Zhang Qing, Chrenek Micah A, Bhatia Shagun, Rashid Alia, Ferdous Salma, Donaldson Kevin J, Skelton Henry, Wu Wenfei, See Thonnie Rose O, Jiang Yi, Dalal Nupur, Nickerson John M, Grossniklaus Hans E
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA.
Department of Mathematics and Statistics, Georgia State University, Atlanta, GA.
Mol Vis. 2019 Feb 7;25:70-78. eCollection 2019.
To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD.
Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images.
Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0–51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60–93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (<63 µm), medium (63.0–124 µm), and large (125 µm) drusen. The RPE was arranged in rosette-like structures overlying small drusen, attenuated overlying medium-sized drusen, and consisted of large multinucleated cells overlying large drusen. The RPE in the area of geographic atrophy was attenuated and depigmented.
Confocal images of flatmounts from eyes with AMD showed RPE patterns overlying various types of drusen and geographic atrophy that correlated with histologic characteristics. We propose RPE repair mechanisms that may result in the patterns that we observed.
可视化并分析年龄相关性黄斑变性（AMD）患者离体平铺的人视网膜色素上皮（RPE）形态，并将该形态与组织学发现进行比较。确定在RPE平铺制剂中正面识别出的RPE下结构是否为具有连续切片组织病理学记录的玻璃膜疣。在AMD患者尸体眼的组织学横切面上检测RPE中正面发现的具有相同结构的特征模式。
对14例患者的28只死后眼（16只AMD眼和12只年龄匹配的对照眼）进行定向和显微解剖，获得RPE - 脉络膜制剂。将组织平铺，用Alexa Fluor 635鬼笔环肽（AF635 - 鬼笔环肽）染色以显示f - 肌动蛋白，用碘化丙啶染色以显示DNA，并使用共聚焦显微镜成像。对黄斑区的部分组织进行电子显微镜检查。共聚焦成像后，将样本重新固定以进行组织学处理，嵌入石蜡，并垂直于RPE - 脉络膜片平面进行连续切片。构建通过组织学横切面发现的玻璃膜疣位置的二维（2D）比例图，并将其与正面共聚焦显微镜图像相关联。
评估了来自14名供体（7名女性和7名男性）的28只死后眼，平均死亡至组织保存时间为23.7小时（范围8.0 - 51小时），平均年龄78岁（范围60 - 93岁）。8名供体患有AMD，6名作为对照。在患有AMD的眼睛和健康眼睛的周边存在散在的小而硬的玻璃膜疣。患有AMD的眼睛黄斑区包含小（<63 µm）、中（63.0 - 124 µm）和大（125 µm）玻璃膜疣。RPE在小玻璃膜疣上方排列成玫瑰花结样结构，在中等大小玻璃膜疣上方变薄，在大玻璃膜疣上方由大的多核细胞组成。地图状萎缩区域的RPE变薄且色素脱失。
AMD眼平铺标本的共聚焦图像显示RPE模式覆盖各种类型的玻璃膜疣和地图状萎缩，与组织学特征相关。我们提出了可能导致我们观察到的模式的RPE修复机制。
