inferior vena cava and the right atrium. Contrast was injected to document position of the catheter. Three fetal lambs were to be euthanized at the end of the procedure to precisely evaluate blood loss. The rest were to deliver vaginally at term, and euthanized postnatally for postmortem examination. Result(s): Percutaneous fetal cardiac access was successful in 7 out of the 8 fetuses. All 4 heart chambers were catheterized in the last 3 fetuses which all survived the procedure. One fetus died during the procedure, post-mortem showed pericardial and peritoneal hemorrhage. The two fetuses dedicated for immediate post-mortem, after having survived the procedure, had hemoperitoneum of 4 and 20ml respectively. Averaged fetal weights were 1027g. To date, thre

Objective: The objective of the study was to explore the maternal-fetal pharmacokinetics of intraamniotic (IA), intravenous (IV), or intramuscular (IM) administration of erythromycin or azithromycin in a pregnant sheep model. Study Design: Pregnant ewes of 115-121 days' gestation received a single maternal IV infusion (5 mg/kg over 60 min), a single IM injection, or a single IA injection (3.2 mg/kg fetal weight) of either erythromycin lactobionate or azithromycin. Maternal/fetal blood and amniotic fluid (AF) samples were collected across 48 h for macrolide assay by liquid chromatography and tandem mass spectrometry. Results: Maternal administration achieved therapeutic maternal plasma macrolide concentrations (<0.5 μg/mL) with low concentrations in AF equivalent to less than 7% transfer; fetal plasma levels were even lower (<1.5% transfer). The IA administration achieved therapeutic concentrations in AF and sustained for 48 h, with poor maternal-fetal transfer (<1% maternal, <0.3% fetal). Modest pharmacokinetic differences were evident between erythromycin and azithromycin. Conclusion: Maternal macrolide administration achieves subtherapeutic concentrations in AF or fetal plasma, whereas a single IA injection achieves therapeutic concentrations in AF but not in maternal-fetal circulations. Combined maternal and single IA administration of macrolides may be a more effective regimen for treatment of intrauterine, but not fetal, infection.

Increasing positive end-expiratory pressure (PEEP) is advocated to recruit alveoli during high-frequency jet ventilation (HFJV), but its effect on cardiopulmonary physiology and lung injury is poorly documented. We hypothesized that high PEEP would recruit alveoli and reduce lung injury but compromise pulmonary blood flow (PBF). Preterm lambs of anesthetized ewes were instrumented, intubated, and delivered by cesarean section after instillation of surfactant. HFJV was commenced with a PEEP of 5 cm H2O. Lambs were allocated randomly at delivery to remain on constant PEEP (PEEPconst, n = 6) or to recruitment via stepwise adjustments in PEEP (PEEPadj, n = 6) to 12 cm H2O then back to 8 cm H2O over the initial 60 min. PBF was measured continuously while ventilatory parameters and arterial blood gases were measured at intervals. At postmortem, in situ pressure-volume deflation curves were recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess inflammation. PEEPadj lambs had lower pressure amplitude, fractional inspired oxygen concentration, oxygenation index, and PBF and more compliant lungs. Inflammatory markers were lower in the PEEPadj group. Adjusted PEEP during HFJV improves oxygenation and lung compliance and reduces ventilator requirements despite reducing pulmonary perfusion.