A hyperspectral approach to RPE fluorophores in AMD

AMD 中 RPE 荧光团的高光谱方法

基本信息

批准号：
10365650
负责人：
Thomas Ach
金额：
$ 53.1万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10365650
关键词：
3-Dimensional Address Adult Age related macular degeneration Algorithms Analytical Chemistry Anterior Apical Area Automobile Driving Biological Biology Blindness Cellular biology Choroid Classification Clinical Clinical Trials Cone Cytoplasmic Granules Detection Development Plans Disease Early Diagnosis Elderly Electron Microscopy Eye Fluorescence Funding Fundus German population Health Histologic Histology Human Image In Situ Ions Knowledge Left Light Lighting Lipids Lipofuscin Liquid Chromatography Measures Melanins Melanosomes Microscopy Molecular Molecular Profiling Morphology Optic Disk Optical Coherence Tomography Organelles Outcome Outcome Measure Pathology Peripheral Photoreceptors Process Resolution Retina Retinal Cone Retinal Diseases Risk Rod Signal Transduction Site Solvents Source Structure Structure of retinal pigment epithelium Sucrose Techniques Technology Thin Layer Chromatography Tissue Donors Tissue imaging Tissues Translating Validation Vertebrate Photoreceptors age related aged base chemical property clinical application clinical imaging data sharing density diagnostic technologies fluorescence lifetime imaging fluorophore follow-up fovea centralis fundus imaging high risk in vivo macula mass spectrometric imaging member microscopic imaging multidisciplinary neovascularization novel optical spectra patient population retinal rods success tandem mass spectrometry

项目摘要

Clinical trials for age-related macular degeneration (AMD) depend on imaging outcomes to measure success. Strong autofluorescence (AF) signal from the retinal pigment epithelium (RPE) has high potential for noninvasive, spatially and molecularly precise early detection, and longitudinal follow-up. Subcellular signal sources of RPE AF are lipofuscin (LF) granules, many also containing melanin (melanolipofuscin, ML). New concepts of human RPE cell biology make fulfilling the potential of fundus AF more possible than ever. Each adult human perifoveal RPE cell has >1400 organelles that generate reflectivity for optical coherence tomography (OCT). Half are LF or ML that also generate fundus AF (FAF) signal to blue-green exciting light. Melanosomes (M) preferentially localize to apical processes and may contribute their own FAF signal. The congruent topographies of FAF and photoreceptors has been confirmed and detailed, with low signal in the fovea and strong signal at a ring of high rod density encircling fovea and optic nerve head. At this ring is also an abundance of well-studied bisretinoid fluorophore A2E. By OCT, hyperreflective foci conferring high progression risk include RPE anteriorly migrating into the retina. A far-reaching new finding from the previous project period is that foveal RPE is dominated by ML, thus imparting a specific molecular signature to RPE at this cone-rich site. Consistent with regional molecular differences in RPE, our imaging mass spectrometry (IMS) studies revealed lipid signals specific to RPE in central macula. Unresolved questions are whether multiple fluorophores localize to each organelle, what organelle emits FAF signal with near-infrared (NIR) excitation, and what molecules are responsible for macular AF. We hypothesize that major fluorophores driving macular FAF signal are bisretinoids localized to specific organelle subtypes, which in turn are regionally distributed in accordance with the distribution of cones and rods. Drs. Curcio, Ach, and Schey, members of the multidisciplinary Hyperspectral Retinal Autofluorescence Team (HYRAFT) with expertise in AMD pathology, clinical imaging and microscopy, and analytic chemistry, respectively, propose studies in human donor eyes to address these knowledge gaps. In RPE-choroid flat mounts and tissue cross-sections of aged normal (N=20) and AMD (N=25) eyes subject to ex vivo OCT, Aim 1 will fortify a regional and morphologic basis of FAF imaging using tissue cross-sections to use imaging mass spectrometry to identify lipid signals including fluorophores. In these tissues Aim 2 will enumerate and determine emission spectra of LF, ML, and F in 3- dimensions using high-resolution structured illumination microscopy and a NIR-sensitive camera. Aim 3 will use discontinuous sucrose gradients to isolate RPE organelles in pooled maculas and peripheries (80 normal eyes, 40 donors) to extract fluorophores, separate them with fluorescent thin layer chromatography, and identify fluorophores using liquid chromatography – tandem mass spectrometry. Results will directly translate to molecularly informed clinical FAF imaging by many technologies, in AMD and other retinal disorders.

年龄相关性黄斑变性 (AMD) 的临床试验取决于影像结果来衡量成功与否。来自视网膜色素上皮 (RPE) 的强自发荧光 (AF) 信号具有很高的潜力无创、空间和分子精确的早期检测以及亚细胞信号的纵向随访。 RPE AF 的来源是脂褐质 (LF) 颗粒，许多还含有黑色素 (melanolipofuscin, ML)。人类 RPE 细胞生物学概念使眼底 AF 的潜力比以往任何时候都更有可能实现。成人中心凹周围 RPE 细胞具有 >1400 个细胞器，可产生光学相干反射率断层扫描 (OCT)。一半是 LF 或 ML，也生成蓝绿色激发光的眼底 AF (FAF) 信号。黑素体 (M) 优先定位于顶端突起，并可能贡献自己的 FAF 信号。 FAF 和光感受器的一致拓扑结构已得到确认和详细说明，在 FAF 和光感受器中信号较低中央凹和围绕中央凹和视神经乳头的高杆密度环处也有强信号。大量经过充分研究的双视黄醇荧光团 A2E 通过 OCT 检测，高反射灶具有高反射率。进展风险包括 RPE 向前迁移到视网膜，这是一项影响深远的新发现。项目期间的特点是中心凹 RPE 由 ML 主导，从而赋予 RPE 特定的分子特征这个富含锥体的位点与 RPE 的区域分子差异一致，我们的成像质谱分析。 (IMS) 研究揭示了黄斑中心 RPE 特异的脂质信号是否存在。每个细胞器都有多个荧光团，哪个细胞器发出近红外 (NIR) 的 FAF 信号激发，以及哪些分子负责黄斑 AF。我们研究了驱动黄斑 AF 的主要荧光团。黄斑 FAF 信号是定位于特定细胞器亚型的双视黄醇信号，而这些亚型又是区域性的根据 Curcio、Ach 和 Schey 博士的分布进行分布。多学科高光谱视网膜自发荧光团队 (HYRAFT) 拥有 AMD 病理学方面的专业知识，临床成像和显微镜以及分析化学分别提出了对人类捐赠者眼睛的研究解决这些知识空白。和 AMD (N=25) 眼睛接受离体 OCT，目标 1 将强化 FAF 的区域和形态学基础使用组织横截面成像，使用成像质谱法识别脂质信号，包括在这些组织中，目标 2 将枚举并确定 3- 中 LF、ML 和 F 的发射光谱。使用高分辨率结构照明显微镜和近红外敏感相机 Aim 3 将测量尺寸。使用不连续的蔗糖梯度来分离汇集的黄斑和外周细胞中的 RPE 细胞器（80 个正常眼睛，40个供体）提取荧光团，用荧光薄层色谱分离它们，并使用液相色谱-串联质谱法识别荧光团结果将直接转化。通过多种技术进行分子信息临床 FAF 成像，用于治疗 AMD 和其他视网膜疾病。