Abstract 3676: MyD88-Dependent Pathway Has a Dual Effect on Brain Injury after Cerebral Ischemia/Reperfusion in Mouse

Abstract

Myeloid differentiation primary-response protein-88 (MyD88) is one of the intracellular adaptors in Toll-like receptor (TLR) signaling pathways, which regulates the activation of nuclear factor kappa B (NF-kappa-B) and transcription of other proinflammatory cytokines. We assessed the hypothesis that MyD88-dependent signaling mediates the inflammatory responses in ischemic brain and is implicated in the process of brain injury. A mouse model of cerebral ischemia/reperfusion (I/R) was induced by transient middle cerebral artery occlusion (tMCAO). MyD88 knockout (KO) mice and wild type control mice (WT, C57BL/6J) were assigned to I/R or sham-operated groups. The mice were killed at 4 hrs or 24 hrs after reperfusion following 60 minutes of MCAO. Using Nissl staining, brain infarct areas were evaluated and expressed as a percentage of the whole brain. Brain edema was evaluated and presented as the percentage of water content in the total weight of the brain tissue. The expression of mRNA was detected by real-time PCR, and the levels of inflammatory cytokines were measured by ELISA and Western Blots. The infarct size was analyzed by t-test. Brain edema and inflammatory response were analyzed by one way ANOVA with post hoc comparisons. Our results showed that the brain infarct size was not significantly different in MyD88KO mice compared with WT mice 24 hrs after cerebral I/R. However, brain edema (water content) significantly increased in MyD88KO mice compared with WT 24 hrs after ischemia (p<0.05). The data also showed that the levels of mRNA of CD14, Pellino-1, cyclooxygenase-2 (COX-2), and Interferon beta-1 (INFb1) increased significantly 4 hrs after cerebral I/R in WT mice compared to sham-operated controls (p<0.05). MyD88 deficiency inhibited the increased expression of Pellino-1 (p<0.05) and COX-2 (p<0.05), but not CD14. Twenty-four hrs after cerebral I/R, the protein levels of Interleukin - 1-beta, Interleukin - 6 (IL-6), p-IkappaB alpha, and the activity of NFkappaB, significantly increased in the ischemic brain in WT mice, and were inhibited in MyD88KO mice. Interestingly, VEGF significantly increased in MyD88KO mice compared with WT mice (p<0.05). In addition, ZO-1, a tight-junction protein on the blood-brain barrier (BBB) decreased in MyD88KO mice compared with WT mice subjected to cerebral I/R. Our data demonstrates that MyD88 contributes to the activation of inflammatory responses after cerebral I/R. However, MyD88 reduces brain edema by mediating the expression of VEGF, and tight-junction proteins, which are important for maintaining BBB integrity. We concluded that the MyD88-dependent pathway has a dual effect on brain injury induced by cerebral I/R. Interpreting the results for the role of MyD88 dependent pathway based on brain injury from whole animal experiments is cautioned.