Bressler Joseph, Clark Katherine, O'Driscoll Cliona
Hugo Moser Laboratory at the Kennedy Krieger, Kennedy Krieger Institute, Baltimore, MD, USA.
Methods Mol Biol. 2013;1066:67-79. doi: 10.1007/978-1-62703-604-7_6.
The impermeability of the blood-brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by their polarity, or oil/water partition coefficient, many lipophilic chemicals are not permeable because of multidrug transporters at the luminal and abluminal membranes. In contrast, many nutrients, which are usually polar, cross the BBB more readily than predicted by their oil/water partition coefficients due to the expression of specific nutrient transporters. In vitro models are being developed because rodent models are of low input and relatively expensive. Isolated brain microvessels and cell culture models each offers certain advantages and disadvantages. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity, whereas cell culture models allow the investigator to measure directional transport and can be genetically manipulated. In this chapter, we describe how to isolate large batches of brain microvessels from freshly slaughtered cows. The different steps in the isolation procedure include density gradient centrifugations and filtering. Purity is determined microscopically and by marker enzymes. Permeability is assessed by measuring the uptake of fluorescein-labeled dextran in an assay that has been optimized to have a large dynamic range and low inter-day variability. We also describe how to evaluate transendothelial cell electrical resistance and paracellular transport in cell culture models.
血脑屏障（BBB）的不透性归因于多种特性，包括相邻内皮细胞间的紧密连接、不存在胞饮小泡以及多药转运蛋白的表达。尽管许多化学物质的通透性可通过其极性或油/水分配系数来预测，但由于管腔膜和管周膜上存在多药转运蛋白，许多亲脂性化学物质并不能通透。相反，许多通常具有极性的营养物质，由于特定营养转运蛋白的表达，其穿过血脑屏障的速度比根据其油/水分配系数预测的要快。由于啮齿动物模型成本高且投入低，体外模型正在被开发。分离的脑微血管和细胞培养模型各有优缺点。分离的脑微血管可用于测量多药转运蛋白和紧密连接的完整性，而细胞培养模型则使研究人员能够测量定向转运，并且可以进行基因操作。在本章中，我们描述了如何从刚屠宰的奶牛中分离出大量脑微血管。分离过程中的不同步骤包括密度梯度离心和过滤。通过显微镜检查和标记酶来确定纯度。通过在经过优化以具有大动态范围和低日间变异性的测定中测量荧光素标记的葡聚糖的摄取来评估通透性。我们还描述了如何在细胞培养模型中评估跨内皮细胞电阻和细胞旁运输。
