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。

所描述的所有程序均已获得亚特兰大退伍军人事务机构动物护理和使用委员会的批准，并符合美国国立卫生研究院关于实验动物护理和使用指南（NIH出版物，第8版， 2011年更新）。
注意:用于开发以下协议的 SD-OCT 系统在 材料表中进行了描述。虽然某些程序特定于该特定系统，但整体方法可以适用于其他OCT设备和动物模型。此外，在我们的实验室中，这些协议通常用于小鼠和大鼠;但是，整体方法可以用于不同的动物模型和SD-OCT设备，前提是个人在其设备上具有正确的镜头和功能。
1. 设置光学相干断层扫描设备
<ol><li>打开 SD-OCT 软件（材料表）。</li>
	<li>定义谁正在服用OCT，研究和治疗组（如果相关）。命名这些类别的方式将有助于研究人员稍后在数据分析期间搜索所需的扫描。<ol><li>在" 患者/ 检查"选项卡中，单击" 测试检查员"。选择考官的姓名。使用 "设置检查员和医生 "按钮添加新的检查员。</li>
		<li>单击"算例名称"以定义 算例 。单击"研究"选项卡以添加新 研究 或修改现有研究中的治疗方法。单击"选择治疗臂"的右侧以 选择治疗臂 。</li>
	</ol></li>
	<li>单击添加 患者 按钮，该按钮用于为整个组添加新的时间点。当窗口出现时，输入 ID 号、名字和姓氏。选择"男性"或"女性"。输入出生日期。</li>
	<li>单击 "添加检查 "按钮以添加单个大鼠。要识别大鼠，请单击检查。单击 编辑考试。在"输入备注"框中 输入 ID 号。单击 保存更改 按钮。</li>
	<li>将正确的镜头安装到设备上（图1B），在软件中选择相应的配置，然后拨入关联的参考臂位置。 注意:所描述的SD-OCT系统具有定制的镜头，预设扫描模式和 参考臂 设置，特定于动物物种和被成像的眼睛区域（视网膜或角膜，小鼠或大鼠）。其中一些细节特定于所描述的SD-OCT系统（见材料表）。例如，并非所有设备都提供 参考臂 路径长度的手动调整。</li>
	<li>在" 患者/ 检查"选项卡中，双击突出显示的检查以进入 " 成像"选项卡并开始成像，或者只需单击 "成像 "选项卡。如果有默认扫描，请右键单击以将其删除。</li>
	<li>通过单击从列表中选择协议按钮加载预设的扫描 协议 。或者，添加单个扫描。</li>
	<li>对于青光眼和糖尿病视网膜病变的大鼠模型以及视网膜变性的小鼠模型，请选择由四个图像组成的预设:2 个 OD 和 2 个 OS 扫描。对于鼠标近视，请选择由 8 张图像组成的预设:4 张 OD 和 4 张操作系统扫描。 注意:预设成像将在第 3 节中更详细地解释。这是每个实验室在现场安装期间为自己或与制造商一起制作的东西。</li>
</ol>2. 麻醉动物
<ol><li>给予麻醉剂。<ol><li>通过腹膜内注射用氯胺酮（60mg / kg）和甲苯噻嗪（7.5mg / kg）麻醉大鼠。</li>
		<li>通过腹膜内注射用氯胺酮（80mg / kg）和甲苯噻嗪（16mg / kg）麻醉小鼠。</li>
		<li>等到动物完全麻醉并且对脚趾捏没有反应。</li>
	</ol></li>
	<li>给予瞳孔散大滴剂（1%托吡卡胺）。等待瞳孔扩张后再进行成像。 注意:瞳孔散大会增加视野，但不是必需的。如果有任何物体会接触到眼睛（例如，如果使用隐形眼镜或使用导板），也应使用局部（角膜）麻醉剂（0.5% 丁卡因）来麻痹眼睛。指南是一种放置在扫描头上的设备，可帮助初学者将眼睛和扫描头对齐。</li>
	<li>麻醉啮齿动物后，将啮齿动物置于可以在3维空间中旋转动物的啮齿动物对齐系统中（图1A，1C和1D）。提供热支持。 注意:目前，我们使用啮齿动物比对系统来设计和销售SD-OCT设备。</li>
	<li>涂抹液体（例如生理盐水或人工泪液）以保持眼睛润滑。确保眼睛在成像过程中不会变干，以便在扫描之间保持眼睛的光学特性（当角膜潮湿时，可以清楚地看到视网膜）。<ol><li>扫描第一只眼睛时，请确保保持对面眼睛的水分，以免其变干。</li>
	</ol></li>
	<li>在成像前，使用精细的工作擦拭来吸走多余的盐水，因为眼睛上的润滑剂过多或过少都会影响图像质量。 注意: 在OCT期间不建议使用无菌润滑凝胶，因为它会干扰成像。如果需要，可以在手术后使用无菌润滑凝胶。也可以使用隐形眼镜以确保在整个测试过程中眼睛有足够的水分。根据我们的经验，隐形眼镜并没有显着改善图像质量，但隐形眼镜确实有助于降低成像过程中角膜干燥的风险。</li>
</ol>3. 啮齿动物OCT成像
<ol><li>从一只眼睛（OS或OD）开始，然后对侧眼睛成像。<ol><li>使用啮齿动物对齐系统的两次旋转运动来定位动物，使视线水平并向下看OCT镜头的轴（图1D）。</li>
		<li>在自由运行模式下使用 OCT 定向视网膜以进行数据收集。最初使用瞄准模式（通过单击瞄准按钮）可实时连续显示水平和垂直B扫描。</li>
		<li>将扫描头移近眼睛，直到视网膜可见（因为小鼠和大鼠视网膜晶状体是定焦的，将晶状体移向眼睛聚焦到视网膜深处）。然后使用啮齿动物对准系统向上/向下调整动物位置并旋转/扭转以将视神经头定位在中心，使水平扫描水平，垂直扫描垂直（图1A）。</li>
		<li>调整工作距离，使视网膜图像平坦且不弯曲。</li>
		<li>调整参考臂位置，使图像靠近显示窗口的顶部。小心不要推得太远，否则眼睛图像会自行翻转。</li>
	</ol></li>
	<li>视网膜成像<ol><li>对于青光眼、视网膜变性和糖尿病视网膜病变模型:定义由 1000 x 100 x 1（A 扫描 x B 扫描 x 重复 B 扫描）组成的体积扫描以进行平均。在大鼠中，进行3 x 3毫米的体积扫描。在小鼠中，进行1.5 x 1.5毫米体积扫描。</li>
		<li>将视神经在水平和垂直通道中居中，以便体积扫描位于中心。花点时间确保视神经头位于扫描的中心，并沿着鼻颞轴和上下轴笔直（图2）。如果需要，扫描并重新居中以确保它正好位于中心。根据需要重复此扫描，直到视神经头居中并沿两个轴对齐。点击 快照 按钮拍摄照片。 注意:某些SD-OCT设备可以选择通过使用 参考臂调整眼睛与光源的距离来光学操纵眼睛的曲率（例如，图像被展平）。我们建议在通过视网膜层进行直接厚度测量时将图像展平并居中，以提高沿前后方向的准确性。</li>
		<li>点击 保存 按钮保存图像。</li>
		<li>以视神经头为中心进行径向扫描，尺寸为1000 x 4 x 20（A扫描x B扫描x重复B扫描）。使用重复的B扫描来提高眼睛或视网膜的图像清晰度，这将有助于在数据分析过程中解释眼睛区域或视网膜层。 注意:同样，在大鼠中，径向扫描为3毫米，而在小鼠中，径向扫描为1.5毫米。</li>
		<li>保存图像。</li>
		<li>在对侧眼中重复步骤 3.1 至 3.2.5。</li>
	</ol></li>
	<li>轴向长度测量<ol><li>对于涉及整只眼睛成像的项目，例如小鼠近视，对整只眼睛进行三次扫描，对每只眼睛进行一次视网膜扫描。选择一个预设，该预设由 500 x 20 x 1 的径向扫描组成，涵盖眼睛的整个直径。 注意:此设置提供从角膜到脉络膜的小鼠眼睛整个长度的图像。</li>
		<li>将眼睛和视网膜的中间置于视野的中心。进行三次径向扫描（全眼扫描）:在同一位置进行一次 1000 x 5 x 2 的线性 B 扫描和两次 1000 x 5 x 2 的附加线性 B 扫描。保存图像。</li>
		<li>然后，如果需要，放大并进行类似于 3.2 中的描述的体积或矩形扫描（视网膜扫描），包括 1000 x 20 A 扫描 x B 扫描。保存卷扫描。</li>
		<li>在对侧眼中重复步骤 3.3 至 3.3.3。 注意:轴向长度测量只能在小眼睛（鼠标或更小）上进行，因为当前系统的成像窗口不足以捕获较大的眼睛。</li>
	</ol></li>
</ol>4. 成像后步骤
<ol><li>将保存的数据存储在云上，这是数据管理的良好做法，并允许轻松访问以供以后分析。使用在数学建模程序中开发的自定义软件（材料表）执行数据分析。</li>
	<li>从啮齿动物对准系统中取出啮齿动物，腹腔注射阿替美唑（大鼠和小鼠为1mg / kg）以逆转甲苯噻嗪的作用，使啮齿动物更快地醒来。</li>
	<li>让啮齿动物在加热垫上用小火恢复。根据需要给予额外的生理盐水滴剂。当啮齿动物恢复完全行走时，将它们送回家笼。</li>
	<li>关闭程序并关闭 OCT。</li>
</ol>5. OCT图像的后处理
<ol><li>使用在数学建模程序中开发的自定义软件处理图像，以满足特定的OCT需求（例如，通过手动标记图像来测量感兴趣区域的厚度）。</li>
	<li>根据图像的目的（小鼠视网膜、大鼠视网膜或近视/轴长），使用以下三种不同程序之一:<ol><li>要处理视网膜，请选择要加载的OCT扫描。首先，通过简单的点击定义视神经头的中心。</li>
		<li>观看程序生成垂直线，定义视神经头两侧的距离。请注意，在大鼠视网膜中，这些线距离视神经头中心0.5毫米和1.2毫米，总共有4条垂直线代表眼睛的鼻颞轴和下上轴，具体取决于当前分析的径向B扫描。 注意:在小鼠视网膜中，这些垂直线距离视神经头中枢分别为0.25毫米和0.5毫米。</li>
		<li>沿每条线描绘以下图层: 视网膜神经纤维层 （RNFL）、内丛状层 （IPL）、内核层 （INL）、外丛状层 （OPL）、外核层 （ONL）、外部限制膜 （ELM）、内段/外段 （IS/OS）、视网膜色素上皮 （RPE） 和总视网膜厚度。 注意:径向扫描打开时通常没有鼻/颞和上/下标签。可以创建扫描，使其具有 n/t 和 s/I 方向，特别是这些扫描将在以后进行分析。</li>
		<li>描绘图像并关闭程序后，将这些测量值导出到电子表格软件中进行数据分析。</li>
	</ol></li>
	<li>使用步骤 5 中的这些长度和厚度值在组之间进行比较，例如，确定是否存在区域差异 （n/t/s/i） 或纵向变化。</li>
	<li>对于视网膜测量，首先确定在0.5毫米和1.2毫米距离处鼻颞轴和下上轴是否存在任何差异。 注意:如果未观察到象限的差异，则可以将0.5 mm和1.2 mm的测量值平均在一起。对于仅在0.25毫米和0.5毫米处进行小鼠视网膜扫描，这是一种类似的方法。</li>
	<li>对于近视研究，请使用该程序评估沿眼睛光轴的眼部参数。打开数学建模程序。首先，选择要加载的图像。<ol><li>加载图像后，手动标记每个扫描（径向和B扫描）。标记角膜、晶状体、玻璃体室和视网膜的前缘和后缘，以便程序计算角膜厚度、晶状体厚度、前室和玻璃体室深度、视网膜总厚度、总轴长。</li>
		<li>标记后，退出程序，提示保存菜单。将划定的值保存在电子表格软件中，并将三个单独的扫描平均在一起。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

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（实际上可以看到视网膜在屏幕上摆动）。如果动物在麻醉后没有完全失去知觉，有时会发生这种情况。为了解决这个问题，可以对多个B扫描进行平均，以帮助可视化视网膜层的边界在哪里，然后可以分析最佳的单个B扫描图像。
另一个常见的错误是眼睛太干或太湿。这可以通过再涂抹一滴盐水，用实验室擦拭将其吸走，并评估图像的清晰度是否提高来轻松检查。在SD-OCT图像上标记视网膜层厚度时要考虑的一个考虑因素是如何标记RNFL。虽然在某些啮齿动物OCT上可以区分RNFL和GCL，但通常这两层是无法区分的。为了保持一致性，我们将整个RNFL区域（RNFL + GCL，如果可见）标记为RNFL。一些研究报告RNFL和GCL作为单独的层或结合GCL和内部丛状层16，17，18，尽管这项研究通常在人类中进行，他们的眼睛比啮齿动物大得多。RNFL厚度的报告在啮齿动物研究中更为典型11，13，19，20。另一个重要问题是，标记的微小变化会导致非常大的变化，特别是在近视中，因为被测量的结构尺寸很小。例如，测量中6μm的差异等于屈光不正变化的屈光度21。由于微小的变化会对测量产生如此大的差异，因此图像清晰度至关重要。
该协议以及一般SD-OCT的局限性是，良好的图像需要清晰的眼部介质。例如，角膜病变、晶状体异常和白内障会阻止用户获得清晰的图像。这是糖尿病视网膜病变成像中的一个问题，特别是因为糖尿病啮齿动物通常会出现白内障22。如果白内障或其他眼部问题很小，有时可以在它周围操纵扫描头。对于较大的眼部介质中断，无法获得视网膜OCT图像。这些视网膜仍然可以使用组织学进行研究，因为视网膜组织学不依赖于清晰的眼介质。
另一个限制是，高反射性病变（例如渗出物和出血）以及主要视网膜血管会导致视网膜结构的阴影，从而丢失潜在形态的细节。在显示脉络膜新生血管膜和糖尿病视网膜病变/黄斑水肿的病例中，视网膜厚度超过400μm，很难辨别潜在的病理和脉络膜23。此外，SD-OCT只能用于评估特定位置的厚度。SD-OCT在脉络膜成像和整只眼睛成像方面也具有有限的穿透深度（整个眼睛可以在小鼠中成像，但不能在较大的动物中成像）。另一个限制是荧光或其他标记物不能与SD-OCT一起使用，就像扫描激光检眼镜（SLO）一样。然而，典型的SLO设备不允许像SD-OCT那样轻松地在横截面上可视化视网膜层。最后，SD-OCT的分辨率并不完美。但是，它比 SD-OCT 开始时可用的分辨率有了很大的改进，并且随着时间的推移会继续改进。
总之，SD-OCT技术的优点和意义在于它允许对眼部结构进行体内成像并定量跟踪眼部尺寸随时间的变化，并且它以快速的扫描速度进行这种成像。由于SD-OCT的高分辨率，它可以用来检测肉眼无法观察到的细微差异（图4和图5）。此外，SD-OCT是在许多疾病和损伤模型中测量眼睛多个参数的有用工具。仅在该协议中，SD-OCT用于测量视网膜变性和糖尿病视网膜病变模型中的视网膜厚度，青光眼模型中的视网膜厚度和拔罐，以及近视模型中的轴向长度。SD-OCT还可用于测量角膜曲率24，评估原始细胞和创伤性脑损伤后的视网膜变化19，25，26，识别年龄相关性黄斑变性的病理学27，并监测眼部注射期间和之后的视网膜健康状况28和视网膜下植入物等假体装置的视网膜放置29。它可用于其他动物模型，如树鼩30和非人灵长类动物31。SD-OCT还可用于根据象限（上象限，下象限，鼻部，颞部）和位置（中央与外围）定位视网膜病变。未来的SD-OCT设备将实现更高的分辨率。此外，OCT血管造影允许通过红细胞表面的激光在通过视网膜脉管系统时从红细胞表面反射来对视网膜和脉络膜微脉管系统进行成像32，33。

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。

<table><tbody><tr><td>1% tropicamide</td><td>Sandoz</td><td>Sandoz #6131403550; NDC- 24208-585-59</td><td></td></tr><tr><td>0.5% tetracaine</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>AIM-RAS G3 120 V</td><td>Leica Bioptigen</td><td>90-AIMRAS-G3-120</td><td>Specialized platform to hold the OCT Scanner Head for mice</td></tr><tr><td>Celluvisc gel</td><td>REFRESH CELLUVISC</td><td>#4554; NDC-0023-4554-30</td><td></td></tr><tr><td>G3 18 mm Telecentric Lens</td><td>Leica Bioptigen</td><td>90-BORE-G3-18</td><td></td></tr><tr><td>G3 Mouse Lens</td><td>Leica Bioptigen</td><td>90-BORE-G3-M</td><td></td></tr><tr><td>G3 Rat Lens</td><td>Leica Bioptigen</td><td>90-BORE-G3-R</td><td></td></tr><tr><td>heating pad</td><td>Fabrication</td><td>11-1130</td><td></td></tr><tr><td>InVivoVue software</td><td>Leica Bioptigen</td><td></td><td>Specialized software that pairs with the Leica Bioptigen SD-OCT system</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td></td><td>mathematical modeling program</td></tr><tr><td>Mouse/Rat Kit</td><td>Leica Bioptigen</td><td>90-KIT-M/R</td><td>Mouse/rat rodent alignment system</td></tr><tr><td>saline</td><td>ADDIPAK</td><td>200-39</td><td></td></tr><tr><td>System Envisu R4300 VHR 120 V</td><td>Leica Bioptigen</td><td>90-R4300-V1-120</td><td>SD-OCT system</td></tr></tbody></table>

in vivo structural assessments of ocular disease in rodent models using optical coherence tomography

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。

如果获得高质量的图像，从而可以可靠地测量眼部尺寸，则认为SD-OCT是成功的。在这里，使用视网膜变性，青光眼，糖尿病视网膜病变和近视的模型说明了SD-OCT的各种用途。
在光诱导的视网膜变性（LIRD）模型中，暴露于强光（10，000勒克斯）会诱导视网膜中感光细胞的变性9。与未受损（对照）小鼠相比，具有代表性的SD-OCT图像显示LIRD BALB / c小鼠视网膜中含有感光细胞体的更薄的外核层（图3A&3B）。量化视网膜层厚度后，观察到未受损小鼠和LORD小鼠在总视网膜厚度（图3C），外核层厚度（图3D）和IS/OS厚度（图3E）方面存在显着差异。
为了对青光眼损伤进行实验建模，我们使用了眼高压（OHT）模型10。简而言之，布朗挪威大鼠（n = 35）接受将高渗盐水注射到一只眼睛的角膜缘静脉中，而对侧眼睛作为内部对照11。对于青光眼研究，我们量化了视网膜神经纤维层（RNFL）的厚度。OHT治疗8周后，我们观察到视神经头有明显的重塑，包括视神经拔罐（图4A&B）。然后，我们量化了RNFL厚度，发现与基线测量相比，OHT治疗8周后RNFL变薄（图4C）。
为了模拟糖尿病视网膜病变，使用了Goto-Kakiza大鼠，这是一种非肥胖的多基因糖尿病模型，早在2-3周龄时就会发生高血糖症，12，13。使用SD-OCT对Goto-Kakizaki大鼠和Wistar大鼠（非糖尿病对照）的视网膜进行成像（图5A&5B）。在6周龄时，与中央视网膜（数据未显示）和周边视网膜（图5C&5D）的Wistar大鼠相比，Goto-Kakizaki大鼠的RNFL和总视网膜厚度减少。在视网膜的下象限和颞象限观察到最大的差异（图5C&5D）。
为了评估近视小鼠模型，测量Bmal1-/-小鼠的轴向长度。Bmal1是感兴趣的时钟基因，因为昼夜节律可能在近视发育中发挥作用14，15。在OCT图像中，Bmal1-/-小鼠眼睛（图6B）的轴向长度明显长于野生型眼睛（图6A）。轴向长度的定量证实，Bmal1-/-小鼠在84日龄时具有显着更长的轴向长度（图6C），表明缺乏时钟基因有助于近视的发展。
该协议在视网膜变性，青光眼，糖尿病视网膜病变和近视模型中生成了眼部结构的图像。图像质量足够高，可以量化眼部尺寸，包括外核层、视网膜神经纤维层、总视网膜厚度和轴向长度。结果表明，使用SD-OCT在体内可以观察到眼部结构尺寸的显着差异。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61588/61588fig01.jpg"> 图 1:SD-OCT 设备的设置。 （A）啮齿动物对齐系统和OCT扫描头的图片。（B）大鼠和小鼠OCT镜头的图片。（C）小鼠啮齿动物对齐系统的图片，说明其在三维空间中移动的能力。（D）啮齿动物对齐系统的特写，特别是控制其运动的旋钮。 <a href="https://www.jove.com/files/ftp_upload/61588/61588fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61588/61588fig02.jpg"> 图 2:SD-OCT 样本扫描。 在进行体积扫描或径向扫描之前对小鼠视网膜进行实时扫描的图片。 （A）描绘了鼻颞对齐，而（B）显示了上下对齐。一旦这两张图像中的视网膜在各自的垂直或水平平面上是直的，并且视神经在两张图像中都居中，我们继续获取SD-OCT图像。 <a href="https://www.jove.com/files/ftp_upload/61588/61588fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61588/61588fig03.jpg"> 图 3:使用 SD-OCT 跟踪视网膜变性小鼠模型中感光层随时间推移的变薄。 （A） 来自 BALB/c 鼠标的未损坏（对照）视网膜的代表性 SD-OCT 扫描。（B）来自光诱导视网膜变性（LIRD）BALB / c小鼠的视网膜的代表性SD-OCT扫描。（中欧）未受损和 LIRD Balb/c 小鼠总视网膜厚度 （C）、外核层 （ONL） 厚度 （D） 和内段/外段 （IS/OS） 厚度 （E） 的定量。平均± SEM。 <a href="https://www.jove.com/files/ftp_upload/61588/61588fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61588/61588fig04.jpg"> 图 4:使用 SD-OCT，我们测量了视网膜神经纤维层厚度的减少，并观察到青光眼大鼠模型中诱导眼高压后的视神经拔罐。 （A）在诱发眼高压之前从大鼠眼睛拍摄的具有代表性的SD-OCT扫描（基线:OHT）。（B）OHT治疗8周后同一大鼠视网膜的SD-OCT扫描（青光眼实验模型）。（C）基线时视网膜神经纤维层（RNFL）厚度与OHT眼睛的量化。平均± SEM。此数据已修改自 Feola 等人.11<a href="https://www.jove.com/files/ftp_upload/61588/61588fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61588/61588fig05.jpg"> 图 5:使用 SD-OCT 观察糖尿病大鼠模型中总视网膜厚度的减少以及特定视网膜层厚度的减少。 （A）来自Wistar（野生型对照）大鼠的视网膜的代表性SD-OCT扫描。（B）来自Goto-Kakizaki（糖尿病）大鼠的视网膜的代表性SD-OCT扫描。视网膜层:视网膜神经纤维层 （RNFL）、内丛状层 （IPL）、内核层 （INL）、外丛状层 （OPL）、外核层 （ONL）、外部限制膜 （ELM）、内段/外段 （IS/OS）、视网膜色素上皮 （RPE） 和总视网膜厚度 （TRT）。（中四）Wistar和Goto-Kakiza视网膜中RNFL（C）和总视网膜厚度（D）的定量，其中中心线是平均值，阴影区域是所有四个象限的SEM（Sup，Superior;温度，时间;Inf，劣等;Nas，鼻）的周边视网膜（距视神经头1.2毫米）。** p < 0.01， *** p < 0.001。此图已修改自 Allen 等人 13<a href="https://www.jove.com/files/ftp_upload/61588/61588fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61588/61588fig06.jpg"> 图 6:使用 SD-OCT 评估近视小鼠模型中的轴向长度。 84日龄野生型（A）和 Bmal1-/ -（B）小鼠眼睛的全眼SD-OCT图像。 Bmal1-/- 小鼠的眼睛具有明显长于野生型眼睛（C）的轴长。AL:轴向长度;RT:视网膜厚度;VCD:玻璃体腔深度;LT:镜片厚度;ACD:前房深度;CT:角膜厚度。长垂直线表示野生型眼的轴向长度边界（顶部和底部由水平线表示）。短箭头表示 Bmal1-/- 眼的后轴长标记。平均± SEM。每个图像中间的中心线（A&B）是垂直饱和伪影。它通常用作使眼睛居中的指南，但如果扫描对齐良好，则可以使其消失。 <a href="https://www.jove.com/files/ftp_upload/61588/61588fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography at JoVE.com

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

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。

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

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

in-vivo-structural-assessments-ocular-disease-rodent-models-using

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.8K 次观看.  Atlanta Veterans Affairs Medical Center. 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。

视频: In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

神经科学

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

医学

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

小鼠视网膜 OCT 纤维图与共聚焦图像的比对

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

作者聚焦：用于改善青光眼诊断和治疗的体内和离体视网膜成像的进展 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

光学相干断层扫描在视网膜病变小鼠模型中的应用

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

<meta charset="utf8"></head><body>图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

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

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在体内图像的Cx3cr1^gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

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光学相干断层扫描在视网膜病变小鼠模型中的应用

图像引导光学相干断层扫描评估啮齿动物视网膜的结构变化