Experts in lung transplantation agree that ischemia is a recognized risk factor for all postlung transplant problems. Better non-invasive methods to detect ischemia post transplant would provide a window to modify therapy, and thereby reduce morbidity of veterans from these complications. Ischemia-reperfusion (IR) lung damage is also encountered clinically in conditions beyond lung transplantation, including necrotizing pneumonias, or crush injury to the chest (e.g Farivar et al, 2005), all injuries that frequently affect veterans. Substantial evidence supports the role of mitochondrial dysfunction and mitochondrially derived reactive oxygen species (mtROS) in IR injuries to the brain and heart (Stowe & Camara, 2009). Our preliminary data support a critical role for mitochondria in a rat model unilateral lung IR injury, a model of lung transplant which eliminates immunological or drug induced pathology in order to focus exclusively on IR injury. We introduce 2 absolutely novel non-destructive methods to track apoptosis and the redox state in intact IR lung, methods that can be rapidly translated into the clinical field. The first is single photon emission computed tomography/computed tomography (SPECT/CT) imaging, using 99m Tc-duramycin (DU) and 99m Tc- hexamethylpropyleneamine oxime (HMPAO) to detect cell death/apoptosis and gluthathione/redox status respectively. The second is fluorescence optical imaging to follow the redox ratio (RR) of lung. We hypothesize that: (i) IR stimulates mitochondrial complex I dysfunction which leads to subsequent apoptosis and decreased cell survival as detected biochemically and histologically (ii) SPECT/CT and optical imaging can detect and quantify apoptosis or altered mitochondrial bioenergetics in vivo. Changes in the values of these imaging endpoints after IR will correlate with histological injury and some indices of mitochondrial dysfunction (iii) treatment with agents that prevent opening of mitochondrial permeability transition pore (mPTP) and those that are used clinically to prevent rejection will protect against lung injury as detected by SPECT/CT or optical imaging, and this protection will correlate to improved indices of mitochondrial function. We will test these hypotheses with three specific objectives/aims. (1) To use SPECT and nuclear medicine probes which target apoptosis/necrosis or oxidoreductive state to detect unilateral IR lung injury in vivo We will correlate serial SPECT data after IR injury to histological and biochemical evidence of apoptosis/necrosis one week after ischemia. (2) To employ optical imaging which detects the oxidoreductive state of tissue to follow IR lung injury in vivo. The mitochondrial metabolic coenzymes Nicotinamide Adenine Dinucleotide (reduced form is NADH) and Flavine Adenine Dinucleotide (FAD) are the primary electron carriers in oxidative phosphorylation. NADH and FAD (oxidized form of FADH2) are autofluorescent and can be monitored by optical techniques without the need for exogenous labels. (3) To investigate the potential of our novel imaging methods to detect protection from IR injury by agents which act on complex I and the mitochondrial permeability transition pore (mPTP) and that are used clinically in lung transplantation. With our novel means to detect apoptosis and redox injury and a survival model of lung IR injury, we are poised to examine the potential of these non-destructive methods to track ischemic Injury. Though not used routinely for detection of lung injury, SPECT imaging with HMPAO is already in clinical use with well established safety profiles. With translational evidence of prognostic value, these imaging modalities can be adapted readily to improve the care of veterans with IR lung damage. We are uniquely positioned to test novel therapeutic interventions to improve IR lung injury with prognostic and mechanistic information in hand.
Many veterans with end stage lung disease are candidates for lung transplantation. In lung transplant, lung cells can be damaged by excessive generation of toxic products called reactive oxygen species that develop as a consequence of lack of blood supply (called ischemia). This injury is correlated with many short and long term complications of lung transplant. We have no means of detecting the extent of these injuries early on the process when interventions would be most effective. Our studies develop two novel imaging techniques which will for the first time measure injury in the lungs of live intact rats. These studies can be quickly translated into the clinical arena, and help decision making of clinicians at the bedside. We will explore the potential of FDA approved agents including an immunosuppressive agent cyclosporin A, steroids, and a sedative amobarbital, in protection against lung cell death with our novel non-invasive imaging methods.
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