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

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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