Pyruvate protects the HT22 neuronal cells treated with rtPA against hypoxia‐reoxygenation damage

Abstract

Recombinant tissue plasminogen activator (rtPA) is the only FDA approved treatment for ischemic stroke, but delayed rtPA treatment may be neurotoxic. Pyruvate has proven neuroprotective in animal models of stroke. We examined pyruvate + rtPA cotreatment of neuronal cells subjected to an in vitro stroke model.MethodsHT22 cells were exposed to 2 or 6 h hypoxia (0.5% O2) + glucose deprivation (OGD), followed by 24 h reoxygenation. Pyruvate (8 mM) and/or rtPA (10 μg/ml) were applied upon reoxygenation. Cell viability was assessed with Calcein AM. DCF‐DA and mitosox staining detected reactive oxygen species (ROS). Matrix metalloproteinase (MMP) activities were measured by gelzymography; MMP, tissue inhibitor of metalloproteinase 2, HIF‐1α, EPO and actin contents were determined by immunoblot.ResultsOGD lowered cell viability by 50% vs. normoxic control (P <0.05). Although rtPA treatment did not increase post‐OGD cell death, it intensified ROS production and activated MMP. Pyruvate‐induced cytoprotection was not compromised by rtPA cotreatment, and pyruvate dampened rtPA‐induced ROS and MMP. Delayed rtPA treatment intensified cell death 2 h after OGD, but pyruvate salvaged 50% of these cells.ConclusionPyruvate attenuates rtPA‐induced MMP activation and ROS formation, thereby protecting reoxygenated HT22 cells. Pyruvate could be a useful cotreatment with rtPA for ischemic stroke. NIH support: R01NS054687, R01NS054651, R01NS076975, T32AG020494