Lymphatic Territories (Lymphosomes) in Swine: An Animal Model for Future Lymphatic Research

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1 EXPERIMENTAL Lymphatic Territories (Lymphosomes) in Swine: An Animal Model for Future Lymphatic Research Ran Ito, M.D., Ph.D. Hiroo Suami, M.D., Ph.D. Houston, Texas Background: The swine is a common preclinical large-animal model for medical research because of the resemblance of its tissue structures to those of humans. However, the lymphatic system in swine is poorly understood. The authors investigated the lymphatic system and defined territories (lymphosomes) in swine using the microinjection technique. Methods: Six swine (two male and four female to 50-kg Sus domesticus) were used. Real-time indocyanine green fluorescence lymphography was performed in four live swine. After the animals were killed, the authors injected a radiocontrast mixture consisting of barium sulfate and hydrogen peroxide with red acrylic dye directly into lymphatic vessels in six swine carcasses. Courses of the lymphatic vessel were analyzed radiographically. The lymphatic vessels were dissected meticulously and chased until they connected to the first-tier (sentinel) lymph node. This procedure was repeated throughout the body until all the relationships between the lymphatic vessels and lymph nodes were defined. Results: The authors successfully mapped the superficial lymphatic vessels and their corresponding lymph nodes. Indocyanine green fluorescence lymphography and subsequent radiography revealed that the swine lymphatic system contained seven lymphosomes: parotid, mandibular, dorsal cervical, ventral cervical, subiliac, inguinal, and popliteal territories. Of note, no lymph nodes existed in the superficial axillary region. Conclusions: The swine could be a useful large-animal model for lymphatic research because of the anatomical consistency of the lymphosomes among animals and the sizable lymphatic vessels. However, swine lack the superficial axillary lymph node found in humans, suggesting that swine may not be a good model for breast cancer related lymphedema. (Plast. Reconstr. Surg. 136: 297, 2015.) The swine is a commonly used large-animal for medical research. Tissue structures of swine closely resemble those of humans, making swine excellent matches for research in gene and cell therapy 1 and xenograft and allograft procedures. 2 4 The emerging technology of decellularization and recellularization has progressed with the use of porcine organs and tissue in research. 3,4 In plastic surgery research, swine are commonly selected for large-animal experiments because From the Department of Plastic Surgery, The University of Texas M. D. Anderson Cancer Center. Received for publication November 26, 2014; accepted February 24, Presented in part at the Third International Symposium on Lymphedema Surgical Treatment, in Barcelona, Spain, March 5, 2014; and at the 10th Australasian Lymphology Association Conference, in Auckland, New Zealand, April 3 through 5, Copyright 2015 by the American Society of Plastic Surgeons DOI: /PRS swine skin is equivalent to human skin with regard to the thickness of the dermis and epidermis and the density of dermal appendages. The size, orientation, and distribution of blood vessels in the dermis of the swine are also similar to those in human skin. Because of these similarities, swine Disclosure: Neither of the authors has any source of financial or other support or any financial or professional relationships that might pose a competing interest. Supplemental digital content is available for this article. Direct URL citations appear in the text; simply type the URL address into any Web browser to access this content. Clickable links to the material are provided in the HTML text of this article on the Journal s Web site ( Journal.com)

2 Plastic and Reconstructive Surgery August 2015 are commonly used for flap research, 5 9 burns and dermal wound healing, and lymphatic research Although swine have been used in many areas of research, very limited information is available concerning the anatomy of the lymphatic system in swine. Investigation of the detailed anatomy of the lymphatic system in swine is crucially important for future lymphatic research. In a literature search, we found only a few reports focused on the lymphatic system in swine. 16,17 In these reports, researchers used an indirect dye injection technique, in which they injected dye into live swine skin at multiple spots, killed the animals, and dissected the carcasses to identify the lymphatic vessels. To the best of our knowledge, no imaging studies have been used for investigation of the lymphatic system in swine carcasses. Suami et al. developed a microinjection technique that allows identification of lymphatic vessels in cadaveric specimens on radiographs. 18,19 This technique has been used to demonstrate anatomical relationships between the lymphatic vessels and corresponding lymph nodes more precisely than the previous indirect dye injection technique. Our published results in human cadavers, canines, and rabbits illustrated that the superficial lymphatic system could be divided into lymphatic skin territories, which we called lymphosomes The aim of the current study was to investigate the lymphatic system in swine using the microinjection technique and to show that the superficial lymphatic system in swine can be divided into lymphosomes. MATERIALS AND METHODS The animal protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee, which is accredited by the Association of Assessment and Accreditation of Laboratory Care International. We obtained six swine (two male and four female Sus domesticus weighing between 17.5 and 50 kg) from Harlan Laboratories, Inc. (Houston, Tx.). Real-Time Indocyanine Green Fluorescence Lymphography We performed real-time indocyanine green fluorescence lymphography in four live swine to delineate the lymph vessels and lymph nodes. When indocyanine green is injected intradermally, it is absorbed by the lymphatic system specifically. The indocyanine green lymphography system (PDE; Hamamatsu Photonics K.K., Hamamatsu, Japan) can identify lymphatic vessels by detecting near-infrared radiation in the tissue at a depth of up to 12 mm from the skin surface. After using an electric shaver to remove the swine s fur, we anesthetized the swine using isoflurane gas. We injected 0.05 ml (2.5 mg/ml) of indocyanine green aqueous solution (IC-Green; Akorn, Lake Forest, Ill.) intradermally at multiple spots into the head, neck, and torso regions along the dorsal midline, ventral midline, and horizontal line at the umbilicus level, and into webs between toes and into the distal tail. We then gently massaged around the injection sites for a few minutes to facilitate dye uptake into the lymphatic system. The swine were then scanned using the indocyanine green fluorescence lymphography system. Indocyanine green lymphographic images were recorded using digital video. Highlighted lines, representing the lymphatic vessels, were visible on a monitor screen, and these lines were traced on the skin using a marker (Fig. 1). (See Video, Supplemental Digital Content 1, which displays indocyanine green fluorescence lymphography, demonstrating the lymphatic vessels and inguinal lymph nodes in the abdominal and medial hind limb in a live swine, lww.com/prs/b363.) After taking the video image and digital photographs, we killed the animals, and the carcasses were used for subsequent radiographic study. Radiographic Lymphatic Mapping We mapped the lymphatic vessels in the swine carcasses using a microinjection technique that was introduced in previous publications. 18,19 Briefly, 0.5 to 1 ml of 3% hydrogen peroxide with 1% blue ink (Professional Acrylic Ink; Liquitex Artist Materials, Piscataway, N.J.) was injected into the skin in the areas being searched for lymphatic vessels (Fig. 2). The injected hydrogen peroxide mixture produced fine oxygen gas bubbles when it reacted with tissue enzymes, and tissue around the injection site was bulging immediately. Increased local interstitial pressure facilitated pigment absorption into the lymphatic system and the lymphatic vessels were stained. A 1-inch incision was made on the distended area. We used a microscope (Discovery V8; Carl Zeiss Microscopy, Thornwood, N.Y.) to carefully remove overlying skin with a scalpel and dissect the stained lymphatic vessels (Fig. 3). The inflated lymphatic vessels in the subcutaneous tissue were distinguishable from arteries and veins because the lymphatic vessels did not contain red blood cells. 298

3 Volume 136, Number 2 Lymphosomes in Swine Fig. 1. (Left) Tracing of the lymphatic vessels in the abdominal and medial hind limb in a live swine, identified by indocyanine green fluorescence lymphography. (Right) Montage of the indocyanine green image of the same region. Arrows indicate the inguinal lymph nodes. Video. Supplemental Digital Content 1 displays indocyanine green fluorescence lymphography, demonstrating the lymphatic vessels and inguinal lymph nodes in the abdominal and medial hind limb in a live swine, We then cannulated a 30-gauge, 1-inch needle into the lymph vessel and injected radiocontrast mixture consisting of 6 g of barium sulfate (Fisher Scientific, Pittsburgh, Pa.) and 30 ml of 3% hydrogen peroxide (room temperature) with 0.3 ml of red ink. The barium mixture was injected manually and slowly so as not to rupture the lymph vessel, using a 1-ml syringe. Resistance of the syringe Fig. 2. Transilluminated photograph showing stained lymphatic vessels in the ear, identified by injections of a mixture of hydrogen peroxide and dye (scale bar = 5 mm). Arrows indicate injection sites. with tactile feedback or overflow from a cannulated hole in the vessel wall was a sign of completion of the injection. The injected lymphatic vessels were dissected meticulously under the microscope and chased proximally until the place where the afferent vessels connected to the first-tier (sentinel) lymph node was reached. This procedure was repeated throughout the body until all of the relationships between the lymphatic vessels and the lymph nodes 299

4 Plastic and Reconstructive Surgery August 2015 Fig. 3. Lymphatic vessels in the right medial forelimb, identified by intradermal injection of a mixture of hydrogen peroxide and dye. A magnified photograph (right) shows stained lymphatic vessels with valvular structures (scale bar = 5 mm). Fig. 4. Radiograph of the left hind limb after injections of radiocontrast mixture into the lymphatic vessels. The lymphatic vessels and popliteal (black arrow), inguinal (white arrow), and subiliac (yellow arrow) lymph nodes are shown. were defined. If we needed to chase the efferent lymphatic vessels from the lymph nodes, hydrogen peroxide was injected into the lymph nodes directly to identify the efferent vessels. After completion of injections into all identified lymphatic vessels, the carcass was radiographed in the anteroposterior position using a portable digital x-ray system (RadPro 40kW; Canon USA, Inc., Melville, N.Y.) to record courses of the lymphatic vessels on digital radiographs. Then, the carcass was eviscerated and cut into sagittal halves using an electric reciprocating saw. The half body was radiographed again to show the lymphatic vessels on the lateral view (Fig. 4). The digital radiographs were transferred to a computer, and we traced the lymphatic vessels and their corresponding sentinel lymph nodes using a graphic software program (Adobe Photoshop CS6 Extended; Adobe Systems, Inc., San Jose, Calif.). We color-coded a group of lymph nodes in accordance with their anatomical regions, and then each afferent lymphatic vessel connected to the group of nodes was color-coded with the same color. We could identify the distribution of the lymphatic vessels underlying the skin, and thus we could define which skin territory connected to which lymph node (i.e., lymphosomes). RESULTS Indocyanine green fluorescence lymphography showed lines extending from the sites of indocyanine green injection; these lines merged and formed larger lines that ran proximally and connected to shiny round structures. Subsequent dissection confirmed that these shiny lines were the lymph vessels and the round structures were lymph nodes. Indocyanine green was specifically taken 300

5 Volume 136, Number 2 Lymphosomes in Swine up by the lymph vessels when it was injected intradermally, and we could delineate the lymphatic pathways by drawing on the skin (Fig. 5). These drawings accurately showed the locations of the lymphatic vessels before we cut the skin and therefore helped to identify the lymphatic vessels during the dissection. The microinjection technique enabled us to inject radiocontrast mixture into the lymphatic vessels. The mixture perfused the vessels and allowed us to identify the first-tier (sentinel) lymph nodes (Fig. 6). We then successfully mapped the superficial lymphatic vessels and their corresponding lymph nodes on radiographs (Fig. 7). The tracing of lymphatic vessels and color coding demonstrated that the superficial lymphatic vessels did not overlap with each other and therefore swine skin was divided into seven distinct lymphosomes: the parotid, mandibular, dorsal cervical, ventral cervical, subiliac, inguinal, and popliteal territories (Fig. 8). The lymphatic vessels were interconnected within the same regional basin but did not interconnect between the neighboring territories. Sizes of the lymphatic vessels varied from 0.3 to 1.0 mm. The number of lymph nodes in each regional lymphatic basin varied from one to three among the specimens. The sizes of the individual lymphosomes varied, but the same seven distinct lymphosomes were identified in all specimens. Of note, no lymph nodes existed in the superficial axillary region, which commonly exists in humans and other mammals The lymphatic vessels in both of the forelimbs and the ventral cranial torso in swine ran through the axillary region and connected to the ventral cervical lymph nodes. Each lymph node was supplied with blood vessels. No lymphatic vessels were found to cross the midlines in either the ventral or dorsal aspects and therefore the midlines were lymphatic watersheds. DISCUSSION We have successfully mapped the lymphosomes in swine using the microinjection Fig. 5. Tracing of the lymphatic vessels on a whole swine carcass, identified by indocyanine green fluorescence lymphography. Fig. 6. Right inguinal lymph node with afferent lymphatic vessels (caudal on the right and midline on the top). Arrows indicate the direction of lymph flow (scale bar = 10 mm). 301

6 Plastic and Reconstructive Surgery August 2015 Fig. 7. Anteroposterior and lateral radiographic images of the swine carcass after lymphatic injections. Fig. 8. Seven lymphosomes were identified in swine: 1, parotid; 2, mandibular; 3, dorsal cervical; 4, ventral cervical; 5, subiliac; 6, inguinal; and 7, popliteal territories. technique, and our results suggest that the swine, with its anatomically consistent lymphosomes and sizable lymphatic vessels, could be a useful large-animal model for lymphatic research. Surgical treatment for lymphedema has been a challenging task for plastic surgeons. The pathophysiology of lymphedema has been investigated in animal experiments, but triggers of lymphedema are still poorly understood. Preclinical large-animal models are needed to evaluate various treatment options for lymphedema. The swine has several advantages as a prospective preclinical large-animal model for lymphatic research, including skin structure similar to that of humans and sizable lymphatic vessels. Currently, however, little information is available regarding the anatomy of the lymphatic system in swine. Knowledge of the locations of lymph nodes and watershed between lymphatic territories can be a fundamental template for future lymphatic research. Using the indirect injection technique, Sabin illustrated the development of the lymphatic system in a swine embryo in (Fig. 9). First, the lymphatic sacs appeared at bilateral neck regions and the lymphatic duct from the sacs spread into three different directions: (1) the posterior neck and shoulder; (2) the face; and (3) the front neck, chest, and forelimbs. Second, additional lymphatic sacs appeared at bilateral iliac regions and the lymphatic duct radiated toward the posterior back and hip and the abdominal wall and hind limbs. Consistent with Sabin s embryonic study, we observed three 302

7 Volume 136, Number 2 Lymphosomes in Swine Fig. 9. The lymphatic system in a swine embryo. (From Sabin FR. The development of the superficial lymphatics in the skin of the pig. Am J Anat. 1904;3: , copyright 1904 Wiley-Liss, Inc.). lymphatic vessels in the forelimbs of other mammals, as reported in our previous studies The absence of the axillary lymph nodes indicates that the swine may not be a suitable model with which to mimic breast cancer related lymphedema in humans, which occurs after axillary lymph node dissection or radiotherapy. Aside from this caveat, we expect that the lymphatic system in swine could serve as a largeanimal model in experimental studies for future lymphatic research. For example, the detailed anatomical information about the lymphatic system in swine provided by our results may allow for swine models to be used for investigation of vascularized lymph node transfer techniques. Vascularized lymph node transfer has shown promising results in clinical settings; however, further scientific evidence is needed to provide a rationale for the procedure. 26 Several vascularized lymph node flaps can be designed on the basis of our study results and angiosomes (Fig. 10). 27 Each flap includes each regional group of lymph nodes supplied by the vascular pedicle. lymphosomes in the upper body: (1) dorsal cervical, (2) parotid, and (3) ventral cervical lymphatic territories (Fig. 8). Although Sabin found primary lymphatic vessels instead of matured lymph collecting vessels, the embryonic development of the lymphatic system in Sabin s study was closely related to the lymphatic system we observed in adult swine. We found that superficial axillary lymph nodes were absent in swine and the lymphatic vessels in the forelimbs directly connected to the ventral cervical lymph nodes. This contrasts with the CONCLUSIONS In summary, we found that the lymphatic system in swine contained seven lymphosomes that correspond to their first-tier lymph nodes. The swine could be a useful large-animal model for lymphatic research because of the anatomical consistency of the lymphosomes and the sizable lymphatic vessels; however, researchers should be aware that swine lack the superficial axillary lymph node, limiting the use of the swine as a model for breast cancer related lymphedema. Fig. 10. Potential vascularized lymph node flaps in swine: 1, superficial cervical; 2, thoracodorsal; 3, deep circumflex iliac; and 4, superficial inferior epigastric artery and vein. 303

8 Plastic and Reconstructive Surgery August 2015 Hiroo Suami, M.D., Ph.D. Department of Plastic Surgery, Unit 1488 The University of Texas M. D. Anderson Cancer Center 1515 Holcombe Boulevard Houston, Texas acknowledgments This work was sponsored by the Kyte Plastic Surgery Research Fund. The University of Texas M. D. Anderson Cancer Center is supported in part by a Cancer Center Support Grant (CA016672) from the National Institutes of Health. references 1. Giordano C, Thorn SL, Renaud JM, et al. Preclinical evaluation of biopolymer-delivered circulating angiogenic cells in a swine model of hibernating myocardium. Circ Cardiovasc Imaging 2013;6: Hammerman MR. Xenotransplantation of embryonic pig pancreas for treatment of diabetes mellitus in non-human primates. J Biomed Sci Eng. 2013;6(5A). 3. Biancosino C, Zardo P, Walles T, Wildfang I, Macchiarini P, Mertsching H. Generation of a bioartificial fibromuscular tissue with autoregenerative capacities for surgical reconstruction. Cytotherapy 2006;8: Wang L, Johnson JA, Chang DW, Zhang Q. Decellularized musculofascial extracellular matrix for tissue engineering. Biomaterials 2013;34: Milton SH. Pedicled skin-flaps: The fallacy of the length: width ratio. Br J Surg. 1969;56: Dorion D, Boyd JB, Pang CY. Augmentation of transmidline skin perfusion and viability in transverse rectus abdominis myocutaneous (TRAM) flaps in the pig. Plast Reconstr Surg. 1991;88: Murphy RX Jr, Sonntag BV. The axillary tree as a source of musculocutaneous and fasciocutaneous flaps in a fixed-skin porcine model. Ann Plast Surg. 1998;40: Zhong A, Pang CY, Sheffield WD, Morris SF, Forrest CR. Augmentation of acute random pattern skin flap viability in the pig. J Surg Res. 1992;52: Massey MF, Gupta DK. The effects of systemic phenylephrine and epinephrine on pedicle artery and microvascular perfusion in a pig model of myoadipocutaneous rotational flaps. Plast Reconstr Surg. 2007;120: Gaines C, Poranki D, Du W, Clark RA, Van Dyke M. Development of a porcine deep partial thickness burn model. Burns 2013;39: Elgharably H, Roy S, Khanna S, et al. A modified collagen gel enhances healing outcome in a preclinical swine model of excisional wounds. Wound Repair Regen. 2013;21: Li J, Topaz M, Tan H, et al. Treatment of infected soft tissue blast injury in swine by regulated negative pressure wound therapy. Ann Surg. 2013;257: Rothkötter HJ, Pabst R. Autotransplantation of lymph node fragments: Structure and function of regenerated tissue. Scand J Plast Reconstr Surg Hand Surg. 1990;24: Blum KS, Radtke C, Knapp WH, Pabst R, Gratz KF. SPECT-CT: A valuable method to document the regeneration of lymphatics and autotransplanted lymph node fragments. Eur J Nucl Med Mol Imaging 2007;34: Blum KS, Hadamitzky C, Gratz KF, Pabst R. Effects of autotransplanted lymph node fragments on the lymphatic system in the pig model. Breast Cancer Res Treat. 2010;120: Baum H. Das Lymphgefäßsystem des Schweines. Berlin: P. Parey; Saar LI, Getty R. The lymph nodes and the lymph vessels of the abdominal wall, pelvic wall and the pelvic limb of swine. Iowa State U Vet. 1963;27: Suami H, Taylor GI, Pan WR. A new radiographic cadaver injection technique for investigating the lymphatic system. Plast Reconstr Surg. 2005;115: Suami H, Taylor GI, O Neill J, Pan WR. Refinements of the radiographic cadaver injection technique for investigating minute lymphatic vessels. Plast Reconstr Surg. 2007;120: Suami H, Shin D, Chang DW. Mapping of lymphosomes in the canine forelimb: Comparative anatomy between canines and humans. Plast Reconstr Surg. 2012;129: Suami H, Yamashita S, Soto-Miranda MA, Chang DW. Lymphatic territories (lymphosomes) in a canine: An animal model for investigation of postoperative lymphatic alterations. PLoS One 2013;8:e Soto-Miranda MA, Suami H, Chang DW. Mapping superficial lymphatic territories in the rabbit. Anat Rec (Hoboken) 2013;296: Suami H, O Neill JK, Pan WR, Taylor GI. Perforating lymph vessels in the canine torso: Direct lymph pathway from skin to the deep lymphatics. Plast Reconstr Surg. 2008;121: Suami H, Chang DW, Matsumoto K, Kimata Y. Demonstrating the lymphatic system in rats with microinjection. Anat Rec (Hoboken) 2011;294: Sabin FR. The development of the superficial lymphatics in the skin of the pig. Am J Anat. 1904;3: Ito R, Suami H. Overview of lymph node transfer for lymphedema treatment. Plast Reconstr Surg. 2014;134: Taylor GI, Minabe T. The angiosomes of the mammals and other vertebrates. Plast Reconstr Surg. 1992;89: