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.

这个技术驱动项目的广泛、长期目标是 (1) 继续开发和 (2) 应用 强大的、新型的、简单的微流体装置（及其使用方法），用于确定过程中的速率 体内大脑细胞外空间的一个具体焦点是特定大脑区域的神经肽的水解。 方法：植入式装置利用电渗流引导溶液。 底物，无论有或没有抑制剂，通过脑组织并将未反应的底物和产物引导至 独特的是，该设备和方法使用自然。 特别关注的是外肽酶，“ecto”意味着细胞表面（膜结合）。 在细胞外空间水解肽的酶 典型的实验注入底物肽并 具有或不具有选择性抑制剂的不可水解的D-氨基酸肽类似物。 代表初始底物浓度。米氏速率参数源自实验数据。 使用基于测量模拟的经过验证的方法需要进行模拟校正。 底物和产物在组织中的扩散 健康相关性：神经肽有很多，而且很多。 重要的大脑功能，例如学习和记忆、疼痛感知、防止缺氧造成的伤害，以及 许多其他方法依赖于细胞外空间中的神经肽浓度，但目前没有办法。 直接测量普遍存在的外肽酶对这些浓度的影响。 微流体装置和模拟提供了测量活性肽水解速率的方法。 所提出的方法允许对细胞外空间中的肽活性（例如，Vmax）进行定量建模 具体目标：有两个平行的、独立的目标，另一个目标是应用该设备。 （一）在现有基础上进行全面改进 功能装置可在选择底物/抑制剂时更灵活地进行实验测量 通过组织的浓度；扩展设备到达更深层大脑区域的能力。 (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 经历轻度慢性疼痛的大鼠和对照组大鼠前扣带皮层的活动。