使用血管通透性标记物评估血脑屏障完整性：伊文思蓝、荧光素钠、白蛋白-Alexa Fluor 偶联物和辣根过氧化物酶。,Methods in Molecular Biology

当前位置： X-MOL 学术 › › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

使用血管通透性标记物评估血脑屏障完整性：伊文思蓝、荧光素钠、白蛋白-Alexa Fluor 偶联物和辣根过氧化物酶。
Methods in Molecular Biology Pub Date : 2021-01-01 , DOI: 10.1007/7651_2020_316
Bulent Ahishali ₁ , Mehmet Kaya _{2,

3}

Affiliation

由脑微血管内皮细胞构成的血脑屏障（BBB）是一个动态界面，它控制和调节包括肽、蛋白质、离子、维生素、激素和免疫细胞在内的各种物质从循环进入脑实质的运输. 某些疾病/病症，如阿尔茨海默氏病、败血症和高血压，可导致不同程度的 BBB 破坏。此外，血脑屏障完整性受损与癫痫等各种神经退行性疾病的发病机制有关。为了探索这些疾病/病症的广泛病理生理机制，在体外细胞培养模型和实验室动物体内针对 BBB 完整性的各种实验性损伤已被证明可以改变 BBB 通透性，从而导致某些示踪剂（例如荧光素钠、尸胺-Alexa 荧光剂、辣根过氧化物酶、FITC-葡聚糖、白蛋白-Alexa 荧光偶联物和穿过屏障的伊文思蓝染料。屏障型内皮细胞的渗透性变化可以通过血管内输注外源性示踪剂和随后检测脑组织中渗出的示踪剂来评估，从而能够对 BBB 完整性进行功能和结构分析。在本章中，我们旨在强调当前关于使用四种最常用示踪剂的知识，即伊文思蓝、荧光素钠、白蛋白-Alexa 荧光素结合物和辣根过氧化物酶。还讨论了我们在实验室中使用的通过肉眼观察、分光光度法、荧光分光光度法、共聚焦激光扫描显微镜和电子显微镜检测这些示踪剂的实验方法。形态学层面的示踪研究主要针对屏障相关细胞和脑实质中示踪剂的鉴定。此外，BBB 对示踪剂的渗透性可以通过共聚焦激光扫描显微镜和电子显微镜使用分光光度法和分光荧光测定法以及图像分析进行量化。我们在各种实验设置下使用上述示踪剂进行的研究结果表明，脑微血管中的屏障型内皮细胞协调了 BBB 中物质的细胞旁和/或跨细胞运输。

"点击查看英文标题和摘要"

Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish Peroxidase.

The blood-brain barrier (BBB) constituted by endothelial cells of brain microvessels is a dynamic interface, which controls and regulates the transport of various substances including peptides, proteins, ions, vitamins, hormones, and immune cells from the circulation into the brain parenchyma. Certain diseases/disorders such as Alzheimer's disease, sepsis, and hypertension can lead to varying degrees of BBB disruption. Moreover, impairment of BBB integrity has been implicated in the pathogenesis of various neurodegenerative diseases like epilepsy. In attempts to explore the wide spectrum of pathophysiologic mechanisms of these diseases/disorders, a variety of experimental insults targeted to the BBB integrity in vitro in cell culture models and in vivo in laboratory animals have been shown to alter BBB permeability causing enhanced transport of certain tracers such as sodium fluorescein, cadaverine-Alexa fluor, horseradish peroxidase, FITC-dextran, albumin-Alexa fluor conjugates, and Evans blue dye across the barrier. The permeability changes in barrier-type endothelial cells can be assessed by intravascular infusion of exogenous tracers and subsequent detection of the extravasated tracer in the brain tissue, which enable functional and structural analysis of BBB integrity. In this chapter, we aimed to highlight the current knowledge on the use of four most commonly performed tracers, namely, Evans blue, sodium fluorescein, albumin-Alexa fluor conjugates, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, confocal laser scanning microscopy, and electron microscopy are also discussed. Tracing studies at the morphological level are mainly aimed at the identification of the tracers both in the barrier-related cells and brain parenchyma. In addition, BBB permeability to the tracers can be quantified using spectrophotometric and spectrophotofluorometric assays and image analysis by confocal laser scanning microscopy and electron microscopy. The results of our studies conducted under various experimental settings using the mentioned tracers indicate that barrier-type endothelial cells in brain microvessels orchestrate the paracellular and/or transcellular trafficking of substances across BBB. These efforts may not only contribute to designing approaches for the management of diseases/disorders associated with BBB breakdown but may also provide new insights for developing novel brain drug delivery strategies.

更新日期：2020-08-13

点击分享查看原文

点击收藏