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.

这个由技术驱动的项目的广泛、长期目标是(1)继续开发和(2)应用 用于测定过程速率的强大的、新的、简单的微流控装置(及其使用方法) 活体脑细胞外间隙。一个特别的焦点是脑部特定区域的神经肽的水解性。 醒着，举止乖巧的动物。方法：该植入器利用电渗流引导溶液 底物，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到 在线测量系统或收集它们以供以后分析。独一无二的是，该设备和方法使用Natural 体内的底物。特别的焦点是外肽酶，“外肽酶”意味着细胞表面(膜结合)。 在细胞外空间中对多肽进行水解酶。一个典型的实验注入底物多肽和 一种不可水解的D-氨基酸肽类似物，带有或不带有选择性抑制剂。不可水解肽 表示初始底物浓度。从实验数据导出了Michaelis-Menten速率参数 使用基于测量模拟的有效方法。需要进行模拟才能进行校正 底物和产物在组织中的扩散。与健康相关：有许多神经肽，而且很多 关键的大脑功能，如学习和记忆、疼痛感知、对缺氧损伤的保护，以及 许多其他依赖于细胞外空间神经肽浓度的细胞。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。建议数 微流控装置和模拟提供了测量活性多肽的水解率的手段。 所提出的方法允许对细胞外空间中的多肽活性(例如，Vmax)进行定量建模 活着。具体目标：有两个平行的、独立的目标。一是对设备进行了改进。另一种方法是应用 现有的新型微流控设备解决了一个关于疼痛感知的问题。(1)在现有的基础上全面改进 功能装置，在选择底态/抑制剂方面具有更大的灵活性进行实验测量 浓度通过组织；以扩展该设备的能力，以达到更深的大脑区域。 (2)应用现有的、功能齐全的微流控装置来确定特定外肽酶的差异 轻度慢性疼痛大鼠和对照组大鼠前扣带回皮质的活动。