Interneuron Function and Survival Following Hypoxia-Ischemia in the Neonatal Rat

Abstract

Despite neonatal hypoxic-ischemic encephalopathy (HIE) being a leading cause of death and disability worldwide, the underlying cellular basis for the associated morbidity and mortality is not well understood. The aim of the present study was to investigate the relationship between experimental hypoxia-ischemia (HI) (Rice Vannucci (RV) model) and hippocampal injury in the neonatal rat, using immunohistochemistry, digital droplet PCR, and live EEG recordings to explore the cellular and molecular changes after injury. Postnatal day (P) 10 Sprague Dawley rats were subject to a unilateral carotid artery ligation procedure followed by systemic hypoxia (8% oxygen) for 80 minutes. Pups were euthanised at 4 h, 1 d, 3 d, or 7 d after injury. We determined a significant reduction in perineuronal nets (PNNs) in the hippocampus at 7 d following injury (P17), with regional vulnerability identified in the CA3 sub-region; and this was significantly normalised to sham after subcutaneous treatment with Tonabersat. Disrupted expression of parvalbumin positive (PV+) interneurons, hyaluronic acid (HA), and glial fibrillary acidic protein (GFAP) was observed as early as 4 h after injury, worsening with each time point. Gene expression analyses revealed no significant changes in the hippocampal expression of GFAP, ACAN, BCAN, NCAN, MMP2, MMP9, HAS2, HYAL1, HYAL2, and HYAL3 after injury. Baseline EEG amplitude, mean EEG intensity, and mean overall EEG intensity were all significantly reduced following HI compared to sham, and ipsilateral seizures were observed in the cortex of injured animals. The results of this study suggest a role for the GABAergic system in the underlying pathogenesis of neonatal HIE, and provide evidence for the therapeutic potential of pharmacological treatment against the spread of HI injury. Further, we lay important groundwork for the implementation of live, continuous EEG recordings in the detection of HI induced seizures and changes in brainwave activity. This study paves the way for future studies into the underlying cellular mechanisms of neonatal HIE.