我们感谢海德堡工程公司为原始眼罩提供仪器和设计，感谢Richard F. Spaide MD介绍基于OCT的多模态成像，感谢Christopher Girkin MD为访问临床成像设备提供便利，感谢David Fisher提供图1。人类供体眼睛用于研究的恢复得到了美国国立卫生研究院（NIH）拨款R01EY06019（C.A.C.），P30 EY003039（Pittler），R01EY015520（Smith），R01EY027948（C.A.C.，T.A.）的支持。R01EY030192（Li），R01EY031209（Stambolian）和U54EY032442（Spraggins），IZKF Würzburg（N-304，TA），阿拉巴马州视力基金会，国际视网膜研究基金会（C.A.C.），阿诺德和梅布尔贝克曼黄斑研究计划（C.A.C.）和预防失明AMD催化剂研究（Schey）。

用于 AMD 研究的基于 OCT 的人类供体眼多模态成像

C.A.C.获得海德堡工程公司的研究支持，并为Apellis，Astellas，Boehringer Ingelheim，Character Bioscience和Osanni提供咨询。T.A.获得诺华的研究支持，并为罗氏、诺华、拜耳、尼德克和Apellis提供咨询。K.B.F.是基因泰克、蔡司、海德堡工程、艾尔建、拜耳和诺华的顾问。

在 COVID 之前的 16 个月内使用基于人群的筛查方法，有可能获得 75 只患有 AMD 的供体眼睛。所有这些都用短DtoP回收，并使用OCT锚定的MMI进行分期。年龄标准（>80 岁）超出了用于可移植角膜的组织恢复的典型年龄范围。尽管年事已高，但我们的标准导致AMD各个阶段的眼睛。许多 RPE 表型在所有 AMD 分期都是通用的，有些是新生血管性 AMD3，46 所独有的。离?...

在光学相干断层扫描（OCT）的指导下，通过抗VEGF治疗管理新生血管年龄相关性黄斑变性（AMD）已有15年的经验，为这种普遍的视力丧失原因的进展顺序和微结构提供了新的见解。一个关键的认知是，AMD是一种三维疾病，涉及神经感觉视网膜，视网膜色素上皮（RPE）和脉络膜。由于试验患者和接受治疗的临床患者的眼睛的OCT成像，现在认识到了几十年来临床标准彩色眼底摄影所看到的病理特征。这些包括视网膜内新生血管形成（3 型黄斑新生血管形成1，以前称为血管瘤增生）、视网膜下玻璃疣沉积物 （SDD，也称为网状假玻璃膜疣）2、RPE 命运的多条途径3，4 和萎缩中的强烈胶质细胞 Müller 细胞5，6。
缺乏黄斑（细胞和动物）的模型系统重建了这种复杂疾病的一些切片7，8，9。减轻AMD负担的进一步成功可能来自发现和探索人眼中的原发性病理学，了解黄斑的独特细胞组成，然后转化为模型系统。本报告描述了学术研究实验室和眼库之间三十年的合作。本文描述的组织表征方法有两个目标：1）通过显微镜证明眼底外观和成像信号源的基础来告知不断发展的诊断技术，以及2）对AMD标本进行分类，用于靶向（免疫组织化学）和非靶向分子发现技术（成像质谱，IMS和空间转录组学），以保留仅锥形中央凹和富含杆的副中央凹和周中心凹。这些研究可以加速向临床OCT的转化，通过眼动追踪可以进行进展序列和纵向随访。这项技术旨在监测治疗效果，使用视网膜血管记录从一次诊所访问到下一次诊所访问的扫描。将眼动OCT与通过破坏性技术获得的实验室结果联系起来可以为分子发现提供新的预后价值水平。
1993年，研究实验室在胶片10上拍摄了死后眼底的彩色照片。这项工作的灵感来自Foos及其同事11，12，13对人类周边视网膜的出色光学显微镜和组织学，以及Sarks等人的广泛AMD临床病理学相关性14，15。从2009年开始，采用锚定在光谱域OCT上的离体多模态成像（MMI）。这种转变的灵感来自其他人的类似努力16，17，特别是认识到萨克斯描述的许多超微结构在诊所18，19中都是三维的。目标是在合理的时间范围内获得附着黄斑的眼睛，以便对视网膜、RPE 和脉络膜中的细胞水平表型进行有力的研究。目的是超越“每只眼睛”的统计数据到“每种病变类型”，这一标准受到心血管疾病20，21的“脆弱斑块”概念的影响。
本报告中的方案反映了近400对捐赠者眼睛在几个流中加入的经验。2011-2014 年，创建了 AMD 组织病理学的 MACULA 项目网站，其中包括来自 142 个存档标本的层厚度和注释。这些眼睛从1996年至2012年保存在戊二醛 - 多聚甲醛固定剂中，用于高分辨率环氧树脂组织学和电子显微镜。所有眼底在收到时都以彩色照片拍摄，并在组织学之前由OCT重新成像。最初设计用于视神经研究的眼支架22用于容纳以中央凹为中心的8毫米直径全层组织冲头。OCT B扫描通过中央凹中心和2毫米的部位，对应于同一水平的组织学，上传到网站，并附有彩色眼底照片。OCT 平面的选择取决于中央凹23 下 AMD 病理学的突出程度以及 SDD 在优于中央凹24，25 的杆富区中的突出性。
从2013年开始，在生命期间用OCT锚定MMI成像的眼睛可用于直接的临床病理相关性。大多数（10名捐赠者中的7名）涉及视网膜转诊诊所的患者（作者：K.B.F.），该诊所为有兴趣在死后捐献眼睛用于研究目的的患者提供了一个高级指导登记处。眼睛由当地眼库回收和保存，转移到实验室，并以与MACULA项目相同的方式制备眼睛。在实验室中无缝读取生前临床OCT卷，从而将生活中看到的病理特征与显微镜下看到的特征对齐26。
从2014年开始，前瞻性眼科采集开始于在没有临床病史的供体眼睛中筛查AMD，但在规定的时间限制（6小时）内保存。为此，对眼睛支架进行了修改，以适应整个地球仪。这减少了以前使用的 8 mm 冲头切割边缘周围脱落的机会。将眼睛保存在4%缓冲的多聚甲醛中用于免疫组织化学，并在第二天转移到1%用于长期储存。在 2016-2017 年（大流行前），来自 90 名捐赠者的 184 只眼睛被收回。此报告中的统计信息和图像是从该系列生成的。在大流行时期（2020 年封锁和后果），转录组学和 IMS 合作的预期收集继续以较低的速度进行，基本上使用 2014 年的方法。
其他供体眼部评估方法也可用。明尼苏达分级系统（MGS）27，28基于AREDS临床系统，用于彩色眼底摄影29。这种方法的局限性包括将萎缩性和新生血管性AMD合并为“晚期AMD”的一个阶段。此外，MGS需要在RPE脉络膜的照片记录之前去除神经感觉视网膜。该步骤在不同程度上将SDD移位30，31，并消除了视网膜外层及其支持系统的空间对应关系。因此，将代谢需求和视网膜信号传导与 RPE 脉络膜病理学联系起来的努力可能会受到阻碍。犹他系统使用离体彩色摄影和OCT实施MMI，将要解剖的眼睛分类为RNA和蛋白质提取区域32。虽然比整个眼罩摘除术更可取，但 AMD 进展风险最高的 3 mm 直径区域33，34 仅占 6 mm 直径中央凹中心冲头的 25%。因此，能够定位中央凹发现的技术，例如用于免疫组织化学的连续切片，是有利的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td># 02-540K</td>
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			<td>Fisher</td>
			<td>Sameco Bio-Tite&#160; 40 mL # 13-711-86</td>
			<td>For preservative</td>
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			<td>contact J. Messinger</td>
			<td>jeffreymessinger@uabmc.edu</td>
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			<td>Adobe</td>
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			<td>Nikon</td>
			<td>SMZ 1000</td>
			<td>for dissection</td>
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			<td>SZX9</td>
			<td>color fundus photography</td>
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			<td>for preservative; dilute for storage</td>
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			<td></td>
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			<td>Heidelberg Engineering</td>
			<td>HRA2</td>
			<td></td>
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			<td>Nikon</td>
			<td>MC-DC2</td>
			<td></td>
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			<td>Spectral Domain OCT device</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA&#38;OCT</td>
			<td>https://www.heidelbergengineering.com/media/e-learning/Totara-US/files/pdf-tutorials/2238-003_Spectralis-Training-Guide.pdf</td>
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</table>

ex vivo oct-based multimodal imaging of human donor eyes for research into age-related macular degeneration

从基于光学相干断层扫描（OCT）的多模态（MMI）临床成像中学习的年龄相关性黄斑变性（AMD）的进展序列可以为实验室结果增加预后价值。在这项工作中，在视网膜组织切片之前将离体OCT和MMI应用于人类供体眼睛。眼睛是从年龄≥80岁的非糖尿病白人捐赠者那里恢复的，死亡保存时间（DtoP）为≤6小时。在现场回收地球仪，用18毫米环钻刻痕以促进角膜去除，并浸入缓冲的4%多聚甲醛中。使用解剖镜和单反相机在三种放大倍率下使用反式、落射和闪光灯照明去除前节后获取彩色眼底图像。将地球仪放置在带有60屈光度镜片的定制设计腔室内的缓冲器中。它们以光谱域OCT（30°黄斑立方体，30μm间距，平均= 25），近红外反射率，488 nm自发荧光和787 nm自发荧光成像。AMD眼睛显示视网膜色素上皮（RPE）发生变化，伴有玻璃膜疣或视网膜下玻璃疣沉积物（SDD），伴或不伴新生血管形成，并且没有其他原因的证据。在2016年6月至2017年9月期间，恢复了94只右眼和90只左眼（DtoP：3.9±1.0小时）。在184只眼睛中，40.2%患有AMD，包括早期中级（22.8%），萎缩性（7.6%）和新生血管性（9.8%）AMD，39.7%患有无明显黄斑。使用OCT鉴定玻璃膜疣，SDD，超反射病灶，萎缩和纤维血管瘢痕。 伪影包括组织混浊，脱离（杆菌，视网膜，RPE，脉络膜），中央凹囊性变化，起伏的RPE和机械损伤。为了指导冷冻切片，使用OCT体积来查找中央凹和视神经头标志和特定病理。通过选择眼动追踪的参考功能，将离体体积与体内体积进行登记。体内可见病理的离体可见度取决于保存质量。在16个月内，使用临床MMI方法恢复和分期了75只处于AMD各个阶段的快速DtoP供体眼睛。

Fifteen years of managing neovascular age-related macular degeneration (AMD) with anti-VEGF therapy under the guidance of optical coherence tomography (OCT) has offered new insights into the progression sequence and microarchitecture of this prevalent cause of vision loss. A key recognition is that AMD is a three-dimensional disease involving the neurosensory retina, retinal pigment epithelium (RPE), and choroid. As a result of the OCT imaging of trial patients and the fellow eyes of treated clinic patients, the features of pathology beyond those seen by color fundus photography, a clinical standard for decades, are now recognized. These include intraretinal neovascularization (type 3 macular neovascularization1, formerly angiomatous proliferation), subretinal drusenoid deposits (SDDs, also called reticular pseudodrusen)2, multiple pathways of RPE fate3,4, and intensely gliotic M&#252;ller cells in atrophy5,6.
Model systems lacking maculas (cells and animals) recreate some slices of this complex disease7,8,9. Further success in ameliorating the burden of AMD could come from the discovery and exploration of primary pathology in human eyes, understanding the unique cellular composition of the macula, followed by translation to model systems. This report portrays a three decade collaboration between an academic research laboratory and an eye bank. The goals of the tissue characterization methods described herein are two-fold: 1) to inform evolving diagnostic technology by demonstrating the basis of fundus appearance and imaging signal sources with microscopy, and 2) to classify AMD specimens for targeted (immunohistochemistry) and untargeted molecular discovery techniques (imaging mass spectrometry, IMS, and spatial transcriptomics) that preserve the cone-only fovea and rod-rich para- and perifovea. Such studies could accelerate the translation to clinical OCT, for which a progression sequence and longitudinal follow-up are possible through eye-tracking. This technology, which is designed to monitor treatment effects, registers scans from one clinic visit to the next using retinal vessels. Linking eye-tracked OCT to laboratory results obtained with destructive techniques could provide a new level of prognostic value to molecular findings.
In 1993, the research laboratory captured color photographs of postmortem fundus on film10. This effort was inspired by the superb photomicroscopy and histology of the human peripheral retina by Foos and colleagues11,12,13&#160;and the extensive AMD clinicopathologic correlations by Sarks et al.14,15. Starting in 2009, ex vivo multimodal imaging (MMI) anchored on spectral domain OCT was adopted. This transition was inspired by the similar efforts of others16,17&#160;and especially by the realization that so much of the ultrastructure described by the Sarks was available in three dimensions, over time, in the clinic18,19. The goal was to acquire eyes with attached maculas in a reasonable time frame for well-powered studies of cellular-level phenotypes in the retina, RPE, and choroid. The intent was to move beyond &#34;per eye&#34; statistics to &#34;per lesion type,&#34; a standard influenced by the &#34;vulnerable plaque&#34; concepts from cardiovascular disease20,21.
The protocol in this report reflects experience with nearly 400 pairs of donor eyes accessioned in several streams. In 2011-2014, the Project MACULA website of AMD histopathology was created, which includes layer thicknesses and annotations from 142 archived specimens. These eyes were preserved from 1996-2012 in a glutaraldehyde-paraformaldehyde fixative for high-resolution epoxy-resin histology and electron microscopy. All the fundi had been photographed in color when received and were reimaged by OCT just prior to histology. An eye holder originally designed for optic nerve studies22&#160;was used to accommodate an 8 mm diameter full-thickness tissue punch centered on the fovea. OCT B-scans through the foveal center and a site 2 mm superior, corresponding to histology at the same levels, were uploaded to the website, plus a color fundus photograph. The choice of the OCT planes was dictated by the prominence of AMD pathology under the fovea23&#160;and the prominence of SDDs in rod-rich areas superior to the fovea24,25.
Starting in 2013, eyes imaged with OCT-anchored MMI during life were available for direct clinicopathologic correlations. Most (7 of 10 donors) involved patients at a retina referral practice (author: K.B.F.), which offered an advanced directive registry for patients interested in donating their eyes after death for research purposes. The eyes were recovered and preserved by the local eye bank, transferred to the laboratory, and prepared in the same way as the Project MACULA eyes. Pre-mortem clinical OCT volumes were seamlessly read in the laboratory, thus aligning the pathology features seen during life with the features seen under the microscope26.
Starting in 2014, prospective eye collection began by screening for AMD in donor eyes without a clinical history but preserved during a defined time limit (6 h). For this purpose, the eye holder was modified to accommodate a whole globe. This reduced the chance of detachment around the cut edges of the previously used 8 mm punch. The eyes were preserved in 4% buffered paraformaldehyde for immunohistochemistry and transferred to 1% the next day for long-term storage. In 2016-2017 (pre-pandemic), 184 eyes from 90 donors were recovered. The statistics and images in this report are generated from this series. During the pandemic era (2020 lockdowns and aftermath), prospective collections for transcriptomics and IMS collaborations continued at a reduced pace, essentially using the 2014 methods.
Other methods for donor eye assessment are available. The Minnesota Grading System (MGS)27,28&#160;is based on the AREDS clinical system for color fundus photography29. The limitations of this method include the combining of atrophic and neovascular AMD into one stage of &#34;late AMD&#34;. Further, the MGS entails the removal of the neurosensory retina before the photo-documentation of the RPE choroid. This step dislodges SDDs to varying degrees30,31&#160;and removes the spatial correspondence of the outer retina and its support system. Thus, efforts to link metabolic demand and signaling from the retina to pathology in the RPE-choroid may be impeded. The Utah System implemented MMI using ex vivo color photography and OCT to categorize eyes destined for dissection into regions for RNA and protein extractions32. Although preferable to whole eyecup extractions, the 3 mm diameter area at the highest risk for AMD progression33,34&#160;represents only 25% of a 6 mm diameter fovea-centered punch. Thus, techniques that can localize findings in reference to the fovea, such as serial sectioning for immunohistochemistry, are advantageous.

作者聚焦：基于离体 OCT 的人类供体眼多模态成像，用于年龄相关性黄斑变性的研究  

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

This protocol enables new treatments and preventions for age-related macular degeneration or AMD. This is the largest disease with a central neurodegeneration and yet we think it's the most approachable because of the precise structure of the eye and the availability of cellular-level clinical imaging of the retina. The neovascular complications of the underlying disease of AMD have been treated successfully for 15 years in the 15%of patients who have it.Recently, the FDA approved the first drug for the end stage of the underlying disease. Single-cell RNA sequencing has revealed the molecular repertoire of the 100-plus cell types in the retina and supporting choroidal vasculature. Eye-tracked optical coherence tomography or OCT has enabled us to glimpse a detailed cellular-level progression sequence in the clinic.Existing cell and animal model systems for AMD research are non-predictive of human pathology and progression risk. This impedes the development of new treatments and preventions. Our lab contributed many top-level findings about AMD, including the early loss of broad photoreceptors required for night vision.It was also observed that better preserved maculas with attached neurosensory retinas are directly relevant to clinical OCT imaging. Many parts of AMD pathology became visible with clinical OCT which shows cross-sectional views of the retina and choroid. The OCT results of human donor eyes can be directly compared to findings in clinical imaging.If laboratories could emulate clinical imaging standards by using OCT to characterize eyes before assays and laboratory findings like gene expression or proteomics and leverage the longitudinal timeline of clinical imaging to focus on the most informative risk indicators.

表1显示，在2016-2017年期间，从94名80岁的白人非糖尿病捐赠者>184只眼睛被回收。平均死亡至保存时间为3.9小时（范围：2.0-6.4小时）。在接受审查的184只眼睛中，75只（40.2%）患有某种AMD。确定了以下类别：不显着（39.7%），可疑（11.4%），早期中级AMD（22.8%），萎缩（7.6%），新生血管（9.8%），其他（8.7%）和未知/未记录/不可分级（<1%）。图2、图3、<strong class="xfig"...

Watch this Scientific Journal Video about Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration at JoVE.com

实验室检测可以利用基于纵向光学相干断层扫描 （OCT） 的年龄相关性黄斑变性 （AMD） 多模态成像的预后价值。在组织切片之前，使用OCT，彩色，近红外反射扫描激光检眼镜和两个激发波长的自发荧光对有和没有AMD的人类供体眼睛进行成像。

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

该协议为年龄相关性黄斑变性或AMD提供了新的治疗方法和预防。这是具有中枢神经变性的最大疾病，但我们认为它是最容易接近的，因为眼睛的精确结构和视网膜细胞水平临床成像的可用性。AMD基础疾病的新生血管并发症已在15%的患者中成功治疗了15年。最近，FDA批准了第一种用于基础疾病终末期的药物。单细胞RNA测序揭示了视网膜和支持脉络膜脉管系统中100多种细胞类型的分子库。眼动追踪光学相干断层扫描或OCT使我们能够在临床上瞥见详细的细胞水平进展序列。用于AMD研究的现有细胞和动物模型系统无法预测人类病理学和进展风险。这阻碍了新疗法和预防措施的发展。我们的实验室贡献了许多关于AMD的顶级发现，包括夜视所需的广泛光感受器的早期损失。还观察到保存得更好的黄斑与附着的神经感觉视网膜与临床OCT成像直接相关。AMD病理的许多部分在临床OCT中变得可见，OCT显示了视网膜和脉络膜的横截面视图。人类供体眼睛的OCT结果可以直接与临床成像结果进行比较。如果实验室可以通过在检测和实验室发现（如基因表达或蛋白质组学）之前使用 OCT 来表征眼睛，并利用临床成像的纵向时间线来关注信息量最大的风险指标，从而模拟临床成像标准。

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Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

2.0K 次观看.  University of Alabama at Birmingham Heersink School of Medicine. 该协议为年龄相关性黄斑变性或AMD提供了新的治疗方法和预防。这是具有中枢神经变性的最大疾病，但我们认为它是最容易接近的，因为眼睛的精确结构和视网膜细胞水平临床成像的可用性。AMD基础疾病的新生血管并发症已在15%的患者中成功治疗了15年。最近，FDA批准了第一种用于基础疾病终末期的药物。单细胞RNA测序揭示了视网膜和支持脉络膜脉管系统中100多种细胞?...