Singh, Ayesha (2022) Activation of Hypoxia Inducible Factor pathway for neuroprotection in in vitro models of ischaemic stroke. Doctoral thesis, Keele University.

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Ischaemic stroke is the most common form of stroke, accounting for appropriate 87% of all strokes. Reperfusion is the only immediate treatment option following ischaemic stroke, however, this option is only applicable to a small percentage of patients. There is an urgent need for intervention to delay or reduce the impact of ischaemia after the stroke. Neuroprotection seeks to restrict injury to the brain parenchyma following an ischaemic insult.

Hypoxia-inducible factors (HIF), is a protein regulated by cellular oxygen tension in mammals. HIF-1 regulates hypoxia inducible genes such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). HIF levels in cells and tissue are tightly controlled by HIF prolyl hydroxylases (PHDs). Pharmacological inhibition of PHD will lead to HIF signalling pathway activation, thus upregulating hundreds of HIF downstream genes.

This study aimed to characterize the effectiveness and underlying mechanism of action of neuroprotective strategies in hypoxic / ischaemic preconditioning with a novel class of PHD inhibitors, as well as by astrocyte conditioned media (ACM) using an in vitro model of ischaemic stroke.

Cerebral ischaemia was modelled in vitro by omission of glucose alone (GD), omission of oxygen alone (OD), and simultaneous omission of glucose and oxygen (OGD) in PC12 cells, primary rat neurons, and astrocytes. Both PC12 cells and primary neurons had similar sensitivities towards OD and OGD. Primary astrocytes were more resistant to OD and OGD insults compared to primary neurons. GD, OD, and OGD altered the HIF1α and HIF2α in all these cells. Preconditioning with sublethal OD and OGD was protective in PC12 cells, primary neurons, and primary astrocytes against subsequent severe OGD (0.3% O2) insults.

The effects of a novel class of PHD inhibitors such as FG4592 and Bayer 85-3934 were applied in the ischaemic tolerant model in PC12 cells and primary neurons along with a non-specific PHD inhibitor such as Dimethyloxalylglycine (DMOG). These PHD inhibitors upregulated HIF1α in normoxia (ambient air) and promoted autophagy. Preconditioning with the clinical PHD inhibitors followed by reversion was protective in PC12 cells and primary neurons against subsequent severe OGD insults by reducing cell death. In addition, the effects of astrocyte hypoxia / ischaemia preconditioning on primary neuronal cultures following OGD were studied. ACM from 6h OGD protected neurons from subsequent OGD damage while ACM from 24 h OGD further damaged the neurons following OGD.

In conclusion, preconditioning strategies that activate HIF1α, HIF2α and / or autophagy were protective in the in vitro model of ischaemic tolerance. The PHD inhibitors possess capabilities to stabilise HIF1α and induced autophagy and are neuroprotective in the cellular models of ischaemic tolerance. Overall, all the interventions explored activated either HIF1α, HIF2α, and / or autophagy in the cells. The link between HIF activation, autophagy, and ischaemic tolerance warrants further investigation.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > RS Pharmacy and materia medica
Divisions: Faculty of Medicine and Health Sciences > School of Pharmacy and Bioengineering
Contributors: Chen, R (Thesis advisor)
Jenkins, SI (Thesis advisor)
Depositing User: Lisa Bailey
Date Deposited: 23 Jun 2022 14:04
Last Modified: 23 Jun 2022 14:04

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