Spectral photoprotection of chronic macular photochemical injury

慢性黄斑光化学损伤的光谱光保护

• 批准号: 7594233
• 负责人: Robert F Bonner
• 金额: $ 26.27万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594233
• 关键词: Academy; Acute; Affect; Age; Age related macular degeneration; Aging; Amber; Animal Model; Automobile Driving; Autopsy; Biological Models; Blindness; Cadaver; Cataract; Cataract Extraction; Cells; Cellular Membrane; Chemicals; Chronic; Clinical; Clinical Research; Collaborations; Complex; Cultured Cells; Cytoplasmic Granules; Dark Adaptation; Dependence; Disease; Early Diagnosis; Early Intervention; Elderly; End Point; Epoxy Compounds; Equilibrium; Excision; Exposure to; Eye; Eye diseases; Fluorescence; Fundus; Generations; Genetic; Genetic Predisposition to Disease; Health; Human; Image; Image Analysis; Individual; Injury; Institutes; Intraocular Lens Implantation; Knockout Mice; Laboratories; Lasers; Lead; Lesion; Light; Link; Lipofuscin; Maryland; Medicine; Membrane; Methods; Microscopic; Modeling; Modification; Molecular; Monitor; National Institute of Child Health and Human Development; Normal Range; Ophthalmology; Ophthalmoscopes; Oxidative Stress; Pathology; Pathology processes; Pathway interactions; Patients; Photosensitization; Pigmentation physiologic function; Population; Population Study; Prevention; Prevention therapy; Primates; Process; Production; Pupil; Rate; Reaction; Reporting; Resolution; Retina; Retinal; Retinal Diseases; Rhodopsin; Russia; Scanning; Series; Singlet Oxygen; Sister; Site; Staging; Structure of retinal pigment epithelium; Sunlight; Testing; Theoretical model; Time; Toxic effect; United States National Institutes of Health; Universities; Validation; Weight; Youth; aged; crosslink; cytotoxic; design; disorder prevention; falls; fluorophore; improved; instrument; lens; light intensity; macula; macular dystrophy; normal aging; novel; oxidation; photoactivation; photoprotection; prevent; reactive oxygen intermediate; receptor; retinal rods; segregation; size; sunglasses; tool; transmission process; vision science

The associations of age-related macular degeneration (AMD) with cataracts, prior cataract surgery, cumulative exposure to sunlight and pigmentation all support the hypothesis that chronic photochemical injury drives macular changes with age and AMD progression. Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) and colocalizes with acute photosensitization of reactive oxygen intermediates (ROI) in the primate retina. We model the normal accumulation of potentially damaging photoproducts with age in the RPE and Bruchs Membrane (BM) complex as well as changes induced by additional spectral filtering of light reaching the macula. Lipofuscin granules contain at least 10 different fluorescent photochemical products includng A2E (N-retinylidene-N-retinylethanolamine), its epoxides and other as yet chemically unidentified A2E-related fluorophores. The precursors (A2PE and A2PEH2) of these fluorophores originate from reactions of all-trans-retinal within the receptor outer segments (ROS) during periods associated with significant rhodopsin bleaching (i.e., normal daylight). Although RPE lysosomal processing enzymatically digests over 99% of the shed ROS contents, A2E and related fluorophores are not digested, but are concentrated into lipofuscin granules. By age 60 years, the average concentration of A2E within RPE cells reaches 400 microM in normal eyes. However, A2E is toxic to cellular membranes at much lower concentrations. We hypothesize that segregation of A2E into lipofuscin granules and prevention of its redistribution into critical membranes is required for RPE health. We developed a biophysical model using normal values of pupil size, lens transmission, and rod dark adaptation time constant trh to determine average retinal spectral irradiance, steady-state concentration of all-trans-retinal, all-trans-retinal photosensitization of oxidative damage, all-trans-retinal reactions to form A2E-related species in the ROS, and A2E photo-oxidation within RPE lipofuscin granules as a function of age and ambient light intensity. Our model predicts a decline of about one third in the action spectra-weighted short-wavelength macular irradiance with each decade and a nearly constant production rate of A2E-related fluorophores in the RPE during the first 60 years (falling significantly thereafter). A similar age dependence of total lipofuscin granule volume and total fluorescence per RPE cell was reported recently in human cadaver eyes. Since the rates of lipofuscin increase with age are slower than the rate of decrease in short-wavelength macular irradiance in the phakic eye with age, ROI photosensitization in the RPE should also fall with increasing age. Photo-oxidative stress in the outer retina might arise from the smaller amounts of A2E-related fluorophores observed in critical membranes of the RPE/BM complex. However, if the RPE/BM complex were the site of photo-oxidative injury driving AMD progression, the magnitude and rate of this oxidative injury would be expected to increase dramatically (not observed) following cataract removal and intraocular lens (IOL) implantation. Consequently, we propose a novel hypothesis that singlet oxygen generation by RPE lipofuscin allows the chemical alteration of accumulating A2E, thereby limiting the steady-state levels of A2E (A2Ess) in the RPE, the redistribution of A2E into retinal membranes, and associated A2E chemical toxicity. Singlet oxygen generated photochemically within the lipofuscin granule reacts with its A2E to form A2E epoxides which then react to form increasingly complex cross-linked molecules. As short-wavelength macular irradiance falls with age, the rate of A2E photo-oxidation falls approximately up to 20-fold, causing A2Ess in the normal phakic eye to increase even as rod bleaching and A2E production decrease. Our theoretical model of macular aging reproduces the normal age dependence of lipofuscin and A2E and provides a primary cytotoxic mechanism in which, once A2E exceeds a cytotoxic threshold concentration in the RPE cell, A2E redistribution into critical membranes causes damage and loss of RPE function with or without additional photo-activation. In our model, it is primarily the yellowing of the lens with age that distorts the original spectral balance between rate of production and rate of photo-oxidation found in youth, and allows the A2Ess to rise with age. We are evaluating noninvasive retinal imaging methods that might permit clinical validation of our predictions of photochemical changes following cataract surgery and our predictions of the benefits of specific spectral photo-protective filters. We have designed and had manufactured bicolored sunglasses with two different specific spectrally selective lenses that provide equivalent spectral photoactivation of lipofuscin photosensitization of singlet oxygen but very different degrees of rod activation in bright ambient light. Our model predicts that in young eyes or elderly pseudophakes the levels of A2PEH2 production in the rods and the A2Ess levels in the RPE will be remarkably divergent in individuals wearing these sunglasses over a period of a few months of significant outdoor daylight activity. In order to distinguish the steady-state levels of different RPE photoproducts in the retina noninvasively, we are developing spectral imaging capabilities by modifying confocal scanning laser ophthalmoscopes and fundus cameras. We are currently evaluating the potential of these modified clinical instruments for noninvasive monitoring of the molecular effects of such filters in patients. Furthermore, we expect that improved spectral separation of the different species in the A2E pathway along with high resolution noninvasive imaging of the human retina, should enable us to quantify early microscopic abnormalities that we believe are the earliest stages in age-related maculopathy leading to AMD as well as genetically -linked diseases such as Stargardts macular dystrophy. Such quantitative early intermediate endpoints may prove critical in developing new more efficient clinical studies of AMD prevention. In collaboration with the NEI and the Eye Institute of the Russian Academy of Medicine, we are designing clinical studies of the effects of such filters on progression of both early and moderate AMD following cataract surgery and IOL implantation and macular changes in subjects with genetic predisposition to generation of increased A2E and lipofuscin at younger ages. In parallel laboratory experimentation, we are studying RPE cell cultures, human autopsy eyes, and animal models to better characterize the pathways and rates of A2E photo-oxidation and chemical modification. In collaboration with our sister NICHD lab (Steve Coon), we are planning to test our hypothesis of spectral imbalance by raising ABCA4 (-)/(-) and (-)/(+) knockout mice (that produce large amounts of A2E) in cyclic ambient light spectrally filtered eitehr with the rod-sparing vermilion filter or the amber filter which does not spare rods but provides the equivalent amount of protection from blue-light photo-oxidation reactions within the retina. This model system should validate our noninvasive spectral imaging separation of components by comparing with HPLC-MS of lipofuscin extracts from the whole retina at sacrifice and provide a stronger experimental rationale for our proposed clinical studies by validate the basic hypothesis that spectral imbalances of retinal irradiance can induce high A2E steady state levels in the RPE.

年龄相关的黄斑变性（AMD）与白内障，先前的白内障手术，累积暴露于阳光和色素沉着的关联都支持了以下假设：慢性光化学损伤随着年龄和AMD的进展，慢性光化学损伤驱动了黄斑变化。 脂肪霉素随着视网膜色素上皮（RPE）的年龄而累积，并随着灵长类动物视网膜中活性氧中间体（ROI）的急性光敏化而共定位。 我们对RPE和BRUCHS膜（BM）复合物中的年龄进行了可能破坏的光产物的正常积累，以及通过到达黄斑的光的其他光谱滤波引起的变化。 脂肪霉素颗粒含有至少10种不同的荧光光化学产品，其中包括A2E（N-替宁二基-N-返回乙酰乙醇胺），其环氧化物和其他尚未化学的A2E与A2E相关的荧光团。这些荧光团的前体（A2PE和A2PEH2）源自与明显的荧光蛋白漂白（即正常日光）相关的时期内受体外部段（ROS）中全反视网膜的反应。尽管RPE溶酶体加工的酶促消化超过99％的脱落玫瑰含量，但A2E和相关的荧光团没有消化，而是浓缩到脂蛋白颗粒中。到60岁时，RPE细胞内A2E的平均浓度在正常眼中达到400 microm。但是，A2E对浓度低得多的细胞膜有毒。我们假设RPE健康需要将A2E分离为脂肪霉素颗粒，并预防其将其重新分布为关键膜。 我们使用学生大小的正常值，透镜传播和杆暗适应时间恒定TRH开发了一个生物物理模型，以确定平均视网膜视网膜，全透明球光敏的稳态浓度，氧化性损伤，全反性损伤，全反性损伤，在A2E中形成了A2E的旋转中的全元素，并在Rosin和A2E中形成了旋转的旋转，并在Rosin和A2E中形成了旋转。作为年龄和环境光强度的函数。我们的模型预测，每十年的动作光谱加权短波黄斑辐照度的下降约为三分之一，并且在头60年中，RPE中与A2E相关的荧光团的生产率几乎恒定（此后大幅下降）。最近在人尸体眼中报道了总脂肪霉素颗粒体积和每个RPE细胞总荧光的年龄依赖性。由于随着年龄的增长，脂肪霉素随着年龄的增长速度的速度慢，而短波长黄斑辐照度的降低速率随着年龄的增长，RPE的ROI光敏化也应随着年龄的增长而下降。外视网膜中的光氧化应激可能是由于在RPE/BM复合物的临界膜中观察到的与A2E相关的荧光团较小。但是，如果RPE/BM复合物是驱动AMD进展的光氧化损伤部位，则该氧化损伤的幅度和速率有望在白内障和眼内透镜（IOL）植入后急剧增加（未观察到）。 因此，我们提出了一个新的假设，即RPE脂肪霉素产生单线氧允许累积A2E的化学改变，从而限制了RPE中A2E（A2ESS）的稳态水平，A2E将A2E重新分布在视网膜膜中，以及相关的A2E化学毒性。脂肪氧在脂肪霉素颗粒内与其A2E反应形成A2E环氧化物的光化学产生，然后反应形成日益复杂的交联分子。 随着短波长黄斑辐照的年龄下降，A2E光氧化的速率大约下降到20倍，从而在正常的Phakic Eye中导致A2ESS增加，即使Rod漂白和A2E产生降低也会增加。 我们的黄斑衰老理论模型再现了脂肪霉素和A2E的正常年龄依赖性，并提供了一种主要的细胞毒性机制，其中一旦A2E超过RPE细胞的细胞毒性阈值浓度，A2E的A2E将重新分配到关键膜中会导致重新分布中的RPE损坏和不带RPE的损坏，而无需其他照片效果。在我们的模型中，主要是随着年龄的增长而变黄的晶状体使原始光谱平衡之间的生产率和年轻人发现的光氧化速率之间的平衡扭曲，并允许A2ESS随着年龄的增长而上升。 我们正在评估非侵入性视网膜成像方法，这些方法可能允许临床验证我们对白内障手术后光化学变化的预测以及我们对特定光谱光保护过滤器的益处的预测。 我们已经设计并制造了两种不同特异性选择性透镜的双色太阳镜，这些镜片可提供脂肪蛋白氧气氧气光敏的光谱光激活，但在明亮的环境光中，杆的杆激活程度非常不同。我们的模型预测，在年轻的眼睛或老年假噬菌体中，杆子中A2PEH2产生的水平和RPE中的A2ESS水平在戴上这些太阳镜的个体中会明显不同，这在几个月的户外日光活动中都会显着。 为了区分视网膜中不同RPE光药物的稳态水平，我们正在通过修改共聚焦扫描激光眼镜和眼底摄像机来开发光谱成像能力。我们目前正在评估这些改良的临床工具的潜力，以无创监测此类过滤器对患者的分子效应。此外，我们期望A2E途径中不同物种的光谱分离以及人类视网膜的高分辨率无创成像，应该使我们能够量化早期的显微镜异常，我们认为这是年龄相关的大量病，导致AMD以及遗传上的疾病，以及像遗传上的callinceasesseases一样，如含糊不清。这种定量的早期中间终点可能对开发对AMD预防的更有效的临床研究至关重要。 在与俄罗斯医学院的NEI和眼科研究所合作，我们正在设计临床研究，了解此类过滤器对白内障手术和IOL植入后的早期和中度AMD进展的影响，以及IOL植入和黄斑变化，以及遗传易感性对增加A2E和脂肪素的产生的遗传易感性。 在平行实验室实验中，我们正在研究RPE细胞培养物，人体尸检和动物模型，以更好地表征A2E光氧化和化学修饰的途径和速率。 In collaboration with our sister NICHD lab (Steve Coon), we are planning to test our hypothesis of spectral imbalance by raising ABCA4 (-)/(-) and (-)/(+) knockout mice (that produce large amounts of A2E) in cyclic ambient light spectrally filtered eitehr with the rod-sparing vermilion filter or the amber filter which does not spare rods but provides the equivalent amount of protection from视网膜内的蓝光光氧化反应。 该模型系统应通过与牺牲时从整个视网膜中的脂肪增生蛋白提取物进行比较来验证组件的非侵入性光谱成像分离，并通过验证基本的临床假设，从而为我们提出的临床研究提供了更强大的实验原理，即视网膜降低的光谱不平衡可以诱导高A2E稳态水平。