A hyperspectral approach to RPE fluorophores in AMD

AMD 中 RPE 荧光团的高光谱方法

• 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
• 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

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.

与年龄相关的黄斑变性（AMD）的临床试验取决于成像结果以衡量成功。 来自视网膜色素上皮（RPE）的强荧光（AF）信号具有很高的潜力。 无创，空间和分子精确的早期检测和纵向随访。亚细胞信号 RPE AF的来源是Lipofoscin（LF）颗粒，许多还含有黑色素（黑色素莫拉替辛，ML）。新的 人类RPE细胞生物学的概念使得能够实现比以往任何时候都更有可能的潜力。每个 成年人类perifoveal RPE单元具有> 1400个细胞器，可为光学连贯性产生反射率 断层扫描（OCT）。一半是LF或ML，它也会产生眼底AF（FAF）信号至蓝绿色令人兴奋的光。 黑色素体（M）优先定位于顶端过程，并可能贡献自己的FAF信号。这 已经确认和详细介绍了FAF和感光器的一致地形，在较低的信号中 Fovea和高杆密度环绕中央凹和视神经头部的强信号。在这个环上也是 研究良好的双素荧光团A2E的抽象。到OCT，高反射焦点会议高 进展风险包括向前迁移到视网膜的RPE。以前的深远的新发现 项目期间是Foveal RPE由ML支配，因此将特定的分子签名授予RPE 这个圆锥丰富的网站。与RPE的区域分子差异一致，我们的成像质谱法 （IMS）研究揭示了中央黄斑中针对RPE的脂质信号。未解决的问题是是否 多个荧光团位于每个细胞器中，哪个细胞器用近红外发射FAF信号（NIR） 兴奋，哪些分子负责黄斑AF。我们假设主要的荧光团驱动 黄斑FAF信号是局限于特定细胞器亚型的双骨类素，又是区域性的 根据锥和杆的分布分布。博士。 Curcio，ACH和Schey， 具有AMD病理学专业知识的多学科高光谱性视网膜自动荧光团队（Hyfraft）， 临床成像和显微镜以及分析化学，分别是人类捐赠者眼中的建议研究 解决这些知识差距。在RPE-螺状平坦的固定座和年龄正常的组织横截面（n = 20） 和AMD（n = 25）的眼睛受到离体OCT的约束，AIM 1将增强FAF的区域和形态学基础 使用组织横截面的成像使用成像质谱法来识别脂质信号 荧光团。在这些组织中，目标2将列举并确定3-中LF，ML和F的发射光谱 使用高分辨率结构化照明显微镜和Nir敏感摄像机的尺寸。目标3意志 使用不连续的蔗糖梯度分离汇总的斑块和外围设备中的RPE细胞器（80正常 眼睛，40个供体）提取荧光团，用荧光薄层色谱分离它们，然后 使用液相色谱 - 串联质谱法鉴定荧光团。结果将直接翻译 通过许多技术，AMD和其他永久性疾病的分子知情的临床FAF成像。