P2.03-34 Fractalkine Attenuate Irradiation-Induced Brain Injury Through Promoting the M2 Polarization of Microglia

Abstract

Radiation-induced brain injury (RIBI) is an unavoidable adverse side-effect induced by cranial radiation therapy. The neuro-inflammation mediated by activated microglia has been proved to be a key role in RIBI by our previous researches. Studies have demonstrated that vascular endothelial cells are damaged by irradiation and Fractalkine (FKN), a crucial mediator modulating the biological activity of microglia, is released. However, the role of FKN in RIBI was poorly understood. The aim of this study was to investigate the effect of FKN on RIBI and its underlying mechanisms. Human Umbilical Vein Endothelial Cells (HUVEC) was subjected to 10 Gy or sham irradiation, FKN expression was detected by Western blotting (WB), qRT-PCR and ELISA, γH2AX formation and nuclear translocation of p65 was analyzed by immunocytochemistry (ICC). BV-2 cells received 10-Gy irradiation after being cultured for 3 h with or without FKN (100ng/ml), or co-cultured with HUVEC. Moreover, the CX3CR1 (the receptor of FKN on microglia) wide-type (CX3CR1WT) and CX3CR1-knockdown (CX3CR1-/-) mice were employed and subjected to lateral ventricular injection (ICV) of 5 μl FKN lentivirus or vector 3 days before 10-Gy whole brain irradiation. The polarization of microglia in vitro or in hippocampus and its inflammatory factors release were evaluated through measuring the signature genes, protein and cytokines of M1/M2 phenotype by RT-PCR, WB and ELISA at different time-points after irradiation. Hippocampus neurogenesis was evaluated through detecting the proliferation marker BrdU/nestin and differentiation marker BrdU/NeuN by immunofluorescence (IF) respectively. Neurological function was evaluated by morris water maze (MWM) at 6 weeks after RIBI, and the relationship between microglia and vascular was explored by IF. Then the in vitro phagocytosis assays were performed to investigate if FKN could promote BV2’ phagocytic function. The expression of FKN in HUVEC was increased by 10 Gy irradiation, simultaneously, γH2AX formation and p65 nuclear translocation were observed. FKN regardless from exogenous or secreted by HUVEC could promote the M2 polarization of microglia and inhibit inflammatory response in vitro， it also enhanced neurogenesis in hippocampus, and improved function recovery in CX3CR1WT mice, but not in CX3CR1-/-mice after RIBI. More interestingly, activated microglia migrated to blood vessels in CX3CR1WT mice was observed in vivo by IF. What’s more, BV2 cells phagocytized more fluorescent microspheres when treated with FKN. The FKN/CX3CR1 axis plays an important role in RIBI, and might be an underlying target for the treatment of radiation-induced cognitive impairment.