Novel Diagnostics to Detect Lung Injury

描述（由申请人提供）： 肺移植专家一致认为，缺血是所有肺移植后问题的公认危险因素。更好的非侵入性方法来检测移植后缺血将提供一个窗口，以修改治疗，从而减少这些并发症的退伍军人的发病率。缺血再灌注（IR）肺损伤在临床上也会出现在肺移植以外的疾病中，包括坏死性肺炎或胸部挤压伤（例如Farivar et al，2005），所有这些损伤都经常影响退伍军人。大量证据 支持线粒体功能障碍和脑源性活性氧（mtROS）在脑和心脏IR损伤中的作用（斯托和卡马拉，2009）。我们的初步数据支持线粒体在大鼠模型单侧肺IR损伤中的关键作用，该模型是一种肺移植模型，其消除了免疫或药物诱导的病理学，以专门关注IR损伤。我们介绍了两种绝对新颖的非破坏性方法来跟踪完整IR肺中的细胞凋亡和氧化还原状态，这些方法可以迅速转化为临床领域。第一种是单光子发射计算机断层扫描/计算机断层扫描（SPECT/CT）成像，使用99 mTc-杜霉素（DU）和99 mTc-六甲基丙烯胺肟（HMPAO）分别检测细胞死亡/凋亡和谷胱甘肽/氧化还原状态。第二种是荧光光学成像，用于跟踪肺的氧化还原比（RR）。我们假设：（i）IR刺激线粒体复合物I功能障碍，这导致随后的细胞凋亡和细胞存活减少，如生化和组织学检测的。（ii）SPECT/CT和光学成像可以检测和量化体内细胞凋亡或改变的线粒体生物能量学。IR后这些成像终点的值的变化将与组织学损伤和线粒体功能障碍的一些指标相关（iii）用防止线粒体渗透性转换孔（mPTP）开放的试剂和临床上用于防止排斥反应的试剂治疗将保护免受肺损伤，如通过SPECT/CT或光学成像检测的，并且这种保护将与线粒体功能的改善指标相关。我们将用三个具体目标来检验这些假设。(1)利用SPECT和核医学探针检测在体单侧IR肺损伤，靶向凋亡/坏死或氧化还原状态。我们将IR损伤后的系列SPECT数据与缺血后一周凋亡/坏死的组织学和生化证据相关联。(2)采用光学成像技术检测组织的氧化还原状态，以跟踪在体IR肺损伤。线粒体代谢辅酶烟酰胺腺嘌呤二核苷酸（还原形式为NADH）和黄素腺嘌呤二核苷酸（FAD）是氧化磷酸化中的主要电子载体。NADH和FAD（FADH 2的氧化形式）是自发荧光的，可以通过光学技术进行监测，而不需要外源性标记。(3)研究我们的新型成像方法检测作用于复合物I和线粒体通透性转换孔（mPTP）的药物对IR损伤的保护作用，以及临床上用于肺移植的药物的潜力。随着我们的新手段来检测细胞凋亡和氧化还原损伤和肺IR损伤的存活模型，我们准备检查这些非破坏性方法来跟踪缺血性损伤的潜力。虽然没有常规用于检测肺损伤，但使用HMPAO的SPECT成像已经在临床上使用，具有良好的安全性。随着预后价值的转化证据，这些成像方式可以很容易地适应，以改善与IR肺损伤的退伍军人的护理。我们处于独特的地位，以测试新的治疗干预措施，以改善IR肺损伤的预后和机械信息在手。

DESCRIPTION (provided by applicant): 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.