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

项目摘要 急性呼吸窘迫综合征（ARDS）是急性肺损伤（ALI）中最严重的一种， 这是成人和儿童中威胁生命的疾病的主要原因。不幸的是，尽管几十年来 在寻求有效治疗的研究中，ARDS的高死亡率基本保持不变， 需要在评估患有ARDS的个体以指导治疗策略的方法学上取得突破。 氧化应激在ARDS（以及许多其他人类疾病）的发病机制中至关重要; 然而，由于活性氧和氮，抗氧化疗法的临床应用很复杂， 在低水平和受控水平下产生的物质也是细胞内稳态所必需的信号分子 以及通过靶向的特异性氧化还原敏感途径适应细胞应激。根据这些发现， “氧化应激”已经从简单的氧化剂和抗氧化剂的不平衡重新定义为还包括 氧化还原信号传导和控制的中断。这一修订后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;这些进展提供了新的机会，了解和治疗 导致ARDS发病的氧化还原调节过程的破坏。一个主要的障碍， 对人类ARDS中氧化还原信号失调的研究缺乏严格的方法学来精确地 确定体内肺的氧化还原状态。该提案通过开发新的 应用电子显微镜在体检测急性肺损伤后氧化还原状态的技术 共振 分子 的 反应 EPR 我们假设电子顺磁的高级应用 使用快速扫描体内EPR成像的临床前模型的共振（EPR）光谱将 提供了新的见解的时间进程和本地化的自由基的生产和氧化还原状态 肺部疾病我们将使用EPR自旋探针，可以区分总细胞和线粒体 超氧化物以及一种新的自旋探针，可以检测细胞内谷胱甘肽的氧化还原状态，提供 精确和专门的测量氧化应激水平不同的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 然后可以很容易地检测和定量的EPR光谱仪，而“自旋探针”分子， 巯基物质如谷胱甘肽可以报告细胞巯基氧化还原状态。重要的是，像MRI一样， 成像光谱仪使用容易穿透组织并使自旋可视化的无线电波 在老鼠的器官里。 . EPR 大胆提出 分配 必不可少的， 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，

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