Limiting brain reperfusion injury by controlling mitochondrial function

 描述(申请人提供)：脑血管疾病，尤其是脑缺血，在美国是导致死亡和长期残疾的主要原因。目前也是唯一的治疗方法是迅速恢复缺血组织的血流量。然而，缺血/再灌流造成的损伤很大一部分发生在再灌流阶段：当缺血组织被复氧时，很快就会产生活性氧物种(ROS)，从复流的早期开始。再灌注损伤被证明很难用药物治疗，可能是因为在再灌注的早期阶段有效的药物浓度没有足够的积累。线粒体电子传递链(ETC)是细胞应激过程中产生ROS的主要部位，由于ETC的过度激活导致线粒体膜电位(ΔΨm)升高，进而引发过量ROS的产生。因此，我们建议理想的治疗方法应该是无创的靶向ETC，以防止复流开始时ROS的产生。因此，我们在这项应用中的总体目标是开发一种新的、非侵入性的治疗方法，以正常化复流期间的线粒体过度活动。我们将利用细胞色素c氧化酶(COX)对红外线(IRL)的感光特性来调节线粒体的活性，从而减弱ROS的产生，从而限制脑缺血/再灌注损伤。细胞色素c氧化酶是IRL的主要细胞光受体，也是ETC的终末酶。我们已经发现了四个特定的IRL波长，它们部分抑制COX(而不是激活COX，即当前的范式)。我们表明，在再灌流时应用抑制性IRL，可提供深刻的神经保护。在这项提案中，我们将以这些令人信服的初步数据为基础，并利用我们研究团队独特的、多学科的专业知识：确定我们的四个IRL波长的组合和能量，以使用分离的大鼠脑COX和线粒体在体外产生最佳的COX和线粒体抑制(目标1)。利用模拟缺血再灌注大鼠原代神经细胞线粒体功能的实时成像，研究IRL介导的保护机制，以支持我们关于IRL在再灌流期间作用的中心假说：IRL→COX活性↓→ΔΨm↓→ROS↓→活力↑(目标2)。开发IRL介导的保护，并使用大鼠全脑缺血模型确定最佳的时间治疗范例，以最大限度地发挥缺血后神经保护作用(目标3)。

 DESCRIPTION (provided by applicant): Cerebrovascular disease, most notably brain ischemia, is a leading cause of death and long-term disability in the US. The current and only treatment is prompt restoration of blood flow to the ischemic tissue. However, a substantial portion of the damage caused by ischemia/reperfusion occurs during the reperfusion phase: as ischemic tissue is reoxygenated reactive oxygen species (ROS) are quickly generated, starting early during reflow. Reperfusion injury has proved difficult to treat pharmacologically, likely because effective drug concentrations have not built up sufficiently during the early phase of reperfusion. The mitochondrial electron transport chain (ETC) is a major site of ROS production during cellular stress due to ETC hyper-activation, which causes high mitochondrial membrane potentials (ΔΨm), which in turn trigger excessive ROS production. We thus propose that the ideal therapy should target the ETC non-invasively to prevent the generation of ROS from the onset of reflow. Accordingly, our overall goal in this application is to develop a new, non-invasive therapy to normalize mitochondrial hyperactivity during reflow. We will capitalize on the photoreceptive properties of cytochrome c oxidase (COX) for infrared light (IRL) to modulate mitochondrial activity, thereby attenuating the production of ROS and, as a result, limit ischemia/reperfusion injury in the brain. Cytochrome c oxidase is the primary cellular photo-acceptor of IRL and the terminal enzyme of the ETC. We have discovered four specific IRL wavelengths that partially inhibit COX (instead of activating COX, i.e., the current paradigm). We show that inhibitory IRL, applied at the time of reperfusion, provides profound neuroprotection. In this proposal, we will build on these compelling preliminary data and capitalize on the unique, multi-disciplinary expertise of our research team to: Identify the combinations and energies of our four IRL wavelengths that yield optimal inhibition of COX and mitochondria in vitro using isolated rat brain COX and mitochondria (Aim 1). Investigate the mechanism of IRL-mediated protection in support of our central hypothesis of IRL action during reperfusion: IRL → COX activity↓ → ΔΨm↓ → ROS↓ → viability↑, using real-time imaging of mitochondrial funtion in rat primary neural cells exposed to simulated ischemia-reperfusion (Aim 2). Develop IRL-mediated protection and identify the optimal temporal treatment paradigm using a rat model for global brain ischemia to maximize post-ischemic neuroprotection (Aim 3).