Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-α and ceramide

Brief "preconditioning" ischemia induces ischemic tolerance (IT) and protects the animal brain from subsequent otherwise lethal ischemia. Identification of the signaling steps most proximal to the development of the IT will allow induction of the resistance to ischemia shortly after the onset of stroke. Animal studies demonstrate a key role of tumor necrosis factor-alpha (TNF-alpha) in induction of IT. The sphingolipid ceramide is known as a second messenger in many of the multiple effects of TNF-alpha. We hypothesized that ceramide could mediate IT. We demonstrate that preconditioning of rat cortical neurons with mild hypoxia protects them from hypoxia and O-2-glucose deprivation injury 24 h later (50% protection). TNF-alpha pretreatment could be substituted for hypoxic preconditioning (HP). HP was attenuated by TNF-alpha neutralizing antibody. HP and TNF-alpha pretreatment cause a two- to threefold increase of intracellular ceramide levels, which coincides with the state of tolerance. Fumonisin B-1, an inhibitor of ceramide synthase, attenuated ceramide upregulation and HP. C-2 ceramide added to the cultures right before the hypoxic insult mimicked the effect of HP. Ceramide did not induce apoptosis. These results suggest that HP is mediated via ceramide synthesis triggered by TNF-alpha.