Abstract W MP40: Pyruvate Protects Post-Hypoxic Neuronal Cells and a Blood Brain Barrier Model From rtPA Toxicity

Abstract

Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved treatment for ischemic stroke. However, rtPA’s therapeutic window is limited to 4.5 h after stroke onset due to hemorrhagic transformation and neurotoxicity. Here, we demonstrated that the intermediary metabolite pyruvate protects neuronal cells and a blood brain barrier (BBB) model from delayed rtPA toxicity in an in vitro oxygen-glucose deprivation (OGD, 0.5% O 2 )-reoxygenation model of ischemic stroke. After 3 or 6 h OGD, neuronal cells were reoxygenated with 11 mM glucose ± 8 mM pyruvate and/or 10 μg/ml rtPA. Cellular viability, reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP2) activity, and cellular contents of NADPH, NADP + , ATP, MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk were measured. Pyruvate treatment after 3 h OGD decreased cell death by 80% in the absence (P < 0.01) and 64% in the presence (P < 0.01) of rtPA. After 6 h OGD, rtPA exacerbated cell death; pyruvate dampened this effect. Three hours OGD and 4 h reoxygenation + rtPA increased ROS formation by 50%. Pyruvate prevented this ROS formation and doubled antioxidant NADPH/NADP + ratio and ATP content. In the BBB model, 3 h OGD and 24 h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and BBB tight junctions from OGD-reoxygenation and rtPA toxicity while reducing ROS and activating anti-apoptotic signaling. These results support the use of pyruvate as an adjuvant to dampen rtPA’s side effects, thereby extending rtPA’s therapeutic window.