Protective and Restorative Mitochondrial Therapeutics in Ischaemic Stroke

Abstract

Background: Ischemic stroke is the most common form of stroke, accounting for 87% of all cases. The only immediate treatment option following ischemic stroke is reperfusion, which can be accomplished through thrombolysis using recombinant tissue plasminogen activator or clot removal by mechanical thrombectomy. However, these therapeutic procedures are only applicable to a small percentage of patients because of the short therapeutic time window. There is an urgent need for ischaemic stroke treatments that limit tissue damage or reduce the consequences of stroke. Neuroprotection aims to limit or reduce injury to the brain parenchyma following ischemic insult by preventing the death of salvageable neurons in the penumbra. Traditional neuroprotection approaches have shown promise in experimental studies but have currently failed to translate into clinical success. Our novel approach involves investigating strategies that improve cellular bioenergetics in ischemic tissue by targeting mitochondria. Optimal mitochondrial function is essential for cellular bioenergetics and health. Numerous studies have shown that mitochondrial function and cellular bioenergetics are disturbed during ischemia, which consequently leads to cellular injury and death. We sought to enhance cellular bioenergetics during and after experimental ischaemia in vitro and in vivo by administration of exogenous therapeutic agents and/ or mitochondrial transplantation. We hypothesized that: 1. Mitochondrial protective molecules will reduce cellular injury and death in ischemic neurons in vitro and will reduce tissue damage in mouse focal ischemia. 2. Transplantation of healthy mitochondria will reduce cellular injury and death in ischemic neurons in vitro and will reduce tissue damage in mouse focal ischemia. 3. Combination treatment (transplantation of healthy mitochondria and mitochondrial protective molecules) will provide synergistic protection against experimental ischemia. Methodology: Two hours of acute oxygen glucose deprivation (OGD) with 1mM glucose and 1% O2 was established and characterized in our lab to study the effect of the proposed therapeutic molecules (nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN)) and/or mitochondrial transplantation. After exogenous molecule administration and/or mitochondrial transplantation, cell proliferation and lactate dehydrogenase (LDH) release was used to assess cellular number and determine cellular injury, respectively. Cellular metabolic function was also determined by measuring ATP, NADH, mitochondrial, and glycolytic bioenergetic function. Confocal microscopy combined with high content imaging analysis (Columbus™) was conducted to assess mitochondrial entry and viability in cultured neurons. Finally, we tested mitochondrial transplantation -/+ NR supplementation in a mouse transient middle cerebral artery occlusion model of stroke to assess the effect on infarction volume. All data underwent normality distribution analysis testing prior to parametric or non-parametric analysis using GraphPad Prism software. Results: NR and NMN improved mitochondrial energy production prior to and during OGD, with NR being more potent showing a significant protective and restorative effect on mitochondrial function post-OGD. We also demonstrated that viable, bioenergetically active mitochondria can be isolated from mouse muscle and taken up by neurones in vitro. Furthermore, in a mouse transient focal ischemia model, NR or intravenous administration of healthy mitochondria alone reduced stroke volume 24 hours post stroke onset. In combination, a synergistic effect was observed. Conclusions: These data show that NR and NMN protect neurons against ischaemia before, during, and after acute OGD. Furthermore, we have shown that NR alone or in combination with mitochondrial administration reduces infarct size in mouse focal ischaemia. These studies raise the exciting possibility that administering molecules that protect mitochondria alone or in combination with healthy mitochondria could be effective treatments for ischemic stroke.