A Microchip-Based Blood Brain Barrier Mimic

描述（由申请人提供）：在本提案中，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 模拟物将包含一个代表循环系统的通道，当三磷酸腺苷 (ATP) 标准品或源自机械变形红细胞 (RBC) 的 ATP 被泵入该通道时。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个通道（构成中枢神经系统（CNS））分开。该方法将用于监测循环通道和次级 CNS 通道中 ATP 刺激的 NO 水平。通过在聚碳酸酯膜上创建内皮模拟物，并使用集成微电极对循环通道和次级 CNS 通道中释放的 NO 进行电流测量，我们将确定红细胞衍生的 ATP 是否是 BBMEC 中 NO 产生的刺激物。该提案提出了这样的假设：光刻衍生的微芯片，其中一个通道通过涂有 BBMEC 的聚碳酸酯膜与第二个通道（构成 CNS）分开，可以用作 BBB 模拟物来确定 ATP 刺激的 NO 产生的作用和命运。在这里，我们打算 1) 证明在聚碳酸酯膜上培养的 BBMEC（将代表血流的通道与代表 CNS 的通道网络分开）可以充当 BBB 的模拟物； 2) 通过电化学测量这些细胞在 ATP（一种已知的内皮源性一氧化氮刺激剂）存在下分泌的一氧化氮，证明 BBB 模拟物中的 BBMEC 具有生物响应性； 3) 通过采用基于微芯片的血脑屏障模拟物，模拟机械变形的兔红细胞释放的 ATP 刺激内皮源性 NO 的生理命运。这些研究的成功完成将使人们更全面地了解红细胞衍生的 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.