An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa. Albumin and EBD readily crossed membrane cell culture inserts with pore diameters of no less than 1 µm in the absence of a cellular barrier, and their permeability was substantially reduced when the membranes were overlaid with a monolayer of BEnd cells. In line with albumin binding, the BEnd permeability of EBD was substantially reduced by the presence of albumin. While EBD at an EBD-to-albumin ratio similar to those typically used in in vivo BBB experiments had little effect on the BEnd permeability of albumin, a much higher concentration of EBD augmented the BEnd permeability of albumin. In conclusion, we investigated the use of EBD as an indicator of albumin permeability in vitro, explored some of its drawbacks and further demonstrated that EBD at the concentration used in vivo does not affect albumin permeability.

中文翻译：

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伊文思蓝染料作为体外脑内皮细胞单层白蛋白渗透性的指标。

血白蛋白的脑内皮 (BEnd) 细胞通透性增加通常被视为血脑屏障 (BBB) 破坏的早期迹象，并且可能先于脑过程中小分子和其他血浆蛋白的 BEnd 通透性增加疾病。因此，埃文斯蓝染料 (EBD) 是一种易于检测和量化的白蛋白结合荧光示踪剂，已被广泛用于研究 BBB 破坏过程中的 BEnd 渗透性。在这里，我们在体外通透性测定中研究了 EBD 是否是小鼠 BEnd 细胞单层（单独或与小鼠皮质星形胶质细胞共培养）白蛋白通透性的合适指标；鉴于 EBD 对白蛋白的强亲和力，我们进一步询问 EBD 是否会影响白蛋白渗透性，反之亦然。在没有细胞屏障的情况下，白蛋白和 EBD 很容易穿过孔径不小于 1 µm 的膜细胞培养插入物，并且当膜覆盖有单层 BEnd 细胞时，它们的渗透性显着降低。与白蛋白结合一致，EBD 的 BEnd 通透性因白蛋白的存在而显着降低。虽然与通常用于体内 BBB 实验的 EBD 与白蛋白比率相似的 EBD 对白蛋白的 BEnd 渗透性影响很小，但更高浓度的 EBD 增加了白蛋白的 BEnd 渗透性。总之，我们研究了使用 EBD 作为体外白蛋白渗透性的指标，探讨了它的一些缺点，并进一步证明了在体内使用的 EBD 浓度不会影响白蛋白渗透性。