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A Trimodal Imaging Platform For In Vivo Cervical Lymph Node Mapping In The Rat: An Anatomical Study To Guide Lymphatic Research
Wan Jiao, MD, PhD1, Anjali C. Raghuram, BA2, Roy P. Yu, BS1, Seungju Bae, MD, PhD1, Alex K. Wong, MD1.
1Keck School of Medicine, Los Angeles, CA, USA, 2Baylor College of Medicine, Houston, TX, USA.

PURPOSE: Of the >500,000 survivors of head and neck cancer in the United States, 50% of patients are at risk for head and neck lymphedema (HNL), a disfiguring morbidity of disease treatment. Animal models of lymphedema are used to examine the role of pharmacologic and microsurgical interventions that could offer these patients improved cosmetic and functional outcomes. Rats are a commonly employed model in plastic surgery given their ease of access in academic institutions, reasonable cost, and appropriately sized vessels for microsurgical procedures. We have previously demonstrated that a mouse Prox1-EGFP bacterial artificial chromosome (BAC) enables the reliable expression of EGFP in Prox1-expressing endothelial cells in rat lymphatic vasculature. In addition, we have recently published the first reproducible head and neck lymphedema model using Prox1-EGFP rats. The aim of the present study is to provide a detailed methodology in order to consistently induce head and neck lymphedema in any strain of rat. Our methodology delineates a trimodal imaging approach to comprehensively map the superficial and deep cervical lymphatic system a rat model. Because of its combination of versatility and precise anatomic visualization, this technique ensures consistency in generating HNL in any strain of rat, thereby eliminating the constraint of using a Prox1-EGFP rat in order to reproduce our previously established model.
METHODS: Indocyanine green (ICG) fluorescence lymphography, methylene blue dye, and GFP-facilitated green fluorescence were employed to visualize the superficial and deep cervical lymph nodes in Sprague-Dawley Prox1-EGFP transgenic rats. Under general anesthesia, 2 ul of ICG and methylene blue, respectively, were injected intradermally at the following locations: the midline and lateral points in the inframandibular region, inferior to the parotid gland, and the lateral tongue. A SPY Elite fluorescence imaging system (NOVADAQ) was used to visualize dye perfusion and pooling intensity in lymph nodes.
RESULTS: GFP-facilitated green fluorescence was able to demonstrate five superficial cervical lymph nodes bilaterally along the linguofacial vein. These nodes can be correspondingly divided into the medial and lateral submandibular lymph nodes, sublingual lymph nodes, and the external jugular vein lymph node. Midline ICG and methylene blue injection resulted in visualization of the medial and lateral submandibular lymph nodes while lateral injection provided visualization of the lateral external jugular vein lymph node. Lateral tongue injection resulted in visualization of the remaining 2 lymph nodes in the superficial cervical lymph node chain, the sublingual lymph nodes, as well as visualization of the deep cervical lymph nodes located inferomedially.
CONCLUSION: When compared to the "gold standard" of green fluorescence lymph node mapping in the Prox1-EGFP rats, ICG and methylene blue yielded comparably precise visualization of cervical lymph nodes when selectively injected at specific locations. As a result, ICG and methylene blue are highly reliable techniques in order to consistently reproduce head and neck lymphedema in any strain of rat. This methodology opens new possibilities for lymphatic research in the head and neck region and can be used to test potential treatments and modalities specific to HNL.


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