Simulated Stroke in a Mouse Neuronal Cell Culture Model

Abstract

INTRODUCTIONDespite many efforts, stroke remains a cause of debilitating illness and death worldwide. The fact that not only ischemia itself, but also the return of blood flow aggravates cellular dysfunction, illustrates a clinical barrier. Hence, catheter‐based interventions, the therapy of ischemic strokes, can add detrimental tissue damage, described as ischemia/reperfusion (I/R) injury. It is therefore crucial to develop potential pharmacological strategies that attenuate reperfusion injury and improve neurological function. Animal in‐vivo and in‐vitro models are vital to determine potential targets for pharmacological therapies. While in‐vivo models better mimic the complexity of the clinical context, in‐vitro studies allow to focus on the pathophysiology and can be performed on individual cell types. This study aimed to establish an in‐vitro stroke model by investigating the effect of simulated I/R injury on mouse brain isolated neurons.METHODSNeurons were plated and grown to confluency. To simulate stroke, cells were then randomized to either control/normoxic (21% O2, 5% CO2, 74% N2) or hypoxic conditions in a hypoxic chamber (0.01% O2, 5% CO2, 94.99% N2) in serum‐ and glucose‐free media. Durations of hypoxia for 2.5, 5 and 10h were compared. After 2h of reoxygenation in regular media and normoxic culture conditions following hypoxia/normoxia, samples were assayed for cell number/viability, cytotoxicity by lactate dehydrogenase (LDH) release and mitochondrial viability. Statistics: Mann‐Whitney Rank Sum Test and regression analysis; alpha=0.05.RESULTSHypoxia significantly impaired cell number/viability after 5 and 10h, whereas 2.5h of hypoxia did not. A large decrease of cell number/viability was observed after 10h of hypoxia compared to 5h and 2.5h. The same observations were made for mitochondrial viability, apart from a slightly lower decrease after 5h hypoxia compared to the cell number/viability. Inverse relations were found for LDH release, except for an already significant increase in cytotoxicity after 2.5h of hypoxia.CONCLUSIONSOur data demonstrate that hypoxic injury in this model is time‐dependent. While 2.5h did not result in sufficient damage and 10h injured neurons too excessively, 5h hypoxia led to a moderate enough damage, still allowing for potential attenuation through protective strategies. Therefore, 5h hypoxia, followed by 2h reperfusion, appears to be a suitable duration for an in‐vitro mouse neuron stroke model to test new treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.