AEA limits leukocyte adhesion to TMEV stimulated brain endothelial cells and leukocyte transmigration through in vitro BBB by CB
involvement. Brain endothelial cell monolayer were stimulated with a combination of TMEV (2 × 105 pfu), AEA (10 μM), SR1 (1 μM) or AM630 (1 μM) for 6 hours. After that, 2.5 × 105 leukocytes stained with AM-calcein (5 μM) were added to the endothelial culture for 20 hours. (A) Representative immunofluorescence microphotographs of the leukocytes stained with AM-calcein adhered to the brain endothelial cell monolayer in each case and phase contrast microphotographs of brain endothelial monolayer merged with immunofluorescence microphotographs of AM-calcein stained leukocytes bring out with arrows. Scale bar 100 μm. (B) Quantification of leukocytes adhered to brain endothelial monolayer in each case normalized to control group (n = 6). (**p < 0.01 vs. vehicle; ##p < 0.01 vs. TMEV; +p < 0.05 vs. TMEV+AEA, ANOVA followed by Tuckey's tests). (C) TMEV (2 × 105 pfu), plus AEA (10 μM), or plus SR1 (1 μM) or AM630 (1 μM) were added to the upper side of the insert (endothelial culture) and IL1-β (10 ng/ml) was added to the bottom side (astrocyte culture) for 6 hours. 2.5 × 105 leukocytes were added to the upper side of the insert for 20 hours and representative phase contrast microphotographs of leukocytes crossed to bottom side of the insert were taken. (D) Quantification of leukocytes in the bottom side of the insert after 20 hours of experiment. (**p < 0.01 vs. vehicle; ##p < 0.01 vs. TMEV+vehicle; ++p < 0.01 vs. TMEV+AEA; &p < 0.05 vs. TMEV+AEA+SR1, ANOVA followed by Tuckey's test; n = 6).