A Microchip-Based Blood Brain Barrier Mimic

描述(申请人提供)：在这项提案中，我们描述了使用光刻衍生的微芯片来模拟完整的血脑屏障(BBB)的开发。当三磷酸腺苷(ATP)标准品或从机械变形的红细胞(RBC)中提取的三磷酸腺苷(ATP)被泵入通道时，基于微芯片的BBB模拟器将包含一个代表循环系统的通道。这个循环通道将被涂有牛脑微内皮细胞(BBMECs)的聚碳酸酯薄膜与第二个通道分隔开来，第二个通道伪装成中枢神经系统(CNS)。这种方法将被用来监测循环通道和次级中枢通道中ATP刺激的NO水平。通过在聚碳酸酯膜上模拟内皮，并用集成微电极安培测量循环通道和次级中枢通道中释放的NO，我们将确定RBC来源的ATP是否刺激BBMECs产生NO。这一建议解决了这样的假设：光刻衍生的微芯片，其中由涂有BBMECs的聚碳酸酯薄膜将一个通道与第二个通道(假装为CNS)分开，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)证明在聚碳酸酯薄膜上培养的BBMECs可以作为BBB的模拟物，该膜将代表血流的通道与代表CNS的通道网络分开；2)通过电化学测量这些细胞在ATP存在下分泌的一氧化氮，证明BBB模拟物中的BBMECs具有生物响应性；以及3)通过使用基于微芯片的血脑屏障模拟物，来近似内皮源性NO的生理命运，由机械变形的兔红细胞释放的ATP刺激。这些研究的成功完成将使我们更全面地了解RBC来源的ATP作为NO刺激的作用，更重要的是，这种NO在血脑屏障中的作用。

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.