Failure and rescue of preconditioning-induced neuroprotection in severe stroke-like insults

Abstract Preconditioning is a well established neuroprotective modality. However, the mechanism and relative efficacy of neuroprotection between diverse preconditioners is poorly defined. Cultured neurons were preconditioned by 4-aminopyridine and bicuculline (4-AP/bic), rendering neurons tolerant to normally lethal (sufficient to kill most neurons) oxygen-glucose deprivation (OGD) or a chemical OGD-mimic, ouabain/TBOA, by suppression of extracellular glutamate (glutamate ex ) elevations. However, subjecting preconditioned neurons to longer-duration supra-lethal insults caused neurotoxic glutamate ex elevations, thereby identifying a ‘ceiling’ to neuroprotection. Neuroprotective ‘rescue’ of neurons could be obtained by administration of an NMDA receptor antagonist, MK-801, just before glutamate ex rose during these supra-lethal insults. Next, we evaluated if these concepts of glutamate ex suppression during lethal OGD, and a neuroprotective ceiling requiring MK-801 rescue under supra-lethal OGD, extended to the preconditioning field. In screening a panel of 42 diverse putative preconditioners, neuroprotection against normally lethal OGD was observed in 12 cases, which correlated with glutamate ex suppression, both of which could be reversed, either by the inclusion of a glutamate uptake inhibitor (TBOA, to increase glutamate ex levels) during OGD or by exposure to supra-lethal OGD. Administrating MK-801 during the latter stages of supra-lethal OGD again rescued neurons, although to varying degrees dependent on the preconditioning agent. Thus, ‘stress-testing’ against the harshest ischemic-like insults yet tested identifies the most efficacious preconditioners, which dictates how early MK-801 needs to be administered during the insult in order to maintain neuroprotection. Preconditioning delays a neurotoxic rise in glutamate ex levels, thereby ‘buying time’ for acute anti-excitotoxic pharmacologic rescue.