A Microchip-Based Blood Brain Barrier Mimic

描述（由申请人提供）：在该提案中，我们描述了使用光刻衍生的微芯片开发完整的血脑屏障（BBB）模拟物。基于微芯片的BBB模拟物将包含一个通道，当三磷酸腺苷（ATP）标准品或来源于机械变形的红细胞（RBC）的ATP被泵送通过该通道时，该通道将代表循环系统。该循环通道将通过涂有牛脑微内皮细胞（BBMEC）的聚碳酸酯膜与第二通道（构成中枢神经系统（CNS））分开。这种方法将被用来监测ATP刺激的NO水平在循环通道和在二级中枢神经系统通道。通过在聚碳酸酯膜上创建内皮模拟物，并使用集成微电极以安培法测量循环通道和次级CNS通道中释放的NO，我们将确定RBC衍生的ATP是否刺激BBMEC中的NO产生。该提案解决了以下假设：光刻衍生的微芯片，其中通道与第二通道（构成CNS）分开，通过涂有BBMEC的聚碳酸酯膜，可以用作BBB模拟物以确定ATP刺激的NO产生的作用和命运。在这里，我们打算1）证明在将代表血流的通道与代表CNS的通道网络分开的聚碳酸酯膜上培养的BBMEC可以起到BBB模拟物的作用; 2）通过电化学测量在存在ATP（一种已知的内皮源性一氧化氮的刺激剂）的情况下由这些细胞分泌的一氧化氮来证明BBB模拟物中的BBMEC是生物响应的;（3）利用基于微芯片的血脑屏障模拟物，模拟机械变形兔红细胞释放ATP刺激内皮源性NO的生理转归。这些研究的成功完成将使我们更全面地了解RBC衍生的ATP作为NO刺激物的作用，以及更重要的是，这种NO在BBB中的作用。

DESCRIPTION (provided by applicant): In this proposal, we describe the development of a complete blood brain barrier (BBB) mimic using lithographically-derived microchips. The microchip-based BBB mimic will contain one channel that will represent the circulatory system, when adenosine triphosphate (ATP) standards or ATP derived from mechanically deformed red blood cells (RBCs) are pumped through the channel. This circulatory channel will be separated from a second channel, which poses as the central nervous system (CNS), by a polycarbonate membrane coated with bovine brain microendothelial cells (BBMECs). This approach will be used to monitor ATP-stimulated NO levels in both the circulation channel and in the secondary CNS channel. By creating an endothelium mimic on the polycarbonate membrane and amperometrically measuring with integrated microelectrodes the released NO in both the circulation channel and the secondary CNS channel, we will determine if RBC-derived ATP is a stimulus for NO production in BBMECs. This proposal addresses the hypothesis that: Lithographically-derived microchips, where a channel that is separated from a second channel, which poses as the CNS, by a polycarbonate membrane coated with BBMECs, may be employed as a BBB mimic to determine the action and fate of ATP-stimulated NO production. Here, we intend 1) To demonstrate that BBMECs, cultured on a polycarbonate membrane that separates a channel representing the blood stream from a channel network representing the CNS, can function as a mimic of the BBB; 2) To demonstrate that BBMECs in the BBB mimic are bioresponsive by electrochemically measuring nitric oxide secreted by these cells in the presence of ATP, a known stimulant of endothelium-derived nitric oxide; and 3) To approximate the physiological fate of endothelium-derived NO, stimulated by ATP released from mechanically deformed rabbit RBCs, by employing the microchip-based blood brain barrier mimic. The successful completion of these studies will lead to a more comprehensive understanding of the role of RBC-derived ATP as a stimulus of NO and, importantly, the effect of this NO in the BBB.