We then used a previously described in vitro model selleck chem of preconditioning to investigate whether treatment with sub lethal concentrations of TWEAK renders neu rons resistant to a subsequent lethal hypoxic injury. Wt cerebral cortical neurons were either left untreated Inhibitors,Modulators,Libraries or incubated with TWEAK 0 to 300 ng mL for 60 minutes followed 24 hours later by exposure to 55 minutes of OGD conditions as depicted in Figure 1B and described in the Methods section. Cell survival was quantified 24 hours later with an MTT assay. Our results indicate that exposure to OGD conditions decreases neuronal survival from 100 0. 11% to 46. 2 2. 8% in non preconditioned cells. Inhibitors,Modulators,Libraries In contrast, cell survi val in neurons preconditioned with 30, 100 or 300 ng mL of TWEAK 24 hours before Inhibitors,Modulators,Libraries exposure to OGD con ditions was 79. 4 3.

1%, 64. 9 2. 0% and 58. 3 4%, respectively. To determine whether the protective effect of precon ditioning with TWEAK is also observed at later time points, we quantified cell survival 24, 48 and 72 hours after exposure to 55 minutes of OGD conditions. We found that cell survival decreased from 100 Inhibitors,Modulators,Libraries 0. 8% in control neurons to 54. 80 2. 4% in neurons exposed to OGD conditions without preconditioning with TWEAK. In contrast, preconditioning with TWEAK 24 hours before exposure to OGD conditions increased neuronal survival to 72. 10 1. 92%, 77 1. 1% and 80 2. 0%, when the MTT assay was performed 24, 48 and 72 hours after exposure to OGD, respectively.

To investigate whether the protective effect of TWEAK is mediated by its interaction with Fn14, we quantified cell survival in Wt and Fn14 cerebral corti cal neurons incubated with TWEAK 100 ng mL for 1 hour followed 24 hours later by exposure Inhibitors,Modulators,Libraries to 55 minutes of OGD conditions. We found that exposure of non preconditioned neurons to OGD conditions decreased cell survival from 100 0. 2% to 50. 3 1. 2% in Wt neu rons and from 100 0. 88% to 40. 8 2. 4% in Fn14 neurons. In contrast, cell survival in Wt and Fn14 neurons preconditioned with TWEAK was 66. 13 1. 8% and 41. 3 1. 8%, respectively, indicating that genetic deficiency of Fn14 abro gates the ability of TWEAK to induce tolerance to the deleterious effects of OGD. Endogenous TWEAK mediates the development of hypoxic and ischemic tolerance It has been demonstrated that exposure to 30 minutes of OGD conditions not only does not induce cell death but instead renders cere bral cortical neurons tolerant to a lethal hypoxic injury applied at later time points. Based on these observations we decided to investigate whether endogenous TWEAK plays a role in the protective effect of hypoxic sellckchem preconditioning. First, we studied the effect of sub lethal hypoxia on TWEAK and Fn14 expression.