Kaya Mehmet, Ahishali Bulent
Departments of Physiology & Histology and Embryology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey.
Methods Mol Biol. 2011;763:369-82. doi: 10.1007/978-1-61779-191-8_25.
Blood-brain barrier (BBB) constituted primarily by the capillary endothelial cells functions to maintain a constant environment for the brain, by preventing or slowing down the passage of a variety of blood-borne substances, such as serum proteins, chemical compounds, ions, and hormones from the circulation into the brain parenchyma. Various diseases such as brain tumors, epilepsy, and sepsis disturb the BBB integrity leading to enhanced permeability of brain microvessels. In animal models, a variety of experimental insults targeted to the BBB integrity have been shown to increase BBB permeability causing enhanced passage of molecules into the brain paranchyma by transcellular and/or paracellular pathways. This alteration can be demonstrated by intravascular infusion of exogenous tracers and subsequent detection of extravasated molecules in the brain tissue. A number of exogenous BBB tracers are available, and they can be used for functional and structural analysis of BBB permeability. In this chapter, we aimed to highlight the basic knowledge on the use of three most commonly performed tracers, namely Evans blue dye, sodium fluorescein, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, and electron microscopy are also discussed. While tracing studies at the morphological level are mainly aimed at the identification and characterization of the tracers both in the barrier related cells and brain parenchyma, spectrophotometric and spectrophotofluorometric assays enable quantification of BBB permeability. The results of our studies that we performed using the mentioned tracers indicate that barrier type of endothelial cells in brain play an important role in paracellular and/or transcytoplasmic trafficking of macromolecules across BBB under various experimental settings, which may provide new insights in both designing approaches for the management of diseases with BBB breakdown and developing novel trans-BBB drug delivery strategies.
血脑屏障（BBB）主要由毛细血管内皮细胞构成，其功能是通过阻止或减缓多种血液传播物质（如血清蛋白、化合物、离子和激素）从循环系统进入脑实质，从而为大脑维持一个恒定的环境。各种疾病，如脑肿瘤、癫痫和败血症，会破坏血脑屏障的完整性，导致脑微血管通透性增强。在动物模型中，针对血脑屏障完整性的各种实验性损伤已被证明会增加血脑屏障的通透性，导致分子通过跨细胞和/或细胞旁途径进入脑实质。这种改变可以通过血管内注入外源性示踪剂并随后检测脑组织中渗出的分子来证明。有多种外源性血脑屏障示踪剂可供使用，它们可用于血脑屏障通透性的功能和结构分析。在本章中，我们旨在突出关于三种最常用示踪剂（伊文思蓝染料、荧光素钠和辣根过氧化物酶）使用的基础知识。我们还讨论了在我们实验室中用于通过宏观观察、分光光度法、分光荧光法和电子显微镜检测这些示踪剂的实验方法。虽然形态学水平的示踪研究主要旨在识别和表征屏障相关细胞和脑实质中的示踪剂，但分光光度法和分光荧光法测定能够定量血脑屏障的通透性。我们使用上述示踪剂进行的研究结果表明，在各种实验条件下，脑内内皮细胞的屏障类型在大分子通过血脑屏障的细胞旁和/或跨细胞质运输中起重要作用，这可能为设计治疗血脑屏障破坏疾病的方法和开发新型跨血脑屏障药物递送策略提供新的见解。
