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

Holly A. Robinson; SunKuk Kwon; Mary A. Hall; John C. Rasmussen; Melissa B. Aldrich; Eva M. Sevick-Muraca

doi:10.3791/4326

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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 imaging takes place.

Place animal in an induction box and sedate with isoflurane.
Once the animal is in a state of profound anesthesia (monitored with toe-pinch maneuver), place sedated animal on a diaper/fluff pad and position nose in a nose cone connected to isoflurane gas.
Clip all hair/fur (if any) around the area to be imaged.
Apply depilatory agent (NAIR) to the clipped area and leave it on the skin for up to 3 min.
Gently wipe off all depilatory agent with warm, damp gauze or paper towel.
Gently rinse the skin with warm water and gently dry the area with gauze or paper towel.
Allow animals to recover on a heating pad or under a heat lamp, and return to their cage.

2. Day of Imaging

Reconstitute imaging agent with sterile water, then dilute using sterile, normal (0.85%) saline to achieve 645 μM (5 μg/10 μl) for ICG or 200 μM (6.8 μg/10 μl) for cABD-IRDye800. Keep solutions in dark conditions and use within 6 hr of reconstitution.
Place animal in an induction box and sedate with isoflurane.
Once the animal is in a state of profound anesthesia (monitored with toe-pinch maneuver), place sedated animal on its side on a diaper/fluff pad and position nose in a nose cone connected to gas isoflurane.
Turn off the lights (so the room is dark). If needed, a small desk halogen light can be used for a small amount of light to see injections.
Using an insulin syringe with a 31-gauge needle, inject ID 5 μl to 50 μl of ICG or cABD-IRDye800 in the dorsal aspect of each hind paw and/or on the left and right side of the base of the tail, depending on the area of interest (see Discussion). Each injected dose may range from 0.083 to 1.25 mg/kg (ICG) or 0.113 to 1.7 mg/kg (cABD-IRDye800). Injection volumes will vary with animal strain and injection site. For athymic mice, the volume of injection can be 5 μl (hind paw) or 10 μl (base of tail). If animal is not under the imaging system for the injection(s), place the animal under the imaging system immediately after the injection(s).
If no dye uptake is seen in the lymphatics, step 2.5 will need to be repeated as needed per animal protocol.
Once lymphatics are seen, cover the injection site with black electrical tape or black paper.
Acquire lymphatic images for up to 1 hr using V++ software and a small animal, NIRF imaging system. (Animals are sedated with isoflurane and respirations are monitored while images are acquiring.) While small animal, NIRF imagers are commercially available, we utilize a customized, small animal NIRF imaging system consisting of a 785-nm laser diode (1005-9mm-78503, Intense, North Brunswick, NJ) outfitted with an aspheric lens (C24TME-B, Thorlabs, Newton, NJ), diffuser (ED1-C20, Thorlabs), and filter (LD01-785/10-25, Semrock, Rochester, NY) to create a uniform excitation field that illuminates the animal at an incident fluence rate of less than 1.4 mW per square centimeter¹⁰. An electron multiplying charged-coupled device (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) camera system with two 830-nm filters (AND11333, Andover Corp., Salem, NH) and a 28-mm Nikkor lens (1992, Nikon, Melville, NY) is used to capture lymphatic images with integration times of 200 msec for dynamic imaging and 800 msec for static imaging⁵. See Figure 1 for system configuration, the Table for additional details of each component, and the Discussion for a brief discussion of key imager properties.
Allow animals to recover on a heating pad or under a heat lamp and return to their cage, or euthanize.
Analyze images using ImageJ or MATLAB software. See Figure 6.