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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Beakers, 250 mL | Fisher | # 02-540K | |
| Bottles, 1 L, Pyrex | Fisher | # 10-462-719 | storage for preservative |
| Bunsen burner or heat source | Eisco | # 17-12-818 | To melt wax |
| Camera, digital | Nikon D7200 | D7200 | |
| Computer and storage | Apple | iMac Pro; 14 TB external hard drive | Image storage |
| Container, insulated | Fisher | # 02-591-45 | For wet ice |
| Containers, 2 per donor, 40 mL | Fisher | Sameco Bio-Tite 40 mL # 13-711-86 | For preservative |
| Crucible, quartz 30 mL | Fisher | # 08-072D | Hold globe for photography |
| Cylinder, graduate, 250 mL | Fisher | # 08-549G | |
| Disinfectant cleaning supplies | https://www.cardinalhealth.com/en/product-solutions/medical/infection-control/antiseptics.html | ||
| Eye holder with lens and mounting bracket | contact J. Messinger | jeffreymessinger@uabmc.edu | custom modification of Heidelberg Engineering original design |
| Face Protection Masks | Fisher | # 19-910-667 | |
| Forceps, Harmon Fix | Roboz | # RS-8247 | |
| Forceps, Micro Adson | Roboz | # RS-5232 | |
| Forceps, Tissue | Roboz | # RS-5172 | |
| Glass petri dish, Kimax | Fisher | # 23064 | |
| Gloves Diamond Grip | Fisher | # MF-300 | |
| Gowns GenPro | Fisher | # 19-166-116 | |
| Image editing software | Adobe | Photoshop 2021, Creative Suite | |
| KimWipes | Fisher | # 06-666 | |
| Lamps, 3 goosenecks | Schott Imaging | # A20800 | |
| Microscope, stereo | Nikon | SMZ 1000 | for dissection |
| Microscope, stereo | Olympus | SZX9 | color fundus photography |
| Paraformaldehyde, 20% | EMS | # 15713-S | for preservative; dilute for storage |
| pH meter | Fisher | # 01-913-806 | |
| Phosphate buffer, Sorenson’s, 0.2 M pH 7.2 | EMS | # 11600-10 | |
| Ring flash | B & H Photo Video | Sigma EM-140 DG | |
| Ruby bead, 1 mm diameter | Meller Optics | # MRB10MD | |
| Safety Glasses 3M | Fisher | # 19-070-940 | |
| Scanning laser ophthalmoscope | Heidelberg Engineering | HRA2 | |
| Scissors, curved spring | Roboz | # RS-5681 | |
| Sharps container | Fisher | # 1482763 | |
| Shutter cord, remote | Nikon | MC-DC2 | |
| Spectral Domain OCT device | Heidelberg Engineering | Spectralis HRA&OCT | https://www.heidelbergengineering.com/media/e-learning/Totara-US/files/pdf-tutorials/2238-003_Spectralis-Training-Guide.pdf |
| Stainless steel ball bearing, 25.4 mm diameter | McMaster-Carr | # 9529K31 | |
| Tissue marking dye, black | Cancer Diagnostics Inc | # 0727-1 | |
| Tissue slicer blades | Thomas Scientific | # 6767C18 | |
| Trephine, 18-mm diameter | Stratis Healthcare | # 6718L | |
| TV monitor (HDMI) and cord for digital camera | B&H Photo Video | BH # COHD18G6PROB | for live viewing and remote camera display features |
| Wax, pink dental | EMS | # 72670 | |
| Wooden applicators | Puritan | # 807-12 |
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