Date of Award

2018

Degree Name

Doctor of Philosophy (College of Health Sciences)

Schools and Centres

Health Sciences

First Supervisor

Associate Professor Bruno Meloni

Second Supervisor

Clinical Professor Neville Knuckey

Third Supervisor

Doctor Ryan Anderton

Abstract

Hypoxic-ischaemic encephalopathy (HIE) is one of the leading causes of mortality and morbidity in infants, globally. This disorder eventuates following a reduction in oxygenated cerebral blood flow to the foetus in utero, leading to excitotoxic-mediated brain cell (e.g. neuron, glia and glial progenitor cell) death. Currently, there is no clinically appropriate neuroprotective treatment to reduce acute brain injury following HIE. Recent studies have demonstrated that poly-arginine and cationic arginine-rich peptides (CARPs; e.g. R18: R = arginine residues) exhibit potent neuroprotective properties in both in vitro and adult animal models of ischaemia, and therefore have the potential to be developed into a neuroprotective treatment to reduce brain injury following HIE. Therefore, the aim of this thesis was to assess the neuroprotective efficacy of CARPs in a model of perinatal HIE in the rat.

To elucidate the neuroprotective efficacy of CARPs, a novel surgical modification to the original in vivo Rice-Vannucci model of perinatal HIE was developed. Using 7- day old Sprague-Dawley rats, brain injury was induced following the permanent ligation of the common and external carotid arteries, followed by a period of transient hypoxia (8% O2/92% N2). Results from this experiment demonstrated that the occlusion of common and external carotid arteries reduced cerebral communicational and/or anastomotic blood flow, reducing variability and improving the reliability in the presence of a cerebral infarct. The demonstration and termination of cerebral communicational and/or anastomotic blood flow improved the pre-clinical assessment of neuroprotective therapies to treat HIE.

The CARPs, R18, R18D (D-enantiomer) and JNKI-1-TATD, were assessed in the modified Rice-Vannucci model of HIE when administered intraperitoneally, immediately after the cessation of hypoxia-ischaemia (HI; 8% O2/92% N2 for 2.5 h). Treatment with R18 and R18D significantly reduced infarct volume and improved behavioural assessments in this model. Surprisingly, the well-characterised neuroprotective peptide JNKI-1-1TATD, used as a positive control and benchmark, did not exhibit any significant neuroprotection. Succeeding positive results obtained following R18D administration immediately after HI, its therapeutic window was further assessed. R18D significantly decreased infarct volume and improved behavioural assessments when administered intraperitoneally up to 1 hour after the cessation of HI; correlating to 3.5 hours since HI onset. To confirm the neuroprotective mechanism of action of CARPs in HIE, an established in vitro primary cortical neuronal excitotoxic injury model was used. Results from this experiment demonstrate that CARPs reduce excitotoxic intracellular calcium influx in a dose-dependent fashion, providing evidence for a role in the reduction of several calcium-dependent pro-cell death cascades. The demonstration of significant neuroprotection following R18 peptide administration provides evidence for a novel therapeutic, which has the potential to reduce brain injury in infants who suffer HIE.

In summary, this thesis has identified a novel surgical modification to improve the reliability and reproducibility of the original Rice-Vannucci model of HIE. In addition, the administration of the R18 and R18D peptides following perinatal HIE, significantly reduces brain injury and improves behavioural assessments when administered up to 3.5 hours after the onset of HI. These findings demonstrate that CARPs provide an exciting and novel approach to reduce brain injury following HIE.

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