非侵入性光学成像淋巴血管的鼠标
Summary
最近开发的成像技术，利用近红外荧光（NIRF）可能有助于阐明淋巴系统在肿瘤转移中发挥的作用，免疫反应，创面修复，以及其他相关的淋巴管疾病。
Abstract
淋巴血管系统的循环系统，保持体液平衡，提供了免疫监视，并介导对脂肪的吸收，在肠道中的重要组成部分。然而，尽管其关键的功能，有相对较少的了解淋巴系统如何适应为这些功能在健康和疾病的1。最近，我们表现出的能力，动态图像淋巴管架构和淋巴结在正常的人类受试者以及患有淋巴功能障碍的近红外荧光（NIRF）染料的使用跟踪管理和自定义的“抽水”行动，第三代强化成像系统2-4。 NIRF成像显示出了巨大的变化，在淋巴管的结构和功能与人类疾病。目前还不清楚这些变化是如何发生的和正在开发的新的动物模型，阐明它们的遗传和分子基础。在这个协议中，我们提出了NIRF淋巴管，S使用吲哚菁绿（ICG），已被用于在人类750年的染料，和一个NIRF染料标记的环状白蛋白结合结构域（CABD-IRDye800）肽，优先结合鼠标和人白蛋白8商场动物成像5,6 。约5.5倍的亮度比ICG，CABD IRDye800有一个类似的淋巴间隙轮廓比，ICG以实现足够的的NIRF信号为成像8，可以在较小的剂量注入。由于组织间隙8白蛋白的的CABD-IRDye800和ICG绑定，它们都可以描绘出活性蛋白运输和淋巴管内。皮内（ID）注射液（5-50微升）的ICG（645μM）或CABD-IRDye800（200μM），在盐水给药每个后爪和/或左，右侧的基极的背侧尾的异氟醚麻醉的小鼠。在动物所得的染料浓度是83-1,250微克/千克的ICG或113-1,700微克/千克CABD，IRDye800。紧随注射剂，功能性淋巴成像长达1小时，使用一个定制的，小动物NIRF成像系统进行。可以描绘整个动物的空间分辨率为100微米或更小的荧光淋巴管，和图像的结构至3厘米的深度可以获取9。图像采集采用V + +软件，并使用ImageJ或MATLAB软件进行分析。在分析过程中，连续绘制地区的利益（投资回报率），覆盖整个容器直径沿淋巴管。每个ROI测量定量评估通过血管淋巴移动的“数据包”，对于一个给定的容器和NIRF强度，每个ROI的尺寸保持不变。
Protocol
所有动物的研究进行了大学，得克萨斯大学健康科学中心（休斯顿，德克萨斯州），比较医学系，分子成像后，审查和批准该协议由各自的机构动物保健中心的标准和使用委员会（IACUC）或动物福利委员会（AWC）。 1。制备动物24小时之前，成像 必须完成下面的步骤中（如需要）的前一天，淋巴造影发生。 将动物的感应箱与异氟醚和稳重。 …
Representative Results
NIRF在小鼠淋巴造影的例子当ICG注入或CABD IRDye800 ID在基地正常小鼠的尾部，在尾巴根部之间的注射部位和腹股沟淋巴结（LN）的淋巴血管应立即可视化。注射后不久（几秒钟到几分钟），淋巴管之间的腹股沟LN和腋下淋巴结应可视化，如在图2中看出。由于小鼠的淋巴管的变化在动物，因为他们在人类，动物之间的体系结构的变化，如在图3中所示，可以?…
Discussion
我们使用一个自定义的，小动物NIRF成像系统捕捉到的图像标记的小鼠淋巴管。为了构建电影淋巴结运动，300或更多个图像被收集。有关从电影淋巴管的功能分析，两个或两个以上的ROI手工画出的沿淋巴容器。的感兴趣区的尺寸中为每个容器中，并保持恒定的直径约为该船只。虽然整个动物的空间分辨率可以描绘荧光淋巴管的100微米或更小，可以采用更精细的分辨率的图像的macrolens 10。为?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的补助伊娃Sevick：NIH R01 CA128919和美国国立卫生研究院R01 HL092923。
Materials
| Solutions, Reagents, and Equipment | Company | Catalog Number | Comments |
| Indocyanine green (ICG) | Patheon Italia S.P.A. | NDC 25431-424-02 | Reconstitute to 645 μM (5 μg/10 μL) |
| Cyclic Albumin Binding Domain(cABD) | Bachem | Custom | Reconstitute to 200 μM (6.8 μg/10 μL) |
| IRDye800 | Li-COR | IRDye 800CW | Reconstitute according to manufacture’s instructions; conjugate with cABD at equilmolar concentrations |
| Sterile Water | Hospira, Inc., Lake Forest, IL | NDC 0409-4887-10 | |
| NAIR | Church & Dwight Co., Inc. | Local Stores | www.nairlikeneverbefore.com |
| Imaging System (components below) | Center for Molecular Imaging | N/A | Custom-built in our laboratories. |
| Electron-multiplying charge-coupled device (EMCCD) camera | Princeton Instruments, Trenton, NJ | Photon Max 512 | |
| Nikon camera lens | Nikon Inc., Melville, NY | Model No. 1992, Nikkor 28mm | |
| Optical filter | Andover Corp., Salem,NH | ANDV11333 | Two 830.0/10.0 nm bandpass filters are used in front of lens |
| 785-nm laser diode | Intense Ltd, North Brunswick, NJ | 1005-9MM-78503 | 500 mW of optical output |
| Collimating optics | Thorlabs, Newton, NJ | C240TME-B | Collimates laser output prior to cleanup filter |
| Clean-up filter | Semrock, Inc., Rochester, NY | LD01-785/10-25 | Removes laser emission in fluorescence band |
| Optical diffuser | Thorlabs, Newton, NJ | ED1-C20 | Diffuses the laser over the animal |
| V++ | Digital Optics, Browns Bay, Auckland, New Zealand | Version 5.0 | Software used to control camera system and save images to computer. http://digitaloptics.net/ |
| Analytic Software Either of the following software packages can be used for image analysis | |||
| ImageJ | National Institutes of Health, Bethesda, MD | Most current version available | Freeware available at http://rsbweb.nih.gov/ij/ |
| MATLAB | MathWorks, Natick, MA | Version 2008a or later | http://www.mathworks.com/ |
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Tags
Non-invasiveOptical ImagingLymphatic VasculatureMouseCirculatory SystemFluid HomeostasisImmune SurveillanceFat AbsorptionLymphatic SystemHealth And DiseaseLymphatic ArchitectureLymph Pumping ActionNear-infrared Fluorescent DyeGen III-intensified Imaging SystemHuman DiseaseAnimal ModelsGenetic BasisMolecular BasisSmall Animal ImagingIndocyanine Green DyeCyclic Albumin Binding Domain PeptideNIRF Signals
Cite This Article
Robinson, H. A., Kwon, S., Hall, M. A., Rasmussen, J. C., Aldrich, M. B., Sevick-Muraca, E. M. Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse. J. Vis. Exp. (73), e4326, doi:10.3791/4326 (2013).