Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Summary

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Abstract

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease¹. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph “pumping” action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system^2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging^5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans⁷, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin⁸. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging⁸. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space⁸, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 μl) of ICG (645 μM) or cABD-IRDye800 (200 μM) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 μg/kg for ICG or 113-1,700 μg/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired⁹. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess “packets” of lymph moving through vessels.

Protocol

All animal studies were performed in accordance with the standards of the University of Texas Health Science Center (Houston, TX), Department of Comparative Medicine, and Center for Molecular Imaging after review and approval of the protocol by their respective Institutional Animal Care and Use Committee (IACUC) or Animal Welfare Committee (AWC). 1. Preparation of Animals 24 Hr Prior to Imaging The steps below must be done (as needed) the day before lymphatic im…

Representative Results

Example of NIRF Lymphatic Imaging in Mice When ICG or cABD-IRDye800 is injected ID at the base of the tail of a normal mouse, the lymphatic vasculature between the injection site at the base of the tail and the inguinal lymph node (LN) should be immediately visualized. Shortly after injection (a few seconds to minutes), the lymphatic vessel between the inguinal LN and the axillary LN should be visualized as seen in Figure 2. Since the lymphatics in mice vary from animal to animal…

Discussion

We use a custom, small animal NIRF imaging system to capture images of labeled lymph vessels in mice. To construct movies of lymph movement, 300 or more images are collected. For functional analysis of lymphatics from movies, two or more ROIs are manually drawn along a lymph vessel. The dimensions of the ROIs are kept constant for each vessel and are approximately the diameter of the vessel. While whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, a macrolens for fine…

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/