Effects Of Activated Retinal Microglia On Blood-retinal Barrier

Objective1 To develop a method of improved efficient rat retinal microglia cell isolation and its purification and to study the function of lipopolysaccharide (LPS)-activated retinal microglia.2 To investigate the effects of activated retinal microglia on the expression of ZO-1 and vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs).3 To investigate the effects of activated retinal microglia on the blood-retinal barrier (BRB) breakdown.Methods:1 Primary microglia culture was modified based on the classic microglia culture method, and microglia were activated with LPS. Immunohistochemistry, flow cytometry and ELISA were applied to observe the morphological characters and functional changes of the microglia.2 Retinal microglia were isolated and cultured from newborn Sprague-Dawley (SD) rats. Cultured retinal microglia were collected and cultured onto Transwell permeable support membrane inserts, then activated by the addition of LPS (100 ng/mL) for 24 h. The previously prepared Transwell inserts containing either untreated or LPS-treated retinal microglia were then placed into wells with HUVECs. Control cultures were incubated with empty inserts without cultured microglia. The cultures were used for experiments after 24 h. Immunocytochemistry was applied to observe ZO-1 expression in these HUVECs. The protein expression of VEGF and ZO-1 was analyzed by Western blot.3 SD adult rats were randomly divided into three groups. Group A: intravitreal injection of 2 HL PBS. Group B:intravitreal injection of 2μL PBS with untreated microglia. Group C:intravitreal injection of 2μL PBS with LPS-treated retinal microglia. Evans blue was injected into vein 7 days after intravitreal injection, then the evans blue leakage from retinas was quantified. The protein expression of VEGF and ZO-1 was analyzed by Western blot.Results:1 The purity of the microglia was 97.6%. Some morphological changes of microglia were observed after treatment with LPS and cytokine TNF-a released from microglia increased significantly.2 HUVECs co-cultured with LPS-treated retinal microglia, compared to either untreated or unexposed HUVECs, expressed lower levels of ZO-1. Western Blot analysis revealed an upregulation of VEGF and a downregulation of ZO-1 in HUVECs which co-cultured with LPS-treated retinal microglia (P<0.05)3 The retinal evans blue leakage in Group C was higher than the other two groups (P<0.05). Western Blot analysis revealed an upregulation of VEGF and a downregulation of ZO-1 in Group C (P<0.05)Conclusions:1 The isolated microglia cells are pure by using this method, which would provide a valuable tool for studying mechanisms of microglia.2 These results indicate that activated microglia can effect the expression of VEGF and ZO-1 in HUVECs, and may play an important role in barrier breakdown.3 These results indicate that intravitreal injection with LPS-activated microglia can reduce BRB permeability of rats and effects the expression of VEGF and ZO-1. Activated microglia may play an important role in BRB breakdown.