Correspondence to: Hiroo Suami, Department of Plastic Surgery, Unit 1488, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Fax: 713–794-5492. E-mail: email@example.com
Our knowledge of the lymphatic system in animals and humans is limited and is one of the least understood aspect of the gross anatomy (Shin et al., 2003; Hadamitzky and Pabst, 2008). A better understanding of the lymphatic system is of the utmost importance in establishing the scientific basis for the clinical management of infectious diseases, lymphedema, and cancer metastasis. Since Aselli described lymphatic vessels as “lacteals” (Aselli, 1627), researchers have used mercury (Nuck, 1692; Cruikshank, 1786; Mascagni, 1787; Sappey, 1874) and dye (Gerota, 1896; Bartels, 1909) in cadaveric studies and radiocontrast medium (Kinmonth, 1952) and radioisotopes (Sherman and Ter-Pogoissian, 1953) in clinical settings to demonstrate the lymphatic system.
Researchers have investigated the anatomy of the lymphatic system in various species: rats, dogs, pigs, and cows (Baum, 1912, 1918, 1938; Ellenberger, 1926; Tilney, 1971; Shesol et al., 1979; Suami et al., 2011a, 2008, 2012). However, despite numerous endeavors, little is known about the anatomy of the lymphatic system in the rabbit, which is commonly used in scientific studies. Indirect dye injection into the skin (Eloesser, 1923), lymphangiography of the hind foot (Wolfe et al., 1983), and lymphoscintigraphy (Sutton et al., 2002) have all been used to visualize the lymphatic system in the rabbit. However, indirect dye injection did not provide a stable means of tracing the entire course of the lymphatic vessel, and lymphangiography was useful only for mapping large lymphatic vessels in the hind limbs along with saphenous vein. Although lymphoscintigraphy could detect the lymph nodes, its low resolution precluded its visualization of individual lymphatic vessels.
We developed a microinjection technique that can be used to map lymphatic vessels as small as 0.1 mm in diameter (Suami et al., 2007, 2005). This technique enabled us to demonstrate anatomic relationships between the lymphatic vessels and lymph nodes more stably than the indirect dye injection. According to our previous studies in human cadavers and canines, the superficial lymphatic system could be divided into cutaneous territories (Suami et al., 2012). The aim of this study was to completely map the lymphatic system in the rabbit to define superficial lymphatic territories in the rabbit.
MATERIAL AND METHODS
The animal protocol for this study was reviewed and approved by The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee, which is accredited by the Association of Assessment and Accreditation of Laboratory Care International. We obtained ten male rabbits weighed 4.5–5.5 kg (New Zealand White) from Harlan Laboratories Inc. (Houston, TX).
Real-Time Indocyanine Green (ICG) Fluorescent Lymphography
Real-time ICG fluorescent lymphography was performed in two rabbits to demonstrate dynamic lymph flow in the living rabbit. We used an ICG fluorescent lymphangiography system (Photodynamic Eye; Hamamatsu Photonics, Hamamatsu, Japan) composed of a camera unit, which includes a black and white charge-coupled device camera for recording video images. ICG is a water-soluble compound and it emits energy in the near-infrared region between 750 and 810 nm when it is bound to protein in tissue (Benson and Kues, 1978). When ICG is injected into the dermal layer of skin, it is bound with protein and the compound is specifically absorbed by the lymphatic system. The lymphography system can identify lymphatic vessels by detecting near-infrared radiation in the tissue at a depth to about 10 mm from the surface (Kitai et al., 2005; Unno et al., 2007; Suami et al., 2011b).
After using an electric shaver to remove the rabbits' fur, rabbits were anesthetized with isoflurane. Under general anesthesia, we injected 0.05 mL of ICG aqueous solution (0.5 mg mL−1) (IC-Green; Akorn, Lake Forest, IL) intradermally into the head, neck, and torso regions along the dorsal and ventral midlines; into webbing in the paws and into the distal tail. We then gently massaged the rabbits for a few minutes to facilitate the dye uptake into the lymphatic system. The rabbits were then scanned using the ICG fluorescent lymphography system which detects the near-infrared radiation emitted from the injected ICG to visualize internal anatomical structures located up to 10 mm from the surface of the skin. Lymphography images were recorded using digital video format (Pinnacle Studio; Pinnacle Systems, Mountain View, CA). Using a monitor screen as a guide, highlighted lines on the monitor were drawn by free-hand on the skin with a marker. After taking the images, the rabbits were euthanized and the carcasses were used for the consecutive microinjection study.
Demonstration of the Lymphatic Vessel With Microinjection
We mapped the lymphatic vessels in eight rabbits using a previously described microinjection technique (Suami et al., 2007, 2005). Briefly, 0.5–1 mL of 3% hydrogen peroxide with 1% ink (Prussian Blue, Professional Acrylic Ink, Liquitex Artist Materials, Piscataway, NJ) was injected into the skin in the areas being searched for lymphatic vessels. Fine oxygen bubbles produced by the reaction of the hydrogen peroxide with tissue enzymes inflated the lymphatic vessels and forced the pigment into the lumen. To directly visualize the vessels, we made a small incision 1 in. from the injection site. We used a microscope (Discovery V8; Carl Zeiss Microscopy, Throunwood, NY) to identify lymphatic vessels stained by the dye.
To determine the extent of each lymphatic vessel and identify their associated lymph nodes, we inserted a 30-gauge needle or attenuated glass tube set with a micromanipulator (MN-153; Narishige International USA, East Meadow, NY) into each lymphatic vessel and injected aqueous blue dye until the first tier of lymph node was found. The rabbits were then meticulously dissected, and the lymphatic vessels and the lymph nodes were photographed using a digital camcorder (HDR-XR500V, Sony Electronics, San Diego, CA) (Fig. 1).
Radiographic Mapping of Arteries
To assess the relationship between the vascular and lymphatic systems in rabbits, we radiographically mapped the arteries of two rabbits and compared these findings to those obtained using the microinjection technique mentioned above. We inserted a 24-gauge cannula into the femoral artery and secured it using 5-0 sutures. We then used a 20-mL syringe to manually inject radiocontrast mixture comprising 200 g of barium sulfate (Fisher Scientific, Pittsburgh, PA), 20 g of gelatin, and 600 mL of 60°C hot water into the arterial system at a rate of 100 g kg−1. The specimen was then stored in a refrigerator at 4°C for 3 hr to allow the injected mixture to solidify. After the mixture solidified, we removed the rabbits' skins by making an incision along the midline and retracting the skin including panniculus carnosus muscle from the underlying tissues. The skins were radiographed using a portable digital X-ray system (FCR Go, Fujifilm Medical Systems USA, Stanford, CT).
Mapping of Superficial Lymphatic Territories
We used a graphic software program (Adobe Photoshop CS 5.5; Adobe Systems, San Jose, CA) to create schematic diagrams of the lymphatic pathways and their corresponding lymph basin. We color-coded each lymph node in accordance with their regional lymph node and then each lymphatic vessel in a retrograde fashion from its lymph node using the same color. Thus, superficial lymphatic territories were defined to reveal which area of the skin drained to which lymphatic basin.
ICG fluorescent lymphography revealed highlighted fine lines extending from the sites of ICG injection that merged and formed larger lines that ran proximally and ultimately connected to a shiny round structure. Subsequent dissection confirmed that these shiny lines were the lymph vessels and the round structure was a lymph node (Fig. 2). Ventral and dorsal lymphography images revealed no lymphatic vessels crossing the midline. ICG fluorescent lymphography also revealed eight lymphatic territories in the preauricular, submandibular, root of the lateral neck, axillary, dorsal lumbar, inguinal, root of the tail, and popliteal regions. The locations of the lymph nodes in the two rabbits subjected to ICG fluorescent lymphography were consistent.
An injection of a mixture of blue dye and hydrogen peroxide into the dermis of the web spaces in the hindfoot and subsequent skin dissection revealed a fine lymph capillary network (Fig. 3). Cannulation of the identified vessel and a direct injection of dye into the lumen revealed two lymphatic vessels that ran along the lateral saphenous vein and connected to the popliteal node (Fig. 4). Using this microinjection technique enabled us to inject blue dye into lymphatic vessels as small as around 0.1 mm in diameter. The blue dye perfused the vessels, which enabled us to follow the routes of the lymphatic vessels were chased and determine the first tier lymph nodes they were connected to (Fig. 5).
We observed two different patterns of lymphatic vessels: a tributary pattern (Fig. 6) in the buttock and lumbar areas, and a straight pattern (Fig. 7) in the rest of the body. Meticulous dissection of the rabbits revealed an intimate relationship between the lymphatic vessels and the vascular system. Arteriography revealed that this correlation was present throughout the body (Fig. 8). Dissection also revealed eight lymph nodes: parotid, cervical, axillary, superficial iliac, lateral sacral, popliteal, inguinal, and mandibular, nodes. The anatomic locations of the lymph nodes among the rabbits were consistent. The superficial lymphatic vessels did not overlap; therefore, eight distinct lymphatic territories could be identified (Fig. 9).
The present study's findings indicate that ICG fluorescent lymphography has several advantages over previously described methods of mapping lymphatic vessels in the rabbit. First, ICG was taken up by the lymphatic vessels. Second, ICG lymphography facilitated the delineation of not only lymph nodes but also lymphatic vessels through the skin. ICG fluorescence could be observed about 10-mm depth from the skin surface. Finally, dissimilar to lymphoscintigraphy, this did not require radioactive isotope and therefore there was no precaution needed.
Lymphatic territories that correspond to individual lymph node (Suami et al., 2012). For prospective applications, mapping lymphatic territories will provide the information needed to determine the lymph basin in which a primary tumor is located. Such findings will facilitate the study of cancer metastasis and could be used as normal controls in analyses of lymphatic changes following lymph node dissection.
The present study's findings add to what little is known about the anatomy of the lymphatic system in rabbits and provide information that could be used to help design animal-based research of the lymphatic system. According to our findings, the lymphatic system of the rabbit has three or four lymphatic vessels in the foot with no clear separation between the superficial and deep lymphatic vessels. This is in contrast to lymphatic vessels in the feet of dogs and humans, which are separated by the deep fascia (Suami et al., 2008, 2011). Also, the tributary pattern of the lymphatic vessel that we observed in the buttock and lumbar regions in the rabbit has not been described in other species. The correlation between the lymphatic and blood vessels throughout the rabbit was also different from that in dogs and humans. Such intimate correlation is present only in the feet in dogs (Suami et al., 2011) and around the cephalic vein and short saphenous vein in humans (Sappey, 1874; Suami et al., 2007).
In conclusion, the lymphatic system of the rabbit includes eight lymphatic territories, each comprising its own lymphatic vessels and lymph node basin. The consistent anatomy of this system suggests that the rabbit could be useful as an animal model in cancer metastatic study and lymphedema research.
The authors thank the personnel in MD Anderson's Department of Veterinary Medicine and Surgery for their technical assistance; Katherine Dixon in MD Anderson's Department of Diagnostic Imaging for her support in this study.