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

Brief "preconditioning" ischemia induces ischemic tolerance (IT) and protec ts the animal brain from subsequent otherwise lethal ischemia. Identificati on of the signaling steps most proximal to the development of the IT will a llow induction of the resistance to ischemia shortly after the onset of str oke. Animal studies demonstrate a key role of tumor necrosis factor-alpha ( TNF-alpha) in induction of IT. The sphingolipid ceramide is known as a seco nd 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-gluco se deprivation injury 24 h later (50% protection). TNF-alpha pretreatment c ould 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 wi th the state of tolerance. Fumonisin B-1, an inhibitor of ceramide synthase , attenuated ceramide upregulation and HP. C-2 ceramide added to the cultur es right before the hypoxic insult mimicked the effect of HP. Ceramide did not induce apoptosis. These results suggest that HP is mediated via ceramid e synthesis triggered by TNF-alpha.