Neonatal seizures are associated with redistribution and loss of GABA_A α‐subunits in the hypoxic‐ischaemic pig

Abstract

Seizures are a common manifestation of hypoxic-ischaemic brain injury in the neonate. In status epilepticus models alterations to GABA R subunit expression have been suggested to contribute to (i) abnormal development of the GABAergic system, (ii) why seizures become self-sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABA R subunit protein expression after neonatal hypoxia-ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α-subunits of the GABA R; α , α and α . Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude-integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post-mortem and GABA R α-subunit protein expression was analysed using western blot and immunohistochemistry. GABA R α and α protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α and α expression, and α redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABA R α reduction. This is the first study to show loss and redistribution of GABA R α-subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.

GABAA receptor expression and white matter disruption in intrauterine growth restricted piglets

“…Brain tissue from the parietal cortex and hippocampus were homogenised in 10× volume distilled water, centrifuged at 1400 g for 5 min at 4 °C and supernatant collected as previously described (Miller et al, 2016). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Victoria, Australia).…”

“…Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Victoria, Australia). Protein samples were separated by 10% SDS‐PAGE, transferred to PVDF membrane and probed with anti‐GABA A receptor α 1 (1:2000, Millipore, USA) and anti‐GABA A receptor α 3 (1:1000, Millipore, USA) as previously described (Kalanjati et al, 2011; Miller et al, 2016). Following incubation with secondary anti‐rabbit IgG‐peroxidase antibody (1:30 000, Sigma Aldrich, USA) for 1 h at RT, proteins were visualised on X‐ray film with ECL reagent (GE Healthcare, Australia) and quantified with Image‐J software.…”

“…Following incubation with secondary anti‐rabbit IgG‐peroxidase antibody (1:30 000, Sigma Aldrich, USA) for 1 h at RT, proteins were visualised on X‐ray film with ECL reagent (GE Healthcare, Australia) and quantified with Image‐J software. A pooled protein sample of all samples was used on every gel (5, 10 and 20 μg) as a control for quantification as previously described (Goasdoue et al, 2016; Miller et al, 2016).…”

Neuropathology in intrauterine growth restricted newborn piglets is associated with glial activation and proinflammatory status in the brain

“…varying degrees of brain maturation at birth. The piglet is regarded an appropriate animal to examine altered brain development arising from comprising perinatal events [28–30]. The newborn piglet is similar to the human newborn infant in size, development, circulation, metabolism and cerebral maturation.…”

Simple and reproducible approaches for the collection of select porcine ganglia

“…These include cystic fibrosis 19–21 , muscular dystrophy 22 , diabetes 23 , cardiovascular disease 24,2526 , diabetes 27 , cancer 28,29 , environmental toxicology 30 , cutaneous wound healing 31 , metabolic syndrome 32 , among others. Porcine models have also been used to investigate traumatic brain injury 3 , neurodegenerative diseases 33,34 , brain development 35 , seizures 36 , cognition 37 , and neurogenesis 38 .…”