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. ? ?
| Hulvey, Matthew K; Martin, R Scott (2009) A microchip-based endothelium mimic utilizing open reservoirs for cell immobilization and integrated carbon ink microelectrodes for detection. Anal Bioanal Chem 393:599-605 |
| Hulvey, Matthew K; Genes, Luiza I; Spence, Dana M et al. (2007) Fabrication and evaluation of a 3-dimensional microchip device where carbon microelectrodes individually address channels in the separate fluidic layers. Analyst 132:1246-53 |
