Limiting brain reperfusion injury by controlling mitochondrial function

 描述（由申请人提供）：脑血管疾病，尤其是脑缺血，是美国死亡和长期残疾的主要原因。目前唯一的治疗方法是迅速恢复缺血组织的血流。然而，由缺血/再灌注引起的大部分损伤发生在再灌注阶段：随着缺血组织再氧化，活性氧（ROS）迅速产生，在再流期间早期开始。再灌注损伤已被证明难以治疗脑缺血，可能是因为有效的药物浓度在再灌注的早期阶段没有充分建立。线粒体电子传递链（ETC）是细胞应激期间由于ETC过度活化而产生ROS的主要位点，其导致高线粒体膜电位（Δ Km），这反过来触发过量ROS产生。因此，我们建议理想的治疗应该以ETC为靶点，以非侵入性方式防止再流开始时ROS的产生。因此，我们在本申请中的总体目标是开发一种新的非侵入性疗法，以使再流期间的线粒体过度活跃正常化。我们将利用细胞色素c氧化酶（考克斯）的红外光（IRL）的光感受特性来调节线粒体活性，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的主要细胞光受体和ETC的末端酶。我们已经发现了四种特定的IRL波长，其部分抑制考克斯（而不是激活考克斯，即，目前的模式）。我们表明，抑制IRL，在再灌注时，提供了深刻的神经保护。在这项提案中，我们将建立在这些令人信服的初步数据，并利用我们的研究团队独特的，多学科的专业知识：确定我们的四个IRL波长的组合和能量，产生最佳的抑制考克斯和线粒体在体外使用离体大鼠脑考克斯和线粒体（目的1）。研究IRL介导的保护机制，以支持我们的再灌注期间IRL作用的中心假设：IRL →考克斯活性↓ → Δ COX ↓ → ROS↓ →活力↑，使用暴露于模拟缺血-再灌注的大鼠原代神经细胞线粒体功能的实时成像（目的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模式，以最大限度地提高缺血后神经保护（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).