Endogenous TWEAK mediates the neuroprotective effect of hypoxic preconditioning. (A) Mean fold increase in TWEAK and Fn14 mRNA expression in Wt cerebral cortical neurons 1, 3 and 6 hours after exposure to 30 minutes of OGD (sub-lethal hypoxia). n = 8, * and ** P < 0.05 compared to TWEAK mRNA expression in sister cultures maintained under normoxic conditions. ^, § and ¶ P < 0.05 compared to Fn14 expression in sister cultures maintained under normoxic conditions. Lines denote SD. (B-D) Mean cell survival in Wt (B), TWEAK-/- (C) and Fn14-/- (D) cerebral cortical neurons exposed to 55 minutes of OGD conditions (lethal hypoxia) 24 hours after 30 minutes of exposure to OGD conditions (hypoxic preconditioning, dark gray bars). A subgroup of neurons was exposed to lethal hypoxia without previous preconditioning (black bar). A sub-set of TWEAK-/- and Fn14-/- neurons was incubated with TWEAK 100 ng/mL during the preconditioning phase (hypoxic preconditioning, light gray bars). n = 12 per experimental group; * in B P < 0.05 compared to cells maintained under normoxic conditions; ** in B P <0.05 compared to neuronal cultures exposed to lethal hypoxia without preconditioning. * in C P < 0.05 compared to TWEAK-/- neurons either exposed to lethal OGD without previous preconditioning or preconditioned in the absence of TWEAK. Lines denote SD. Fn14: fibroblast growth factor-inducible 14; HPC: hypoxic preconditioning; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; Ns: non-significant; OGD: oxygen-glucose deprivation; TWEAK: tumor necrosis factor-like weak inducer of apoptosis.