2-ClHDA impairs barrier function in vitro and in vivo.

<p>(A) BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of HC-induced BMVEC was continuously monitored by impedance sensing at 4 kHz in the presence of the indicated 2-ClHDA concentrations or vehicle (DMSO). (B) BMVEC were cultured on coverslips until confluence. After incubation for 3 h in the presence of vehicle (DMSO; upper panel) or 15 µM 2-ClHDA (lower panel) immunofluorescence labeling of ZO-1 (red) and VE-cadherin (green) was performed. Sites of nuclear ZO-1 redistribution (arrows) and frizzy-like structures (arrowheads) are indicated. Scale bars: 20 µm. (C, D) The left common carotid artery of anesthetized rats was exposed and cannulated. After sectioning of jugular veins animals were perfused (3 ml/min) with Ringer solution for 5 min. Subsequently, perfusion was switched for 90 min to Ringer solution containing vehicle (DMSO) or 25 µM 2-ClHDA. This was followed by perfusion with Ringer solution supplemented with EB and SF for 5 min and a washout with Ringer solution (without dyes) for 7 min. Animals were decapitated and the brains were immediately removed. (C) Macroscopic evaluation of EB extravasation, and (D) determination of SF fluorescence intensity in brain homogenates are shown. Results represent mean values ± SD from 4 animals (**p<0.01, Students t-test).