Monitoring rapid guanosine signaling during ischemia

项目总结 随着时间的推移，测量局灶性缺血部位的动态鸟苷信号仍然具有挑战性 现有技术尚未了解当地鸟苷的动态、调节机制和功能 脑缺血期间的波动将积极影响我们对大脑即刻局部 神经保护性反应。鸟苷是一种核苷嘌呤，被认为是一种有效的恢复剂。 在脑缺血损伤后的作用；然而，到目前为止，鸟苷在脑中的作用机制和动力学仍然存在 悬而未决。此外，了解鸟苷信号作为缺血的函数发生变化的程度 持续时间和严重程度将为鸟苷作为神经保护者的作用提供关键的洞察力。我们建议解决 在局灶性脑缺血期间鸟苷信号动力学的理解上存在显著差距 在脑片的子区域启动持续局部缺氧-葡萄糖剥夺的微流控平台和使用 以毫秒至秒时间分辨率记录鸟苷的快速扫描循环伏安法(FSCV) 提供对鸟苷调节机制的关键洞察。测量局部鸟苷的动态变化 时空分辨率显著提高的损伤部位将提供大脑的关键信息 立即对当地的损坏做出反应。这一建议符合我们的长期目标，即开发分析工具来检测 并了解损伤期间大脑中动态的神经化学调节炎症。这样做的理由是 建议是这些工具将提供关于快速鸟苷的动力学、机制和功能的知识 首次在脑缺血时发出信号，这可能进一步通知鸟苷的发育。 针对神经损伤的靶向治疗。该提案将通过以下三个具体目标完成：(1) 开发用于时空控制和持续性局灶性脑缺血的微流控平台 脑片，(2)表征海马区鸟苷快速释放和清除的机制 缺血的严重程度和部位，以及(3)表征快速鸟苷信号对局部腺苷的影响 局灶性脑缺血时的变化。我们将以一种创新的方法实现这些目标，使用新型微流控技术 与快速电化学记录相结合的脑片时间控制的缺血传输平台 鸟苷信号转导首次与FSCV。这项工作意义重大，因为这些研究将使 对大脑在不同缺血持续时间内对缺血的即时反应的非凡机械洞察力 以及严重程度，这将直接影响未来的脑损伤治疗策略。这些工具可翻译为 研究局部组织反应的任何生物系统。预期的结果是一个新的平台来快速调查 首次在脑内发现内源性鸟苷信号及对鸟苷的深入理解 缺血时的调节和神经调节。这项工作将对鸟苷的研究产生积极的影响 这将大大提高人们对鸟苷在大脑直接损伤反应中的作用的了解。

PROJECT SUMMARY Measuring dynamic guanosine signaling at the site of focal ischemia over time remains challenging to probe with existing technology yet knowledge of the dynamics, regulatory mechanisms, and function of local guanosine fluctuations during ischemia would positively impact our understanding of the brain’s immediate local neuroprotective response. Guanosine is a nucleoside purine which has been postulated to play a potent restorative role after ischemic injury; however, to date, the mechanism and dynamics of guanosine action in the brain remains unresolved. Additionally, knowledge of the extent to which guanosine signaling changes as a function of ischemia duration and severity would provide critical insight into guanosine’s role as a neuroprotector. We propose to solve a significant gap in the understanding of guanosine signaling dynamics during focal ischemia by developing a microfluidic platform to initiate sustained local oxygen-glucose deprivation in a sub-region of a brain slice and using fast-scan cyclic voltammetry (FSCV) recording of guanosine with millisecond-to-second temporal resolution to provide critical insight into the mechanisms of guanosine regulation. Measuring local guanosine dynamics at the site of injury with significantly improved spatiotemporal resolution will provide critical information of the brain’s immediate local damage response. This proposal fits within our long-term goal to develop analytical tools to detect and understand dynamic neurochemical-regulated inflammation in the brain during injury. The rationale for this proposal is that these tools will provide knowledge of the dynamics, mechanism, and function of rapid guanosine signaling in the brain during ischemia for the first time which could further inform the development of guanosine- targeted therapies for neurological injury. The proposal will be completed by the following three specific aims: (1) Develop microfluidic platforms for delivery of spatiotemporally controlled and sustained focal ischemia to brain slices, (2) Characterize the mechanism of rapid guanosine release and clearance in the hippocampus as a function of ischemia severity and location, and (3) Characterize the impact of rapid guanosine signaling on local adenosine changes during focal ischemia. We will pursue these aims with an innovative approach by using novel microfluidic platforms for time-controlled delivery of ischemia to brain slices coupled to rapid electrochemical recording of guanosine signaling with FSCV for the first time. This work is significant because these studies will enable extraordinary mechanistic insight into the brain’s immediate response to ischemia over varying ischemia durations and severities which will directly impact future therapeutic strategies for brain injury. The tools are translatable to any biological system to study local tissue responses. The expected outcome is a new platform to investigate rapid endogenous guanosine signaling in the brain for the first time and an in-depth understanding of guanosine regulation and neuromodulation during ischemia. This work will have a positive impact on how guanosine is studied and will significantly advance knowledge of guanosine’s role in the brains immediate damage response.