Examining the GABA<sub>A</sub> receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

Abstract

Hypoxic ischaemic encephalopathy (HIE) is the most common cause of mortality and morbidity in the newborn. Reduced blood flow to the foetus, and consequently the foetal brain, due to maternal factors (e.g. hypertension) or foetal factors (e.g. umbilical cord compression); leads to significant brain injury and neurodevelopmental disability. HIE remains the leading cause of seizures in the term and preterm infant, accounting for over 60% of all seizures in the newborn period. Current antiepileptic drugs (AEDs) are largely ineffective in the newborn, however there has been little change to treatments for neonatal seizures over the last 50 years. AEDs achieve good seizure control in children and adults but these same AEDs have limited efficacy in the neonatal brain. Despite evidence that the AED phenobarbital is effective in only 30-50% of babies, it remains the standard first-line treatment for neonatal seizures in neonatal intensive care units around the world.In the perinatal hypoxic-ischaemic (HI) brain, over activation of excitatory neurotransmitter systems plays a key role in the generation of seizures and excitotoxic neuronal cell injury and cell death. In the mature brain GABA (g-aminobutyric acid) hyperpolarises neurones and inhibits neuronal firing, thus providing a protective mechanism by reducing excitability. However, in the immature brain GABA depolarises the neurone due to differences in the chloride (Cl-) gradient across the membrane, and instead creates excitation when GABA binds to the GABAA receptor. Understanding the physiological consequences of activation of the GABA system in the HI newborn brain and in the presence of seizures, is critical to identifying the mechanisms behind the apparent failure of AEDs in the newborn as well as driving development of appropriate drugs for the treatment of neonatal seizures.The general aim of this thesis was to investigate changes to the GABAergic system following perinatal hypoxia and seizures, by examining alterations in expression of the GABAA receptor (GABAAR) a-subunit in the pig brain. The piglet brain growth trajectory most closely mirrors that of the human neonate, with respect to timing of myelination, neuronal and glial growth spurts, and the proportion of grey to white matter. Normal developmental expression of the a-subunits a1, a2, and a3 was characterised across gestation and multiple brain regions revealing the crossover in expression of a3 to a1 expression corresponding to the transition from foetal to postnatal life. There was a significant peak in a3 expression observed at 100d gestation (87% gestation), that coincides with a previously reported peak in prenatal pig brain growth.nIt has been suggested that the transition of the GABAergic neurotransmission system from excitatory, during development, to the mature inhibitory system coincides with the developmental transition of various GABAAR subunits including the a-subunit.A clinically relevant piglet model of perinatal asphyxia was established that mimics an intrapartum HI event, and results in brain injury similar to that seen clinically in the HIE neonate. Animals that developed seizures had significantly more injury, with greater MR abnormalities and histopathology, compared with HI animals that did not develop seizures. GABAAR a1 and a3 expression was reduced in several brain regions at 24 h and 72 h post-HI, particularly in animals that developed seizures after neonatal HI. Regression analysis revealed that loss of the a3 protein was independently associated with seizures and was not solely due to loss of neurones. IHC revealed that, although a1 was not significantly reduced, there was evidence of redistribution of this subunit isoform from the cell surface to the cytoplasmic compartment. During the course of this project a splice variant of GABAAR a3 was identified through western blotting and RNA sequencing. Initial characterisation of this variant showed its expression to be developmentally regulated, with higher expression in the immature brain. In summary, this thesis details that the use of the neonatal piglet is a clinically relevant research model to investigate HI injury in term-born human neonates and, details changes in expression of the predominant a-subunits of the GABAAR; both across development and after neonatal HI. The perinatal period is a critical transition period for GABAAR subunit expression particularly in cortical brain regions- areas that are substantially affected after perinatal asphyxia in the human neonate. Treatment of neonatal seizures involves the administration of AEDs that target the inhibitory actions of the GABAAR. Significant alterations to the expression of GABAAR a- subunits after neonatal seizures may have immediate implications in the ability of GABAergic anticonvulsants to bind to the GABAAR limiting their treatment efficacy. In the long term, altered a-subunit protein expression after neonatal seizures may disturb the developmental transition of the GABA neurotransmitter system to the mature inhibitory system, and increase susceptibility to enhanced excitation in the mature brain that has been documented in various neurological disorders such as post-neonatal epilepsy.