TLR4-mediated MyD88-dependent signaling pathway is activated by cerebral ischemia–reperfusion in cortex in mice

To study whether the signaling pathway is activated in the inflammatory reaction of cerebral ischemia–reperfusion and its mechanism. The mice were randomly divided into sham group, ischemia–reperfusion group and TLR4-blocked group with different time points of reperfusion 12 h, 24 h, 48 h and 72 h group. We observed the different expression of TLR4 mRNA and MyD88 mRNA, activation of NF-κB and the TNF-α and IL-1β protein levels in each group at different time point after ischemia–reperfusion. Mice cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. TLR4 signaling pathway could be inhibited by specific anti-TLR4 binding protein to prevent TLR4 from interacting with its receptors. We determined the result of TLR4 antibodies-blocking and mice cerebral ischemia–reperfusion injuries by Western blot, and evaluated neuronal damage in cortex. We also determined the expression of TLR4 mRNA and MyD88 mRNA by in situ hybridization (ISH), the activation of NF-κB by EMSA, and the expression of TNF-α protein by Western blot. Anti-TLR4 binding TLR4 receptors before reperfusion was effective; There was distinct difference among each group respecting neuronal damage; The expression of TLR4 mRNA and MyD88 mRNA, the activation of NF-κB, and the expression of TNF-α protein showed clear difference as well. LR4-mediated MyD88-dependent signaling pathway activated by ischemia–reperfusion may be involved in the mechanism of ischemia–reperfusion through upregulation of NF-κB and TNF-α.