Our read FAQ results indi cate that sub lethal hypoxia induces a rapid and transi ent increase in TWEAK and Fn14 mRNA expression in cerebral cortical neurons that is maximal at 1 hour for TWEAK and 3 hours for Fn14. We then quantified cell survival in Wt, TWEAK and Fn14 cerebral cortical neurons exposed to sub lethal hypoxia followed 24 hours later by lethal Inhibitors,Modulators,Libraries hypoxia. Sister cultures were exposed to lethal hypoxia without previous preconditioning as controls. A sub group of TWEAK and Fn14 neurons was incubated with TWEAK 100 ng mL during the preconditioning phase. Our results indicate that hypoxic preconditioning induces a 23. 44% increase in cell survival in Wt neurons and that this effect is abro gated in neurons genetically deficient in either TWEAK Inhibitors,Modulators,Libraries or Fn14.

Importantly, incubation with TWEAK during the preconditioning phase had a rescue effect in TWEAK but not in Fn14 neurons. To investigate whether treatment with TWEAK also has a neuroprotective effect in vivo, we measured the Inhibitors,Modulators,Libraries volume of the ischemic lesion in Wt and Fn14 mice intraperitoneally injected with either TWEAK or a com parable volume of saline solution 24 hours before tMCAO, as described in the Methods section. We found that preconditioning with TWEAK decreases the volume of the ischemic lesion from Inhibitors,Modulators,Libraries 69. 3 7. 2 mm3 to 46. 41 3. 3 mm3 and 54. 31 4. 8 mm3 24 and 48 hours after tMCAO, respectively. In contrast, we failed to observe a significant decrease in the volume of the ischemic lesion in Fn14 mice preconditioned with TWEAK.

TNF a mediates the neuroprotective effect of TWEAK Because it has been reported that TNF a mediates some of the biological effects of TWEAK, we investigated whether TNF a also mediates TWEAK induced toler ance. First, we used an ELISA to study the expression of TNF a in the culture media of Wt cerebral cortical neu rons incubated for 1 to 60 minutes Inhibitors,Modulators,Libraries with TWEAK 100 ng mL. Our results indicated that TWEAK induces a rapid increase in the expression of neuronal TNF a and that this effect is maximum at 30 minutes of incubation. We then used the MTT assay to study cell survival in Wt cerebral cortical neurons incubated for 60 minutes with TWEAK 100 ng mL alone or in combination with neutralizing antibodies against either TNF a 0. 04 ug mL or TNFR1 100 ug mL, or an immunoglobulin G isotype control, followed 24 hours later by exposure to 55 min utes of OGD conditions.

We found that, whereas treatment with TWEAK rendered certainly neurons tol erant to a lethal hypoxia insult applied 24 hours later, this preconditioning effect was abrogated by incubation with either anti TNF a or anti TNFR1 antibodies. To further study the role of TNF a on TWEAK induced neuroprotection, we quantified cell survival in TNF a cerebral cortical neurons incubated for 1 hour with TWEAK 0 to 300 ng mL followed 24 hours later by exposure to 55 minutes of OGD condi tions. Our results indicated that TWEAK fails to induce hypoxic tolerance in TNF a neurons.