Thomas Riya, Diaz Kazandra, Koster Kevin P, Tai Leon M
Department of Anatomy and Cell Biology, University of Illinois at Chicago.
Department of Anatomy and Cell Biology, University of Illinois at Chicago;
J Vis Exp. 2017 Jun 20(124):55846. doi: 10.3791/55846.
Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-β (oAβ), which is an important contributor to Alzheimer's disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oAβ disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.
血脑屏障（BBB）覆盖在中枢神经系统（CNS）的稳态中起着核心作用。血脑屏障由星形胶质细胞、周细胞和脑内皮细胞（BECs）动态维持。在此，我们详细介绍了使用永生化人BECs的单培养物、原代小鼠BECs的单培养物以及血脑屏障的人源化三重培养模型（BECs、星形胶质细胞和周细胞）来评估血脑屏障覆盖的方法。为了突出这些测定方法在疾病状态下的适用性，我们描述了寡聚淀粉样β（oAβ）对血脑屏障覆盖的影响，oAβ是阿尔茨海默病（AD）进展的一个重要因素。此外，我们利用表皮生长因子（EGF）来阐明这些技术的药物筛选潜力。我们的结果表明，在基础条件下单培养和三重培养的BECs形成网状结构，oAβ破坏这种细胞网状结构的形成并使预先形成的网状结构退化，但EGF可阻止这种破坏。因此，所描述的技术对于剖析调节血脑屏障覆盖的基本和与疾病相关的过程很重要。
