Developing EPR tools for preclinical interrogation of redox regulation mechanisms contributing to acute lung injury

项目总结 急性呼吸窘迫综合征(ARDS)是最严重的急性肺损伤(ALI)形式， 这是成人和儿童患上危及生命的疾病的主要原因。不幸的是，尽管几十年来 在寻找有效治疗方法的研究中，ARDS的高死亡率基本保持不变， 需要在评估ARDS患者的方法学上取得突破，以指导治疗策略。 氧化应激在ARDS(以及许多其他人类疾病)的发病机制中起关键作用； 然而，抗氧化剂疗法的临床效用是复杂的，因为活性氧和氮 在低水平和受控水平产生的物种也是细胞内稳态所必需的信号分子。 以及通过靶向的特定氧化还原敏感通路来适应细胞压力。基于这些发现， “氧化应激”已从简单的氧化剂和抗氧化剂失衡重新定义为也包括 氧化还原信号和控制的中断。这一修订的定义正在推动改进的工具和治疗 氧化还原生物学领域的方法；这些进展为理解和治疗提供了新的机会 参与ARDS发病的氧化还原调节过程的破坏。一个主要的障碍是 对人类ARDS氧化还原信号异常的研究缺乏严格的方法学来准确地 测定活体肺的氧化还原状态。这项提案通过开发新的 体内急性肺损伤肺组织氧化还原状态的电子学检测技术 共振 分子 那 反应 EPR 我们假设电子顺磁的高级应用 使用体内快速扫描EPR成像的临床前模型的EPR波谱将 为自由基生成和氧化还原状态的时间进程和定位提供新的见解 在肺部疾病中。我们将使用EPR自旋探针，它可以区分总细胞和线粒体 以及一种新型的自旋探针，它可以检测细胞内谷胱甘肽的氧化还原状态，提供 精确和专门测量不同程度氧化应激小鼠的氧化还原状态。我们 顺磁 (EPR)成像光谱学是测量自由基的金标准： 被称为“自旋陷阱”的探针与特定的短寿命自由基反应，形成更稳定的自由基。 然后可以很容易地被EPR光谱仪检测和量化，而“自旋探测器”分子 含有硫醇的物种，如谷胱甘肽，可以报告细胞内硫醇的氧化还原状态。重要的是，像核磁共振一样， 成像光谱仪使用无线电波，可以很容易地穿透组织，并使脊柱可视化 在老鼠的器官里。 。EPR 大胆求婚 作业 必不可少的， 研究ARDS的内表型，并为未来的临床研究和治疗决策提供信息。 这将提供以前无法获得的信息，指导

PROJECT SUMMARY Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), is a principal cause of life-threatening illness in both adults and children. Unfortunately, despite decades of research in search of effective treatments, the high mortality of ARDS has remained largely unchanged, requiring breakthroughs in the methodologies to assess individuals with ARDS to guide therapeutic strategies. Oxidative stress is critical in the pathogenesis of ARDS (as well as numerous other human diseases); however, the clinical utility of antioxidant therapies is complicated because reactive oxygen and nitrogen species produced at low and controlled levels are also signaling molecules essential for cellular homeostasis and adaptation to cellular stress through targeted specific redox-sensitive pathways. Based on these findings, “oxidative stress” has been redefined from a simple imbalance in oxidants and antioxidants to also include a disruption in redox signaling and control. This revised definition is driving improved tools and therapeutic approaches in the field of Redox Biology; these advances provide new opportunities to understand and treat the disruption of redox-regulated processes that contribute to pathogenesis of ARDS. One major barrier to the study of dysregulated redox signaling in human ARDS is the lack of rigorous methodologies to precisely determine the redox status of the lung in vivo. This proposal addresses this major gap by developing new technology to measure the redox status of the acutely injured lung in vivo using electron resonance molecular that react EPR probes We hypothesize that advanced applications of Electron Paramagnetic Resonance (EPR) spectroscopy to preclinical models using rapid scan in vivo EPR imaging will provide novel insight into the time-course and localization of free radical production and redox status in lung disease. We will use EPR spin probes that can differentiate between total cell and mitochondrial superoxide as well as a novel spin probe that can detect the redox status of intracellular glutathione, providing precise and specialized measures of the redox status in mice with different levels of oxidative stress. We paramagnetic (EPR) imaging spectroscopy is the gold-standard for the measurement of free radicals: probes known as “spin traps” react with specific short-lived free radicals to form more stable radicals can then be easily detected and quantified by the EPR spectrometer, while “spin probe” molecules that with thiol species such as glutathione can report cellular thiol redox status. Importantly, like MRI, the imaging spectrometer uses radio waves that readily penetrate tissue and enable visualization of the spin within the mouse organs. . EPR boldly propose assignment essential, of endophenotypes in ARDS and inform future clinical studies and therapeutic decisions. that this will provide previously unattainable information that will guide the