In our study, we observed a slight increase in IL-6 after IPC in astrocytes. The release of small amounts of cytokines from cells may partly contribute to TLR3 signal activation during preconditioning and then induce expression of a range of neuroprotective mediators. It has been reported previously that several downstream products of IRF3, such as TRIM30-a, negatively regulate the NF-kB signaling pathway. However, the exact molecular mechanisms by which TRIF and IRF3 mediate downregulation of the NF-kB pathway require further study. Poly I:C activation of TLR3, which signals through a TRIFdependent pathway, induces expression of various neuroprotective mediators and anti-inflammatory cytokines in human astrocytes. Borysiewicz et al. reported that TLR3 ligation with Poly I:C up to 2 mg/mL protects TCS PrP Inhibitor 13 astrocytes against oxidative stress. Another study reported that acute Poly I:C treatment up to100 mg/mL significantly reduced OGD�Cmediated cell death in mixed cortical cultures from mice. We and others have shown that Poly I:C preconditioning SA 47 provides neuroprotection against cerebral ischemia in vivo. Here, we show that Poly I:C also induces ischemic resistance in astrocytes. Preconditioning with 5 or 10 mg/mL Poly I:C significantly reduced OGD-induced cell death and LDH release, increased TRIF and pIRF3 protein expression, enhanced IFNb release, and decreased IL-6 release. Poly I:C activation of astrocytes triggered a 2.9-fold increase in interferon regulatory factor-1 expression, and Poly I:C activation of monocytes triggered a 100-fold increase in IFNb production. We found that IFNb increased approximately twofold over the control level after Poly I:C treatment. These discrepancies may be the result of species specificity or differences in sensitivity of detection methods. Because Poly I:C activates not only TLR3 but also at least two other cytosolic receptors, MDA-5 and Rig-I, we confirmed involvement of TLR3 signaling in Poly I:C-induced ischemic tolerance by using TLR3 neutralizing antibody. Poly I:C preconditioning-induced protection may be related to activation of TRIF-pIRF3 signaling via TLR3 in astrocytes, which, in turn, would enhance production of antiinflammatory cytokines in the ischemic astrocytes. Additionally, Gesuete et al. indicated that Poly I:C preconditioning might attenuate blood�Cbrain barrier dysfunction through induction of IFNb. IPC in the brain is a natural phenomenon that likely protects against ischemic brain injury by preventing inflammation.