使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估
Summary
在这里，我们描述了使用光谱域光学相干断层扫描（SD-OCT）在视网膜变性，青光眼，糖尿病视网膜病变和近视模型中可视化体内视网膜和眼部结构。
Abstract
光谱域光学相干断层扫描（SD-OCT）可用于可视化体内视网膜和眼部结构。 在研究中，SD-OCT是评估和表征各种视网膜和眼部疾病和损伤模型变化的宝贵工具。在光诱导的视网膜变性模型中，SD-OCT可用于跟踪感光层随时间推移的变薄。在青光眼模型中，SD-OCT可用于监测视网膜神经纤维层减少和视网膜总厚度，并观察诱发眼高压后的视神经拔罐情况。在糖尿病啮齿动物中，SD-OCT帮助研究人员观察到总视网膜厚度减少以及特定视网膜层厚度减少，特别是视网膜神经纤维层随着疾病进展而减少。在近视小鼠模型中，SD-OCT可用于评估轴向参数，例如轴向长度变化。SD-OCT的优势包括眼部结构的活体成像，定量跟踪眼部尺寸随时间变化的能力，以及快速的扫描速度和高分辨率。在这里，我们详细介绍了SD-OCT的方法，并展示了其在我们的实验室中用于视网膜变性，青光眼，糖尿病视网膜病变和近视模型的示例。方法包括麻醉，SD-OCT成像和处理用于厚度测量的图像。
Introduction
光谱域光学相干断层扫描（SD-OCT）是一种精确的高分辨率成像方式，允许临床医生和研究人员无创地检查眼部结构。这种成像技术基于干涉测量法，以微米尺度1，2捕获体内三维视网膜图像。它已成为视觉研究和临床中最常用的成像方式之一，因为它易于检测和准确检测病理特征，例如结构缺陷和/或视网膜层和视网膜下液变薄3。在使用视觉相关疾病的动物模型的研究中，SD-OCT提供了关于结构和功能与其组织病理学起源之间关系的基本无创分析4。由于其分辨率（高达2-3微米，取决于眼睛的深度5），SD-OCT能够检测到视网膜层厚度的微小变化。这种类型的分析可以为疾病进展提供基本信息，并评估神经保护方法和治疗视力相关疾病的疗效。
SD-OCT是组织学检查结构的无创替代方法，两者已被证明是相关的6。虽然SD-OCT不能达到细胞分辨率，但它确实允许在动物中进行纵向研究。这是有利的，因为随着时间的推移，可以在个体动物中跟踪疾病进展，而不是必须在特定时间点对动物实施安乐死。随着成像技术的不断改进，SD-OCT技术也将不断发展，提供增强的图像质量以及详细评估视网膜血管功能等生物过程的能力。自 1991 年问世以来，SD-OCT 技术在分辨率、速度和灵敏度方面都取得了巨大进步7。
本研究利用SD-OCT系统量化视网膜变性、青光眼和糖尿病视网膜病变啮齿动物模型中视网膜层的变化。这里使用的SD-OCT系统是一种傅里叶域OCT系统，它利用低功耗、近红外光实时采集、处理和存储深度分辨图像。SD-OCT 系统在 800 nm 波段具有扩展的深度成像能力，可提供 8 mm 深度和 4 μm 分辨率。在傅里叶域检测中，来自组织的散射光与参考路径之间的干涉信号被傅里叶变换，以构建散射强度8的轴向扫描和/或轴向深度剖面。对于这里的研究，OCT束在所需的视网膜结构上扫描，同时连续获取轴向扫描。通常，扫描图案将二维网格（B扫描）作为线性一维扫描线（A扫描）的集合获取，这些线对应于使用光栅扫描图案的2D横截面图像。对于专注于小鼠近视的研究，该系统还用于测量眼部结构的尺寸（例如角膜厚度、晶状体厚度、玻璃体室深度和轴向长度）。
目前的系统允许用户设计自己的协议，创建可以根据感兴趣的眼部结构进行定制和选择的扫描。这些用户定义的协议中具有的主要扫描功能使这种成像技术用户友好。对于图像分析，我们在数学建模程序中开发了定制编程。SD-OCT是一种强大的工具，可以无创地识别和量化眼部结构的病理形态变化，并监测与视力相关的疾病进展。
Protocol
Representative Results
Discussion
体内眼部结构的高分辨率成像可以评估视网膜和眼部随时间的变化。在该协议中，SD-OCT被证明可以捕获视网膜变性，青光眼，糖尿病视网膜病变和近视模型中体内眼部结构的差异。
执行SD-OCT时最关键的方面是获得视网膜或其他感兴趣的眼部结构的清晰图像。重要的是要花时间确保视网膜完全居中并具有出色的清晰度。啮齿动物的沉重呼吸会导致嘈杂的图像（实际上可以看到?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了退伍军人事务部康复研发服务职业发展奖（CDA-1，RX002111;CDA-2;RX002928）授予RSA，优异奖（RX002615）和研究职业科学家奖（RX003134）授予MTP，职业发展奖（CDA-2，RX002342）授予AJF，EY028859授予MTP，NEI核心资助P30EY006360，预防失明研究和基金会抗盲。
Materials
| 1% tropicamide | Sandoz | Sandoz #6131403550; NDC- 24208-585-59 | |
| 0.5% tetracaine | Alcon | NDC 0065-0741-12 | |
| AIM-RAS G3 120 V | Leica Bioptigen | 90-AIMRAS-G3-120 | Specialized platform to hold the OCT Scanner Head for mice |
| Celluvisc gel | REFRESH CELLUVISC | #4554; NDC-0023-4554-30 | |
| G3 18 mm Telecentric Lens | Leica Bioptigen | 90-BORE-G3-18 | |
| G3 Mouse Lens | Leica Bioptigen | 90-BORE-G3-M | |
| G3 Rat Lens | Leica Bioptigen | 90-BORE-G3-R | |
| heating pad | Fabrication | 11-1130 | |
| InVivoVue software | Leica Bioptigen | Specialized software that pairs with the Leica Bioptigen SD-OCT system | |
| MATLAB | Mathworks | mathematical modeling program | |
| Mouse/Rat Kit | Leica Bioptigen | 90-KIT-M/R | Mouse/rat rodent alignment system |
| saline | ADDIPAK | 200-39 | |
| System Envisu R4300 VHR 120 V | Leica Bioptigen | 90-R4300-V1-120 | SD-OCT system |
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