Oxidative Stress in Neurodegeneration & Neuroprotection in Glaucoma

描述(申请人提供)：我们最近对人类青光眼和体内、体外实验模型的研究共同支持氧化应激是青光眼神经变性过程中细胞事件的主要组成部分。基于这些研究的结果，我们假设(1)氧化应激降低视网膜神经节细胞(RGC)和轴突应对青光眼高眼压(IOP)相关应激的能力，并促进神经退行性损伤(通过产生直接的神经毒性和继发性神经退行性后果，如胶质功能障碍和神经炎症)；以及(2)通过抑制或消除氧化应激的有害后果，抗氧化剂治疗可以保护神经退行性损伤。拟议的项目将在一只小鼠的青光眼模型中测试这一假设的有效性，该模型通过在一只眼内注射前房微珠/粘弹性来实验诱导高眼压。我们将使用两种实验范式来分析氧化应激增加或减少对青光眼神经元损伤的时间效应。在第一个实验范式中，我们将分析在实验诱导的超氧化物歧化酶1(SOD1)-/-与C57BL/6J(野生型)小鼠的青光眼中，“缺乏抗氧化反应”对神经元损伤的影响。这项分析将确定SOD1-/-小鼠的过载氧化应激是否会增加实验性青光眼神经元损伤的易感性。在第二个实验范式中，我们将分析药物“抗氧化治疗”对实验性青光眼野生型小鼠神经元损伤的影响。动物将接受Tempoll(一种抗氧化剂，防止多种氧化剂，如超氧化物、过氧亚硝酸盐和羟基自由基)或载体(生理盐水)，使用皮下植入的微型渗透泵，通过持续输注给药。这项分析将确定氧化应激的药物抑制是否能打破导致神经退行性损伤的事件循环，以及抗氧化剂治疗是否有可能作为青光眼的神经保护策略。我们将使用一种基于成像的改进方法，通过计算RGC轴突和SoMAS的整体数量来确定神经元损伤，而不是采样偏差。我们还将确定Sod1-/-和Tempol对青光眼氧化应激相关主要分子结果的影响，包括反映氧化修饰的视网膜和视神经蛋白羰基和HNE水平，以及调节青光眼神经胶质免疫/炎症反应的“氧化还原敏感”转录因子--NF-κB。这些数据将在长达12周的实验期间，在不同时间点的累积眼压暴露(以及每组中的轴突丢失)匹配的实验组之间进行统计比较。该项目有望确定氧化应激对青光眼神经变性的致病作用，以及抗氧化剂治疗作为青光眼神经保护策略的潜力。这一新信息也将对其他眼部和神经退行性疾病产生广泛的影响。

DESCRIPTION (provided by applicant): Our recent studies of human glaucoma and in vivo and in vitro experimental models collectively support that oxidative stress is a major component of cellular events during glaucomatous neurodegeneration. We hypothesize based on the findings of these studies that (1) oxidative stress reduces the ability of retinal ganglion cells (RGCs) and axons to cope with the elevated intraocular pressure (IOP)-related stress in glaucoma and facilitates neurodegenerative injury (by generating direct neurotoxicity and also secondary neurodegenerative consequences, such as glial dysfunction and neuroinflammation); and (2) by suppressing or eliminating the harmful consequences of oxidative stress, antioxidant treatment can protect against neurodegenerative injury. Proposed project will test the validity of this hypothesis in a mouse model of glaucoma with experimentally induced ocular hypertension by anterior chamber microbead/viscoelastic injections in one eye. We will use two experimental paradigms to analyze the temporal effects of increased or decreased oxidative stress on glaucomatous neuronal injury. In the first experimental paradigm, we will analyze the effects of "deficient antioxidant response" on neuronal injury in experimentally induced glaucoma in superoxide dismutase 1 (SOD1)-/- versus C57BL/6J (wild-type) mice. This analysis will determine whether overloaded oxidative stress in SOD1-/- mice increases the susceptibility to neuronal injury in experimental glaucoma. In the second experimental paradigm, we will analyze the effects of a pharmacological "antioxidant treatment" on neuronal injury in wild-type mice with experimental glaucoma. Animals will receive Tempol (an antioxidant protecting against multiple oxidants, such as superoxide, peroxynitrite, and hydroxyl radicals) or vehicle (saline) using subcutaneously implanted osmotic mini-pumps for drug delivery by constant infusion. This analysis will determine whether pharmacological inhibition of oxidative stress acts to break the cycle of events leading to neurodegenerative injury, and whether antioxidant treatment has any potential as a neuroprotective strategy for glaucoma. We will determine neuronal injury by counting RGC axons and somas in their entirety using an imaging-based improved methodology free from sampling bias. We will also determine SOD1-/- and Tempol effects on oxidative stress-related major molecular outcomes of glaucoma, including retina and optic nerve protein carbonyl and HNE levels reflecting oxidative modifications and a "redox- sensitive" transcription factor, NF-κB that regulates glial immune/inflammatory responses in glaucoma. The data will be statistically compared between experimental groups matched for the cumulative IOP exposure (and axon loss within each group) at different time points during an experimental period of up to 12 weeks. This project is expected to determine the pathogenic importance of oxidative stress for glaucomatous neurodegeneration and the potential of antioxidant treatment as a neuroprotection strategy in glaucoma. The new information will also have broad implications useful for other ocular and neurodegenerative diseases.