A novel microfluidic system for studying brain chemistry and application to study of enkephalin-degrading enzymes in pain perception

一种用于研究脑化学的新型微流体系统及其在疼痛感知中脑啡肽降解酶研究中的应用

• 批准号: 10504385
• 负责人: STEPHEN G. WEBER
• 金额: $ 53.2万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10504385
• 关键词: Address; Affective; Amino Acids; Animals; Anoxia; Anterior; Brain; Brain Chemistry; Brain region; Cell Surface Proteins; Cell surface; Communication; Data; Devices; Diabetes Mellitus; Diffusion; Dimensions; Disease; Drug Design; Enkephalins; Enzymes; Extracellular Space; Goals; Head; Health; Hydrolysis; Image; In Vitro; Injury; Kidney Diseases; Kinetics; Knowledge; Lasers; Learning; Liquid substance; Measurement; Measures; Membrane; Memory; Methods; Michigan; Microdialysis; Microfluidic Microchips; Microfluidics; Modeling; Moods; Neuropeptides; Opioid Receptor; Pain; Pain Disorder; Peptides; Perfusion; Preparation; Process; Publishing; Rattus; Reaction; Reaction Time; Reproduction; Research; Sampling; Sensory; Signal Transduction; Source; Stroke; System; Technology; Testing; Tissues; Trauma; Writing; awake; base; brain tissue; chronic pain; chronic pain management; cingulate cortex; design; endogenous opioids; experience; experimental study; extracellular; flexibility; healing; implantable device; improved; in vivo; inhibitor; lithography; mind control; neuropsychiatric disorder; novel; pain perception; peptide analog; photopolymerization; receptor; simulation; two-photon

The broad, long-term objectives of this technology-driven project are (1) to continue developing and (2) apply a powerful, new, simple, microfluidic device (and method for its use) for determining rates of processes in the extracellular space of brain in vivo. A specific focus is hydrolysis of neuropeptides in specific brain regions in awake, behaving animals. Method: The implantable device uses electroosmotic flow to direct solutions of substrates, with or without inhibitors, through brain tissue and direct unreacted substrate and products to an online measurement system or collect them for later analysis. Uniquely, this device and method uses natural substrates in vivo. The particular focus is ectopeptidases, the “ecto” implying cell-surface (membrane-bound) enzymes that hydrolyze peptides in the extracellular space. A typical experiment infuses a substrate peptide and an unhydrolyzable D-amino acid peptide analog with or without a selective inhibitor. The unhydrolyzable peptide represents initial substrate concentration. Michaelis-Menten rate parameters are derived from experimental data using a validated approach based on a simulation of the measurement. The simulation is required to correct for diffusion of substrate and products in the tissue. Health relatedness: There are many neuropeptides, and many critical brain functions such as learning and memory, pain perception, protection from injury due to anoxia, and many others that rely on neuropeptide concentrations in the extracellular space. But there is currently no means to directly measure the impact of the ubiquitous ectopeptidases on those concentrations. The proposed microfluidic device and the simulation provide the means to measure the rate of hydrolysis of active peptides. The proposed method permits quantitative modeling of peptide activity (e.g., Vmax) in the extracellular space in vivo. Specific Aims: There are two parallel, independent aims. One improves the device. The other applies the existing novel microfluidic device to a question about pain perception. (1) Improve upon the existing, fully functional device to make experimental measurements with more flexibility in choosing substate/inhibitor concentrations being passed through the tissue; to extend the device’s capability to reach deeper brain regions. (2) Apply an existing, fully functional microfluidic device to determine differences in specific ectopeptidase activities in the anterior cingulate cortex between rats experiencing mild chronic pain and controls.

这个技术驱动的项目的广泛的长期目标是（1）继续开发和（2）应用 一种用于确定微流控系统中的过程速率的强大的、新的、简单的微流控装置（及其使用方法）， 脑细胞外间隙的细胞外间隙。一个具体的焦点是水解的神经肽在特定的大脑区域， 醒着的动物方法：植入式装置使用电渗流来引导 底物，有或没有抑制剂，通过脑组织，并直接未反应的底物和产物， 在线测量系统或收集它们用于以后的分析。独特的是，该装置和方法使用天然的 体内底物。特别关注的是外肽酶，“外”意味着细胞表面（膜结合） 在细胞外空间水解肽的酶。一个典型的实验注入底物肽， 含有或不含有选择性抑制剂的不可水解的D-氨基酸肽类似物。不可水解肽 表示初始底物浓度。根据实验数据导出了米氏速率参数 使用基于测量模拟的经验证的方法。需要模拟来校正 基质和产物在组织中的扩散。健康相关性：有许多神经肽， 关键的大脑功能，如学习和记忆，疼痛感知，保护免受缺氧损伤，以及 许多其他的依赖于细胞外空间中的神经肽浓度。但目前还没有办法 直接测量无处不在的外肽酶对这些浓度的影响。拟议 微流控装置和模拟提供了测量活性肽水解速率的手段。 所提出的方法允许肽活性的定量建模（例如，Vmax）在细胞外间隙中的 vivo.具体目标：有两个平行、独立的目标。一个改进了设备。另一个应用了 现有的新型微流体装置来解决关于疼痛感知的问题。(1)充分改进现有的 功能性装置，在选择底物/抑制剂方面具有更大的灵活性， 通过组织的浓度;以扩展设备到达更深大脑区域的能力。 (2)应用现有的、功能齐全的微流体装置来确定特定外肽酶的差异 在经历轻度慢性疼痛的大鼠和对照组之间的前扣带皮层的活动。