Theranostics of Photoreceptor-RPE-Choroid Neurovascular Unit in Eye Diseases:

标题：眼科疾病中光感受器-RPE-脉络膜神经血管单位的病理学 PI：Robert J.Zawadzki，博士 摘要 遗传性和老年性黄斑变性(AMD)目前是严重视力障碍的原因 在200多万美国居民中，随着人口老龄化，患病率预计到2040年将翻一番。 变性发生在光感受器细胞、视网膜色素上皮(RPE)细胞和脉络膜 血管系统，眼球后部紧密相互依赖的组织的复合体。这个项目将继续我们的 光感受器-视网膜色素上皮-脉络膜复合体形态和功能的活体研究 视网膜变性，包括RPE线粒体功能障碍模型和Doyne Honeycomb视网膜 营养不良。这些模型概括了遗传性和老年性黄斑变性的主要特征： RPE细胞退变和RPE与Bruch‘s之间细胞外沉积的增龄性增加 膜，它将RPE与脉络膜毛细血管床分开。该项目将使用创新的蜂窝- 视网膜结构和功能的活体水平分辨率探测结合额外的功能测试 光感受器-RPE-脉络膜神经血管单位主要成分的年龄相关性变化特征 (中华人民共和国-NVU)。这些研究将在具有遗传缺陷和野生动物的小鼠队列中纵向进行。 类型对照组，以高脂饮食喂养(以加速疾病进展)。这些研究将检验这一假设 RPE-Bruch膜的原发缺陷会导致光感受器的继发性退化和 脉络膜毛细血管系统。研究包括光感受器结构和功能的测量(使用 时间散斑平均-光学相干层析成像(TSA-OCT)，基于OCT的光学视网膜成像 (Org)和全场视网膜电图(FfERG))，RPE马赛克和Bruchs膜的标测 水驱动的BRM散射、RPE细胞自发荧光强度和RPE细胞的形态随时间的瞬时变化 TSA-OCT的发射光谱、RPE细胞的氧化还原状态和脉络膜毛细血管的形态 血管造影术。我们将分析这些多维纵向数据，以发现潜在的结构和 PRC-NVU退变的功能生物标志物并评价其对疾病进展的预测价值。在… 在活体实验结束后，每只小鼠一只眼的脉络膜RPE将被成像 配备光学相干显微镜(OCM)的定制高分辨率体外共焦显微镜 单元和LiveCell Stage Top孵化系统，可对平装在一起的RPE细胞进行成像 巩膜(以验证活体观察和评价RPE细胞器的荧光寿命)； 每只小鼠第二只眼睛的视网膜将通过常规的组织学和生化方法进行评估。 在光学或电子显微镜上进行测量，以验证体内的形态发现。 一种通过近红外光降解纳米颗粒将药物输送到RPE的新方法将被用于 局部靶向RPE细胞的治疗药物，以及空间分辨成像将用于确定 这些治疗剂可以延缓和阻止疾病的发展。通过结合纵向、活体成像和 光学纳米疗法，这些研究将为在人眼内局部靶向给药奠定基础 疾病。

TITLE: Theranostics of Photoreceptor-RPE-Choroid Neurovascular Unit in Eye Diseases PI: Robert J. Zawadzki, Ph.D. SUMMARY Inherited and age-related macular degeneration (AMD) are currently responsible for severe vision impairment in over 2 million US residents, with prevalence expected to double by 2040 as the population ages. Degeneration occurs in photoreceptor cells, retinal pigment epithelial (RPE) cells, and the choroidal vasculature, a complex of tightly interdependent tissues in the posterior eye. This project will continue our in vivo investigations of morphology and function of the photoreceptor-RPE-choroid complex in animal models of retinal degenerations, including the RPE mitochondrial dysfunction model and Doyne Honeycomb Retinal Dystrophy. These models recapitulate major hallmarks of inherited and age-related macular degeneration: degeneration of RPE cells and age-related increase in extracellular deposits between the RPE and Bruch’s membrane, which separates the RPE from the choroidal capillary bed. The project will use innovative, cellular- level resolution in vivo probing of retinal structure and function combined with additional functional tests to characterize age-related changes in major components of the photoreceptor-RPE-choroid neurovascular unit (PRC-NVU). These studies will be performed longitudinally in cohorts of mice with genetic defects and wild- type controls, fed with a high-fat diet (to accelerate disease progression). The studies will test the hypothesis that primary defects in RPE-Bruch’s membrane cause secondary deterioration of photoreceptors and choriocapillaris vasculature. The studies include measures of photoreceptor structure and function (using Temporal Speckle Averaging - Optical Coherence Tomography (TSA-OCT), OCT-based Optoretinography (ORG)), and full-field Electroretinography (ffERG)), mapping of RPE mosaic and Bruch’s membrane morphology with water driven transient changes in BrM scattering, RPE cell autofluorescence intensity and emission spectrum, the redox status of RPE cells, and choriocapillaris vascular morphology using TSA-OCT Angiography. We will analyze these multidimensional longitudinal data to discover potential structural and functional biomarkers of PRC-NVU degeneration and evaluate its predictive value for disease progression. At the termination of in vivo experiments, the choroid-RPE from one eye of each mouse will be imaged with a custom high-resolution ex vivo confocal microscope equipped with an Optical Coherence Microscopy (OCM) unit and a LiveCell stage top incubation system allowing imaging of a “live” RPE cells flat-mounted with the sclera (to allow validation of in vivo observations and evaluation of Fluorescence Lifetime of RPE organelles); the retina from the second eye of each mouse will be evaluated by conventional histological and biochemical measurements performed on light or electron microscopes, to allow validation of morphological in vivo findings. A novel method for delivering drugs to the RPE via near-infrared light-degradable nanoparticles will be used to locally target therapeutic agents to ailing RPE cells, and spatially resolved imaging will be used to determine if the therapeutic agents slow and stop disease progression. By combining longitudinal, in vivo imaging and optical nanotherapies, these studies will lay a foundation for locally targeted drug delivery in human ocular disease.