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.

年龄相关性黄斑变性（AMD）的临床试验依赖于成像结果来衡量成功。 来自视网膜色素上皮（RPE）的强自体荧光（AF）信号具有高的潜在性， 无创、空间和分子精确的早期检测和纵向随访。亚细胞信号 RPEAF的来源是脂褐素（LF）颗粒，许多还含有黑色素（黑素脂褐素，ML）。新 人类RPE细胞生物学的概念使得实现眼底AF的潜力比以往任何时候都更有可能。每个 成年人视网膜色素上皮细胞具有>1400个产生光学相干反射的细胞器 断层扫描（OCT）。一半是LF或ML，也产生眼底AF（FAF）信号，以蓝绿色激发光。 黑素体（M）优先定位于顶端突起，并可能贡献自己的FAF信号。的 FAF和光感受器的一致性形貌已经得到证实和详细说明，在FAF和光感受器中具有低信号。 中心凹及视乳头周围高密度杆状环信号强。在这枚戒指上， 大量经过充分研究的双维A酸荧光团A2 E。通过OCT，高反射病灶 进展风险包括RPE向前迁移到视网膜中。一个影响深远的新发现， 在项目期间，中心凹RPE由ML主导，因此在ML时赋予RPE特定的分子特征， 这个富含松果的地方与RPE的区域分子差异一致，我们的成像质谱 (IMS)研究揭示了中央黄斑中RPE特异性的脂质信号。未解决的问题是， 多个荧光团定位于每个细胞器，哪个细胞器用近红外（NIR）发射FAF信号 激发，以及什么样的分子是黄斑AF的原因。我们假设，主要的荧光团驱动 黄斑FAF信号是定位于特定细胞器亚型的双视黄酸，这些细胞器亚型又在区域上 根据锥体和杆的分布来分布。Curcio、Ach和Schey博士， 多学科高光谱视网膜自体荧光团队（HYRAFT），具有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，分别提出了在人类供体眼中的研究， 填补这些知识空白。在RPE-脉络膜平面标本和正常老年人（N=20）的组织横截面中 和AMD（N=25）眼进行离体OCT，目的1将加强FAF的区域和形态学基础 使用组织横截面成像以使用成像质谱法来识别脂质信号，包括 荧光团。在这些组织中，目标2将列举并确定3- 10中LF、ML和F的发射光谱。 使用高分辨率结构照明显微镜和NIR敏感相机测量尺寸。目标3将 使用不连续蔗糖梯度分离混合黄斑和周边中的RPE细胞器（80例正常人 眼睛，40个供体）提取荧光团，用荧光薄层色谱法分离它们， 使用液相色谱-串联质谱法鉴定荧光团。结果将直接转化为 在AMD和其他视网膜疾病中，通过许多技术进行分子信息临床FAF成像。