Flavoprotein Autofluorescence Imaging in AMD

 描述（由申请人提供）：在发达社会中，年龄相关性黄斑变性（AMD）是65岁以上人群中法律的失明的最常见原因。新出现的证据表明视网膜色素上皮（RPE）线粒体功能障碍在干性AMD病理学中的重要作用。线粒体功能障碍的特征在于超氧化物的慢性过量产生和ATP的减少产生，并且其促进RPE细胞中损伤反应途径的活化，导致亚RPE存款形成（富脂玻璃疣的前体）和细胞外基质更新的失调，这是干性AMD的标志性特征。因此，视网膜靶向药物正在成为一类有吸引力的潜在治疗干性AMD的药物。鉴定活体眼睛中RPE线粒体功能障碍的存在的技术是有限的。在体内检测线粒体功能障碍将能够选择可能受益于AMD靶向药物的个体，并将加速干性AMD疾病模型中线粒体功能障碍的机制研究。本项目的目标是开发RPE黄素蛋白自发荧光作为线粒体功能障碍的成像生物标志物。线粒体功能障碍伴随着黄素蛋白（特别是FAD+）的氧化还原形式的比例增加，这是ATP产生的电子传递链的重要组成部分。氧化黄素蛋白的增加在自发荧光发射光谱中产生特征性偏移，这在分离的细胞或组织中容易测量。然而，在活体动物或人类的组织（尤其是眼睛）中非侵入性地检测这种变化更具挑战性。它需要一个专门的设备来成像的RPE和分析方法来检测特定的自发荧光签名归因于黄素蛋白。在这个项目中，我们将设计和建立原型多光谱成像设备使用快速可调激发激光源，声光可调屏障滤波器，和共焦扫描激光检眼镜视网膜成像仪，结合基于同步荧光光谱的分析软件，检测和定量RPE黄素蛋白自发荧光。我们将使用细胞培养和动物模型实验系统来开发和验证这项技术，我们将采用集成的多光谱成像设备，在没有视网膜疾病的人类受试者和AMD受试者中启动RPE黄素蛋白自发荧光的试点临床研究。

 DESCRIPTION (provided by applicant): In developed societies, age-related macular degeneration (AMD) is the most common cause of legal blindness in people over age 65. Emerging evidence demonstrates an important role for retinal pigment epithelium (RPE) mitochondrial dysfunction in dry AMD pathobiology. Mitochondrial dysfunction is characterized by chronic overproduction of superoxide and diminished production of ATP, and it promotes activation of injury response pathways in RPE cells, leading to sub-RPE deposit formation (precursors of lipid-rich drusen) and dysregulation of extracellular matrix turnover, the hallmark features of dry AMD. Thus, mitochondrial-targeting drugs are emerging as an attractive class of potential therapeutics for dry AMD. Technologies to identify the presence of RPE mitochondrial dysfunction in living eyes are limited. Detecting mitochondrial dysfunction in vivo would enable selection of individuals who might benefit from mitochondria-targeting drugs and would accelerate mechanistic studies of mitochondrial dysfunction in dry AMD disease models. The goal of this project is to develop RPE flavoprotein autofluorescence as an imaging biomarker for mitochondrial dysfunction. Mitochondrial dysfunction is accompanied by an increased ratio in the oxidized to reduced forms of flavoproteins (especially FAD+), which are important components of the electron transport chain for ATP production. Increase in oxidized flavoprotein produces a characteristic shift in the autofluorescence emission spectra, which is easily measured in isolated cells or tissues. However, detecting this shift noninvasively in tissues of living animals or humans, especially the eye, is much more challenging. It requires both a specialized device to image the RPE and an analytical approach to detect the specific autofluorescence signature attributable to flavoprotein. In this project, we will design and build prototype multispectral imaging device using a rapidly tunable excitation laser source, an acousto-optical tunable barrier filter, and a confocal scanning laser ophthalmoscope retinal imager, integrated with analytical software based on synchronous fluorescence spectroscopy, to detect and quantify RPE flavoprotein autofluorescence. We will use cell culture and animal model experimental systems to develop and validate this technology, and we will adapt the integrated multispectral imaging device to initiate a pilot clinical study of RPE flavoprotein autofluorescence in human subjects without retinal disease and in subjects with AMD.