The edaravone concentrations used were in the millimolar range similarly to another independent work on cultured human brain R428 endothelial cells. For long-lasting protection suprapharmacological concentration of edaravone was needed in our culture study. However, the applied methylglyoxal levels were also comparably high, as in other in vitro methylglyoxal studies. Although we use higher concentrations in cultured cells, importantly, the ratio of the methylglyoxal to edaravone used in our study is the same as the ratio of the pathological plasma methylglyoxal concentrations to clinical concentrations of edaravone. Originally, edaravone has been described as a drug to treat ischemic stroke by protecting against oxidative stress. Its antioxidant effect was observed in our experiment, too. In a recent independent study edaravone suppressed methyglyoxal-induced ROS production in human brain endothelial cells by two possible mechanisms. Pre-treatment with edaravone decreased methylglyoxal-induced AGE accumulation and activation of its receptor RAGE, and the subsequent production of ROS. Furthermore, edaravone inhibited protein-glycation by methylglyoxal in a cell free system, therefore, it decreased ROS generated as by-products during protein glycation. All these results together indicate that the antioxidant mechanisms induced by edaravone contribute to its protective effect against methylglyoxal-induced oxidative stress. However, it remained unanswered whether edaravone can also protect against methylglyoxal-induced barrier dysfunction in brain endothelial monolayers. Therefore, this study focused on the protective effect of edaravone against methylglyoxal-induced barrier damage. We found that co-treatment with edaravone restored barrier properties of endothelial cells and protected against methylglyoxalinduced decrease of resistance and increase in permeability for paracellular and transcellular markers. Moreover, we also demonstrated that edaravone treatment alone tightened the brain endothelial barrier. Our data expand and further support previous observations on barrier enhancing effect of edaravone. Increased endothelial permeability was coupled with disturbed localization of junctional proteins claudin-5 and b-catenin after incubation with methylglyoxal, while co-treatment with edaravone restored distribution of both proteins along the cell borders. Similar observation was made in a previous study, where edaravone treatment enhanced b-catenin at cell-cell contact area and the cortical arrangement for its linked protein, actin on half confluent endothelial monolayer. Our holographic phase contrast microscopic data are in accordance with these observations: edaravone completely prevented methylglyoxal-induced changes in cell morphology, no sign of detachment and cellular morphological change was observed, indicating there was no cytoskeletal rearrangement. Our results have answered the question that edaravone can protect against methylglyoxal-induced barrier dysfunction in brain endothelial cells.