Metformin Improves Cell Viability after in vitro Ischemia‐Reperfusion and Improves Survival with Neuroprotection after Rodent Cardiac Arrest

Abstract

Introduction Cardiac arrest (CA) causes ischemic-reperfusion injury that results in multiorgan damage. Despite successful restoration of circulation, many patients die from neurological injury. Metformin is a widely used drug with multiple affects, such as a modulator of mitochondrial function through reversible inhibition of complex I, attenuating oxidative stress, and activating the AMPK pathway. In the current study, we examined the effects of metformin on single-cultures of neurons and astrocytes after in vitro ischemia-reperfusion injury, while exploring the effects of metformin on survival and neuroprotection in a rodent model of CA. Hypothesis Administering metformin during reperfusion will preserve mitochondrial function and help to improve viability and survival of cells and rodents, respectively. Methods In vitro cell viability analysis (WST-8) was performed after 6 h of oxygen-glucose deprivation (OGD) followed by 20 h of reperfusion (OGD-R) and metformin administration in single-cultures of neurons (mouse cell line HT-22) and astrocytes (mouse cell line C8-D1A). In vivo study was performed in rats after 10 min asphyxial-CA followed by resuscitation and immediate intravenous administration of either metformin (100 mg/Kg in saline), or vehicle (n=16 each). Survival and modified neurological deficit scores (mNDS) were monitored for 72 h. Brains were harvested from the surviving rats for histological evaluation using TUNEL stain on the hippocampus CA1 region. Plasma was taken to measure oxidative stress using Amplex Red assay. Brain mitochondria were isolated for cytochrome C release as a surrogate marker of mitochondrial damage after 2 h post-resuscitation. Results HT-22 neurons showed improvement in survival after OGD-R in a dose dependent manner with metformin treatment without much impact observed in C8-D1A astrocytes. In rodent CA, metformin treatment demonstrated an improved survival at 72 h from 43.8% to 68.8% (p= 0.0692) as well as significantly improved neurological status at 24, 48, and 72 h when compared with vehicle. Metformin also reduced apoptotic neuronal damage when compared with vehicle. Metformin treatment was associated with significantly lower mitochondrial cytochrome C release and decreased oxidative stress compared with vehicle suggesting reduced mitochondrial damage (Figure). Conclusion Our results suggest that metformin improves cell viability after ischemia-reperfusion and survival with better neurologic outcomes after CA in both in vitro and in vivo models, respectively. Overall, metformin could be a potential therapeutic intervention for improving survival and preventing neuronal death after CA.