LCN2 Regulates The Dynamic Changes Of Glial Phenotypes In Retinal Ischemia/Reperfusion Injury

Objectives: Pro-inflammatory polarization of glial cells contributes to the excessive neuroinflammatory response.The dynamic changes of glial phenotypes after retinal ischemia/reperfusion(RI/R)injury and its underlying regulatory mechanisms remain unclear.The aims of this study are going to explore the dynamic patterns of glial phenotypes after RI/R injury.In addition,the roles of LCN2 in regulating the phenotypic switching of glial cells are going to be clarified in the study.Methods: Western blotting and immunofluorescence were used to detect the morphological and functional phenotypes of glial cells at different time points(2,6,12 hours and 1,3,7 days)post RI/R injury;Rats were intravitreally injected with a recombinant adeno-associated virus(AAV)expressing the short hairpin RNA targeting LCN2 m RNA(AAVDJ.sh LCN2)to knockdown the retinal LCN2 expression;Western blotting,immunofluorescence and flash-visual evoked potentials(f-VEP)were used to evaluate the effect of LCN2 knockdown in regulating the morphological and functional phenotype of glial cells as well as in RGC survival and visual function after RI/R injury.Results: Both MHC-Ⅱ(pro-inflammatory marker)positive and Arg1(anti-inflammatory marker)positive microglia were significantly increased at the early phase(within 1 day)after RI/R injury.The expression of proinflammatory astrocyte marker complement C3 and anti-inflammatory marker S100A10 were consistently increased in retinal astrocytes.We further investigated that the upregulation of pro-inflammatory markers(MHC-Ⅱ and C3)was higher than anti-inflammatory markers(Arg1 and S100A10).Knockdown of LCN2 inhibited the upregulation of MHC-Ⅱ and C3,and enhanced the S100A10 expression at the early phase(12 hour)after injury.LCN2 knockdown improved the RGCs survival and f-VEP P2 amplitude after injury.Conclusions: The upregulation of pro-inflammatory glial phenotype markers is significantly higher than anti-inflammatory phenotype markers at the early phase(within 1 day)after RI/R injury,which suggests glial cells may transform into pro-inflammatory phenotypes.LCN2 might promote the pro-inflammatory polarization of glial cells after RI/R injury,which further induces or aggravates the retinal neurodegeneration.