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The developing brain is particularly vulnerable to foetal growth restriction (FGR) and abnormal neurodevelopment is common in the FGR infant ranging from behavioural and learning disorders to cerebral palsy. No treatment exists to protect the FGR newborn brain. Recent evidence suggests inflammation may play a key role in the mechanism responsible for the progression of brain impairment in the FGR newborn, including disruption to the neurovascular unit (NVU). We explored whether ibuprofen, an anti-inflammatory drug, could reduce NVU disruption and brain impairment in the FGR newborn. Using a preclinical FGR piglet model, ibuprofen was orally administered for 3 days from birth. FGR brains demonstrated a proinflammatory state, with changes to glial morphology (astrocytes and microglia), and blood-brain barrier disruption, assessed by IgG and albumin leakage into the brain parenchyma and a decrease in blood vessel density. Loss of interaction between astrocytic end-feet and blood vessels was evident where plasma protein leakage was present, suggestive of structural deficits to the NVU. T-cell infiltration was also evident in the parenchyma of FGR piglet brains. Ibuprofen treatment reduced the pro-inflammatory response in FGR piglets, reducing the number of activated microglia and enhancing astrocyte interaction with blood vessels. Ibuprofen also attenuated plasma protein leakage, regained astrocytic end-feet interaction around vessels, and decreased T-cell infiltration into the FGR brain. These findings suggest postnatal administration of ibuprofen modulates the inflammatory state, allowing for stronger interaction between vasculature and astrocytic end-feet to restore NVU integrity. Modulation of the NVU improves the FGR brain microenvironment and may be key to neuroprotection.

The foetal brain is particularly vulnerable to the detrimental effects of foetal growth restriction (FGR) with subsequent abnormal neurodevelopment being common. There are no current treatments to protect the FGR newborn from lifelong neurological disorders. This study examines whether pure foetal mesenchymal stromal cells (MSC) and endothelial colony-forming cells (ECFC) from the human term placenta are neuroprotective through modulating neuroinflammation and supporting the brain vasculature. We determined that one dose of combined MSC-ECFCs (cECFC; 106 ECFC 106 MSC) on the first day of life to the newborn FGR piglet improved damaged vasculature, restored the neurovascular unit, reduced brain inflammation and improved adverse neuronal and white matter changes present in the FGR newborn piglet brain. These findings could not be reproduced using MSCs alone. These results demonstrate cECFC treatment exerts beneficial effects on multiple cellular components in the FGR brain and may act as a neuroprotectant.

Fetal brain development is particularly vulnerable to the effects of fetal growth restriction (FGR) and abnormal neurodevelopment is common in the FGR infant. Adverse outcomes range from behavioural and learning disorders through to cerebral palsy. Unfortunately, no treatment exists to protect the FGR newborn brain. Recent evidence suggests inflammation may play a key role in the mechanism responsible for the progression of brain impairment in the FGR newborn, including disruption to the neurovascular unit (NVU). We explored whether ibuprofen, a non-steroidal anti-inflammatory drug, could reduce NVU disruption and brain impairment in the FGR newborn. We used a preclinical piglet model of growth restriction in which FGR occurs spontaneously. Newborn FGR (birthweight th centile) and normally grown piglets were collected on day of birth and oral ibuprofen was administered daily for 3 days (20mg/kg/day, day 1 and 10mg/kg/day days 2 and 3). FGR brains demonstrated an inflammatory state, with changes to glial morphology (astrocytes and microglia), and blood brain barrier disruption, assessed by IgG and albumin leakage into the brain parenchyma and a decrease in blood vessel density. Loss of interaction between astrocytic end-feet and blood vessels was evident in the microvasculature where leakage was present, suggestive of structural deficits to the NVU. Ibuprofen treatment reduced the pro-inflammatory response in FGR piglets, reducing levels of pro-inflammatory cytokines and number of activated microglia and astrocytes associated with blood vessels. Ibuprofen treatment also attenuated albumin and IgG leakage. There were no alterations to angiogenesis, or blood vessel proliferation in treated-FGR piglets. These findings suggest postnatal administration of ibuprofen on day of birth modulates the inflammatory state, allowing for stronger interaction between vasculature and astrocytic end-feet to restore NVU integrity. These changes to the FGR brain microenvironment may be key to neuroprotection.

Preterm infants are at higher risk of adverse neurodevelopmental outcomes. Inadequate cerebral oxygen delivery resulting from poor cardiovascular function is likely to be a significant contributor to preterm brain injury. In this context, improved support of cardiovascular function is integral to improving preterm outcomes. Many of the treatments used to support preterm cardiovascular function are based on adult physiology and may not be appropriate for the unique physiology of the preterm infant. The preterm heart is structurally immature with reduced contractility and low cardiac output. However, there is limited evidence that inotropic support with dopamine and/or dobutamine is effective in preterm babies. Hypovolemia may also contribute to poor preterm cardiovascular function; there is evidence that capillary leakage results in considerable loss of plasma from the circulation of newborn preterm babies. In addition, the vasoconstrictor response to acute stimuli does not develop until quite late in gestation and is limited in the preterm infant. This may lead to inappropriate vasodilatation adding to functional hypovolemia. The first line treatment for hypotension in preterm infants is volume expansion with crystalloid solutions, but this has limited efficacy in the preterm infant. More effective methods of volume expansion are required. Effective support of preterm cardiovascular function requires better understanding of preterm cardiovascular physiology so that treatments can target mechanisms that are sufficiently mature to respond.

Intrauterine growth restriction (IUGR) is one of the most common causes of perinatal mortality and morbidity. White matter and neuronal injury are major pathophysiological features of the IUGR neonatal brain. GABAA (γ-aminobutyric acid type A) receptors have been shown to play a role in oligodendrocyte differentiation and proliferation in the neonatal brain and may be a key factor in white matter injury and myelination in IUGR neonates. Whether there are impairments to the GABAergic system and neuronal cytoskeleton in IUGR brain has yet to be elucidated. This study aims to examine GABAA receptor α1 and α3 subunit protein expression and distribution in parietal cortex and hippocampus of the IUGR piglet at four different ages (term = 115 d – days gestational age), 100 d, 104 d, birth (postnatal day 0–P0) and P7 and to examine neuronal and myelination patterns. Significant alterations to GABAA receptor α1 and α3 protein expression levels were observed in the IUGR piglet brain of P7 IUGR piglets with significantly greater α3 expression compared to α1 expression in the hippocampus while there was virtually no difference between the two subunits in the parietal cortex. However a significantly lower α1/α3 ratio was evident in P7 IUGR cortex when compared with P7 NG cortex. Neuronal somatodendrites studied using MAP2 immunohistochemistry showed reduced and disrupted somatodendrites while MBP immunolabelling showed loss of axonal fibres from gestational day 104 d through to P7. These findings provide insights into the effects of IUGR on the development of the GABA system, altered developmental maturation of GABAA receptor subunit expression in the IUGR brain may influence myelination and may partly explain the cognitive disabilities observed in IUGR. Understanding the mechanisms behind grey and white matter injury in the IUGR infant is essential to identifying targets for treatments to improve long-term outcomes for IUGR infants.

GABA is a major neurotransmitter in the mammalian brain. In the mature brain GABA exerts inhibitory actions via the GABAA receptor (GABAAR); however, in the immature brain GABA provides much of the excitatory drive. We examined the expression of 3 predominant GABAA α-subunit proteins in the pig brain at various pre- and postnatal ages. Brain tissue was collected from piglets born via caesarean section at preterm ages 91, 97, 100, and 104 days' gestational age (GA), at term equivalent (114 days' GA, caesarean section) and at term, postnatal day 0 (P0) (spontaneous delivery, term = 115 days). Tissue was obtained from piglets at P4 and P7. Adult tissue from sows was collected postmortem after caesarean section. In all cortical regions and basal ganglia (1) α3 exhibited a significant increase in protein expression at 100 days' GA, (2) α3 expression decreased with age after 100 days' GA, (3) α1 increased with age, with peak expression at P7 in cortices, hippocampus, and thalamus, and (4) α2 protein expression remained relatively constant across the ages examined. The subunit expression of α3 was most abundant at preterm ages, with α1 the predominant subunit expressed postnatally. Immunofluorescent labelling revealed α1 expression on the somatic membranes of pyramidal cells in the cortex and hippocampus, and in the cerebellar Purkinje cells. Positive α3 labelling was apparent on interneurones in the cortex and hippocampus. The switch between dominant α-subunits may coincide with the functional change in GABAergic neurotransmission from excitation to inhibition. Brain growth in the pig closely reflects that in the term human, making the pig a valuable non-primate model for studying development and the effects of insults on the perinatal brain.

Seizures in the neonatal period are most often symptomatic of central nervous system (CNS) dysfunction and the most common cause is hypoxic-ischaemic encephalopathy (HIE). Seizures are associated with poor long-term outcomes and increased neuropathology. Blood-brain barrier (BBB) disruption and inflammation may contribute to seizures and increased neuropathology but are incompletely understood in neonatal HIE. The aim of this study was to investigate the impact of seizures on BBB integrity in a preclinical model of neonatal hypoxic-ischaemic (HI) injury. Piglets (age

The principal function of the γ-aminobutyric acid (GABA) system in the adult brain is inhibition; however, in the neonatal brain, GABA provides much of the excitatory drive. As the brain develops, transmembrane chloride gradients change and the inhibitory role of GABA is initiated and continues throughout juvenile and adult life. Previous studies have shown that GABAA receptor subunit expression is developmentally regulated, and it is thought that the change in GABA function from excitation to inhibition corresponds to the changeover in expression of ‘immature’ to ‘mature’ subunit isoforms. We examined the protein expression pattern and distribution of GABA type A (GABAA) receptor α1-, α3- and β2-subunits in the parietal cortex and hippocampus of the developing piglet brain. Four perinatal ages were studied; 14 days preterm (P–14), 10 days preterm (P–10), day of birth (P0) and at postnatal day 7 (P7). Animals were obtained by either caesarean section or spontaneous birth. Protein expression levels and subunit localization were analysed by Western blotting and immunohistochemistry, respectively. In the cortex and hippocampus, GABAA receptor α1-subunit showed greatest expression at P7 when compared to all other age groups (p < 0.05). In contrast, α3 expression in the cortex was elevated in preterm brain, peaking at P0, followed by a significant reduction by P7 (p < 0.05); a similar trend was observed in the hippocampus. GABAA receptor β2-subunit protein expression appeared relatively constant across all time points studied in both the cortex and hippocampus. Immunolabelling of the α1-, α3- and β2-subunits was observed throughout all cortical layers at every age. GABAA receptor α3-subunit appeared to show specific localization to layers V and VI whilst labelling for the β2-subunit was observed in layer IV. In the hippocampus of all animals, the α1- and β2-subunits were shown to immunolabel various cells and processes in the dentate gyrus (DG), CA1 and CA3; the α3-subunit was barely observed except at the stratum moleculare of the DG. We report for the first time the ontogenesis of GABAA receptor subunits α1, α3 and β2 in the perinatal pig brain.

Hypoxia-ischemia is a significant cause of brain damage in the human newborn and can result in long-term neurodevelopmental disability. The loss of oxygen and glucose supply to the developing brain leads to excitotoxic neuronal cell damage and death; such over-excitation of nerve cells can also manifest as seizures. The newborn brain is highly susceptible to seizures although it is unclear what role they have in hypoxic-ischemic (H/I) injury. The aim of this study was to determine an association between seizures and severity of brain injury in a piglet model of perinatal H/I and, whether injury severity was related to type of seizure, i.e. sub-clinical (electrographic seizures only) or clinical (electrographic seizures+physical signs). Hypoxia (4% O(2)) was induced in anaesthetised newborn piglets for 30 min with a final 10 min period of hypotension; animals were recovered and survived to 72 h. Animals were monitored daily for seizures both visually and with electroencephalogram (EEG) recordings. Brain injury was assessed with magnetic resonance imaging (MRI), (1)H-MR spectroscopy ((1)H-MRS), EEG and by histology (haematoxylin and eosin). EEG seizures were observed in 75% of all H/I animals, 46% displayed clinical seizures and 29% sub-clinical seizures. Seizure animals showed significantly lower background amplitude EEG across all post-insult days. Presence of seizures was associated with lower cortical apparent diffusion coefficient (ADC) scores and changes in (1)H-MRS metabolite ratios at both 24 and 72 h post-insult. On post-mortem examination animals with seizures showed the greatest degree of neuropathological injury compared to animals without seizures. Furthermore, clinical seizure animals had significantly greater histological injury compared with sub-clinical seizure animals; this difference was not apparent on MRI or (1)H-MRS measures. In conclusion we report that both sub-clinical and clinical seizures are associated with increased severity of H/I injury in a term model of neonatal H/I.

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

Background Large animal models are an essential tool in the development of rationally-based new clinical therapies for preterm infants. We provide a description of the newborn pig as a model of the preterm neonate in terms of growth parameters, physiology and the requirement for intensive care over a range of gestational ages. Methods Twenty-nine litters of piglets (n = 298) were delivered by caesarean section at six timepoints during gestation from 91d to 113d (term = 115d). Two groups, at 91 and 97d gestation, also received maternal glucocorticoid treatment. At four of these timepoints, piglets (n = 79) were ventilated, sedated and monitored using standard neonatal intensive care techniques for up to 8 h in various experimental protocols. Results Body weight increased from mean 697 g (SD 193) at 91d gestation to 1331 g (SD 368) at 113d gestation. Piglets delivered at 97d gestation were able to be resuscitated and kept alive for at least 8 h on respiratory support after surfactant administration. Maternal glucocorticoid treatment 48 h and 24 h hours prior to delivery reduced the requirement for ventilator support and improved cardiovascular stability. Conclusion The pig provides a relevant model for the study of human preterm physiology and for investigation of novel therapies to improve outcomes.

Background and aims: Current therapeutic options for neonatal hypoxia/ischemia (HI) are limited to hypothermia and treatment of seizures. The principal function of the GABA system in mature brain is inhibition, however in neonatal brain GABA provides much of the excitatory drive. Thus whilst anticonvulsants augment GABA's inhibitory actions in mature brain, administration of GABAergic drugs to neonates may potentially exacerbate seizures and worsen HI brain injury. Furthermore changes in GABAA receptor expression will influence receptor pharmacology. We aimed to assess changes in protein expression of the GABAA receptor in the neonatal HI piglet. Methods: Newborn piglets were subjected to a 30 min HI insult and euthanased at 72 h. Brain tissue was collected from several brain regions and GABAA receptor α3 protein expression levels analysed by western blot. Results: Expression of α3 was significantly elevated in frontal cortex of HI animals without seizure; animals with seizure trended toward lower a3 expression. In occipital cortex, α3 expression was found to be diminished in all HI animals however presence of seizures was associated with significantly lower α3 expression. Conclusions: GABAA receptor α3 expression was significantly altered following neonatal HI; presence of seizures further changed this expression. Efficacy of anticonvulsants in neonatal brain may not only depend on regional and temporal maturation of GABAergic inhibitory function but also on GABAA receptor subunit expression following both HI and seizures. There is a critical need to develop effective treatment strategies specific to the neonatal HI brain.

Background and Aims: Antiepileptic drugs (AEDs) do not effectively control seizures in newborn brain and there is growing concern that use of standard AEDs such as phenobarbital may exacerbate seizures and brain injury. Recent data suggests this may be due to the changing role of the GABAA receptor (an AED target) during normal neurodevelopment, a process tightly coupled to developmental expression of the chloride co-transporter proteins NKCC1 and KCC2. The timing of the 'switch' in GABAA receptor function from excitation to inhibition is unclear and in human may not be completed until 3-4 months of age. The aim of this project was to establish the developmental profile of NKCC1, KCC2 and the GABAA receptor µ1 and µ3 subunits in piglet brain. Methods: Preterm and term piglets of several different gestational ages around birth were obtained by caesarean section or spontaneous delivery. Brain tissue was collected from several brain regions and expression analysed by western blot. Results: In parietal cortex the switch between NKCC1 and KCC2 and, GABAA receptor µ1 and µ3 expression was evident by P3. The switch in GABAA receptor µ1 and µ3 expression had not yet occurred in the frontal, temporal or occipital cortex. NKCC1, KCC2 analysis is ongoing. Conclusions: Efficacy of AEDs in neonatal brain may depend on regional and temporal maturation of inhibition. Furthermore changes in GABAA receptor expression as we have demonstrated following HI and seizures will influence receptor pharmacology. There is a critical need to develop effective treatment strategies specific to the neonatal brain.