Name: A Murine Tail Lymphedema Model (Video) | JoVE Journal
Uploaded: 2021-02-10T13:00:00.000+00:00
Duration: 4 min 38 s
Description: Learn a reproducible murine tail lymphedema model using surgical excision and near-infrared laser angiography. This method enables tissue nanotransfection for genetic cargo delivery and therapeutic development.

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5 minutes

This work was supported by grant funding provided by the American Association of Plastic Surgeons Academic Scholarship and the Department of Defense W81XWH2110135&#160;to AHH. Aesthetic Surgery Education and Research Foundation grant to MS. NIH U01DK119099, R01NS042617 and R01DK125835 to CKS.

The protocol follows the guidelines of the institution&#39;s animal research ethics committee. All animal experiments were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. Animals were housed under a 12-hour light-dark cycle with food and water ad libitum.
1. Surgical Disruption of Mouse Tail Lymphatics
<ol>
	<li>Use eight week-old C57BL/6 mice of equal gender distribution.</li>
	<li>Place a mouse under general anesthesia in an induction chamber with 3-4% isoflurane in 100% oxygen followed by maintenance sedation at 1-3% during the procedure.</li>
	<li>Administer 0.5 mg/kg sustained-release (SR) buprenorphine subcutaneously for pain control. 
	NOTE: Additional analgesic drugs administered post-op: Carprofen&#160;once every 24 h for at least 48 h and Bupivacaine once either after the incision was made or before closing the incision, applied by dripping onto skin edges (lasts up to 4 &#8211; 6 h).</li>
	<li>Position the mouse dorsally and prep the tail with 70% isopropyl alcohol.</li>
	<li>Measure the tail diameter prior to the procedure at 5 mm increments starting 20 mm from the base of the tail using a caliper. These measurements will be used to calculate volume using the truncated cone equation37.</li>
	<li>Mark a 3 mm circumferential excision on the tail 20 mm from the base.</li>
	<li>Perform a meticulous 3 mm full-thickness skin excision with a sterile surgical blade (size 15), leaving all the underlying vasculature intact under surgical microscopic magnification. Incise the superior circumferential mark (20 mm from tail base) first through the dermis followed by a circumferential full thickness incision 3 mm distlal to the first incision.
	<ol>
		<li>Make a perpendicular full thickness vertical incision to connect the two incisions. Use a toothed fine pickup to grasp a leading edge and use microscissors to carefully dissect deep within the avascular plane to the dermis and superficial to the vein adventitia.</li>
	</ol>
	</li>
	<li>Inject 0.1 mL of isosulfan blue (1%) subcutaneously proximal to tip of the tail.</li>
	<li>Identify the two lymphatic channels adjacent to the lateral tail veins under the surgical microscope. The lymphatics will appear blue because of isosulfan injection. Transect the lymphatics using straight microsurgical scissors. Use the scissors to carefully dissect a plane between the lateral vein and the lymphatic. Then pass the tip of one scissor blade between the lymphatic vessel and the lateral vein and close the blades to transect the lymphatic vessel.</li>
	<li>Dress the tail wound with a sterile&#160;adherent clear dressing.&#160;Check post-op incisions daily to ensure that they are not infected or bleeding and provide wound care for 2 weeks.&#160;</li>
	<li>House the animals singly to prevent any further injury to the tail and to prevent the animals from biting each other, which would lead to surgical complications.&#160;</li>
</ol>
2. Tail vascular assessment with laser speckle contrast imaging
<ol>
	<li>Anesthetize the mouse as in step 1.2.</li>
	<li>To use laser speckle contrast imaging to visualize tail vascularity, set the width to 0.8 cm, height to 1.8 cm, point density to high, frame rate to 44 images/second, time to 30 seconds, and color photo to 1 per 10 seconds.</li>
	<li>Evaluate the venous and arterial perfusion for patency. Qualitatively, continuity of flow should be visualized.</li>
</ol>
3. Functional lymphatic evaluation with near infrared laser angiography
<ol>
	<li>Anesthetize the animal as in step 1.2</li>
	<li>Reconstitute indocyanine green (ICG) (25 mg/10 mL) and administer 0.1 mL subcutaneously into the distal mouse tail near the tip.</li>
	<li>Dim the room lights. Place near-infrared laser angiography in buffering setting followed by live capture.</li>
</ol>
4. Focal delivery of nucleic acid cargo to mouse tail using TNT
<ol>
	<li>Anesthetize the animal as in step 1.2.</li>
	<li>Exfoliate the mouse tail using topical skin exfoliation cream.</li>
	<li>Immerse the mouse tail in collagenase solution (10 mg/mL) at 37 &#176;C for 5 minutes.</li>
	<li>Load DNA into the TNT2.0 chip reservoir35.</li>
	<li>Place the TNT2.0 silicone chip device over the desired focal site of delivery on the tail with nanoneedles in contact with the tail.</li>
	<li>Place a positive electrical probe in the reservoir. Attach the negative probe to a 30 G needle and insert the needle subcutaneously into the tail to the site of delivery.</li>
	<li>Apply square wave pulse electric stimulation (10 x 10 ms pulses, 250 V, 10 mA).</li>
</ol>

<ol><li>Kataru, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrosis+and+secondary+lymphedema%3A+chicken+or+egg%5BTitle%5D)+AND+%22Translation+Research%22%5BJournal%5D)">Fibrosis and secondary lymphedema: chicken or egg.</a> Translation Research. 209, 68-76 (2019).</li>
<li>Brayton, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphedema+prevalence+and+treatment+benefits+in+cancer%3A+impact+of+a+therapeutic+intervention+on+health+outcomes+and+costs%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Lymphedema prevalence and treatment benefits in cancer: impact of a therapeutic intervention on health outcomes and costs.</a> PLoS One. 9 (12), 114597(2014).</li>
<li>Mendoza, N., Li, A., Gill, A., Tyring, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Filariasis%3A+diagnosis+and+treatment%5BTitle%5D)+AND+%22Dermatology+and+Therapy%22%5BJournal%5D)">Filariasis: diagnosis and treatment.</a> Dermatology and Therapy. 22 (6), 475-490 (2009).</li>
<li>Rockson, S. G., Rivera, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimating+the+population+burden+of+lymphedema%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">Estimating the population burden of lymphedema.</a> Annals of the New York Academy of Sciences. 1131, 147-154 (2008).</li>
<li>Soran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breast+cancer-related+lymphedema--what+are+the+significant+predictors+and+how+they+affect+the+severity+of+lymphedema%5BTitle%5D)+AND+%22Breast+Journal%22%5BJournal%5D)">Breast cancer-related lymphedema--what are the significant predictors and how they affect the severity of lymphedema.</a> Breast Journal. 12 (6), 536-543 (2006).</li>
<li>Hayes, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Upper-body+morbidity+after+breast+cancer%3A+incidence+and+evidence+for+evaluation%2C+prevention%2C+and+management+within+a+prospective+surveillance+model+of+care%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Upper-body morbidity after breast cancer: incidence and evidence for evaluation, prevention, and management within a prospective surveillance model of care.</a> Cancer. 118, 8 Suppl 2237-2249 (2012).</li>
<li>Carl, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+Review+of+the+Surgical+Treatment+of+Extremity+Lymphedema%5BTitle%5D)+AND+%22Journal+of+Reconstructive+Microsurgery%22%5BJournal%5D)">Systematic Review of the Surgical Treatment of Extremity Lymphedema.</a> Journal of Reconstructive Microsurgery. 33 (6), 412-425 (2017).</li>
<li>Garza, R., Skoracki, R., Hock, K., Povoski, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+overview+on+the+surgical+management+of+secondary+lymphedema+of+the+upper+and+lower+extremities+related+to+prior+oncologic+therapies%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">A comprehensive overview on the surgical management of secondary lymphedema of the upper and lower extremities related to prior oncologic therapies.</a> BMC Cancer. 17 (1), 468(2017).</li>
<li>Hassanein, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+Inferior+Epigastric+Artery+Vascularized+Lymph+Node+Transfer%3A+A+Simple+and+Safe+Option+for+Lymphedema%5BTitle%5D)+AND+%22Journal+of+Plastic%2C+Reconstructive%2C+Aesthetic+Surgery%22%5BJournal%5D)">Deep Inferior Epigastric Artery Vascularized Lymph Node Transfer: A Simple and Safe Option for Lymphedema.</a> Journal of Plastic, Reconstructive, Aesthetic Surgery. 73 (10), 1897-1916 (2020).</li>
<li>Hassanein, A. H., Sacks, J. M., Cooney, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+perioperative+lymphatic-venous+anastomosis+localization+using+transcutaneous+vein+illumination%2C+isosulfan+blue%2C+and+indocyanine+green+lymphangiography%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Optimizing perioperative lymphatic-venous anastomosis localization using transcutaneous vein illumination, isosulfan blue, and indocyanine green lymphangiography.</a> Microsurgery. 37 (8), 956-957 (2017).</li>
<li>Chang, D. W., Masia, J., Garza, R., Skoracki, R., Neligan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphedema%3A+Surgical+and+Medical+Therapy%5BTitle%5D)+AND+%22Plastic+and+Reconstructive+Surgery%22%5BJournal%5D)">Lymphedema: Surgical and Medical Therapy.</a> Plastic and Reconstructive Surgery. 138, 3 Suppl 209-218 (2016).</li>
<li>Gould, D. J., Mehrara, B. J., Neligan, P., Cheng, M. H., Patel, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymph+node+transplantation+for+the+treatment+of+lymphedema%5BTitle%5D)+AND+%22Journal+of+Surgical+Oncology%22%5BJournal%5D)">Lymph node transplantation for the treatment of lymphedema.</a> Journal of Surgical Oncology. 118 (5), 736-742 (2018).</li>
<li>Cook, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immediate+Lymphatic+Reconstruction+after+Axillary+Lymphadenectomy%3A+A+Single-Institution+Early+Experience%5BTitle%5D)+AND+%22Annals+of+Surgical+Oncology%22%5BJournal%5D)">Immediate Lymphatic Reconstruction after Axillary Lymphadenectomy: A Single-Institution Early Experience.</a> Annals of Surgical Oncology. , (2020).</li>
<li>Cook, J. A., Hassanein, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ASO+Author+Reflections%3A+Immediate+Lymphatic+Reconstruction%3A+A+Proactive+Approach+to+Breast+Cancer-Related+Lymphedema%5BTitle%5D)+AND+%22Annals+of+Surgical+Oncology%22%5BJournal%5D)">ASO Author Reflections: Immediate Lymphatic Reconstruction: A Proactive Approach to Breast Cancer-Related Lymphedema.</a> Annals of Surgical Oncology. , (2020).</li>
<li>Johansson, K., Branje, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arm+lymphoedema+in+a+cohort+of+breast+cancer+survivors+10+years+after+diagnosis%5BTitle%5D)+AND+%22Acta+Oncologica%22%5BJournal%5D)">Arm lymphoedema in a cohort of breast cancer survivors 10 years after diagnosis.</a> Acta Oncologica. 49 (2), 166-173 (2010).</li>
<li>Johnson, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphedema+Incidence+After+Axillary+Lymph+Node+Dissection%3A+Quantifying+the+Impact+of+Radiation+and+the+Lymphatic+Microsurgical+Preventive+Healing+Approach%5BTitle%5D)+AND+%22Annals+of+Plastic+Surgery%22%5BJournal%5D)">Lymphedema Incidence After Axillary Lymph Node Dissection: Quantifying the Impact of Radiation and the Lymphatic Microsurgical Preventive Healing Approach.</a> Annals of Plastic Surgery. 82, 4S Suppl 3 234-241 (2019).</li>
<li>Gartner, R., Mejdahl, M. K., Andersen, K. G., Ewertz, M., Kroman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+in+self-reported+arm-lymphedema+in+Danish+women+treated+for+early-stage+breast+cancer+in+2005+and+2006--a+nationwide+follow-up+study%5BTitle%5D)+AND+%22Breast%22%5BJournal%5D)">Development in self-reported arm-lymphedema in Danish women treated for early-stage breast cancer in 2005 and 2006--a nationwide follow-up study.</a> Breast. 23 (4), 445-452 (2014).</li>
<li>Shin, W. S., Rockson, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+for+the+molecular+and+mechanistic+study+of+lymphatic+biology+and+disease%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">Animal models for the molecular and mechanistic study of lymphatic biology and disease.</a> Annals of the New York Academy of Sciences. 1131, 50-74 (2008).</li>
<li>Tobbia, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphedema+development+and+lymphatic+function+following+lymph+node+excision+in+sheep%5BTitle%5D)+AND+%22Journal+of+Vascular+Research%22%5BJournal%5D)">Lymphedema development and lymphatic function following lymph node excision in sheep.</a> Journal of Vascular Research. 46 (5), 426-434 (2009).</li>
<li>Olszewski, W., Machowski, Z., Sokolowski, J., Nielubowicz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+lymphedema+in+dogs%5BTitle%5D)+AND+%22Journal+of+Cardiovascular+Surgery%22%5BJournal%5D)">Experimental lymphedema in dogs.</a> Journal of Cardiovascular Surgery. 9 (2), 178-183 (1968).</li>
<li>Rutkowski, J. M., Moya, M., Johannes, J., Goldman, J., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secondary+lymphedema+in+the+mouse+tail%3A+Lymphatic+hyperplasia%2C+VEGF-C+upregulation%2C+and+the+protective+role+of+MMP-9%5BTitle%5D)+AND+%22Microvascular+Research%22%5BJournal%5D)">Secondary lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9.</a> Microvascular Research. 72 (3), 161-171 (2006).</li>
<li>Tabibiazar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+manifestations+of+experimental+lymphatic+insufficiency%5BTitle%5D)+AND+%22PLoS+Medicine%22%5BJournal%5D)">Inflammatory manifestations of experimental lymphatic insufficiency.</a> PLoS Medicine. 3 (7), 254(2006).</li>
<li>Slavin, S. A., Van den Abbeele, A. D., Losken, A., Swartz, M. A., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Return+of+lymphatic+function+after+flap+transfer+for+acute+lymphedema%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">Return of lymphatic function after flap transfer for acute lymphedema.</a> Annals of Surgery. 229 (3), 421-427 (1999).</li>
<li>Zampell, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-like+receptor+deficiency+worsens+inflammation+and+lymphedema+after+lymphatic+injury%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Cell+Physiology%22%5BJournal%5D)">Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury.</a> American Journal of Physiology-Cell Physiology. 302 (4), 709-719 (2012).</li>
<li>Gardenier, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diphtheria+toxin-mediated+ablation+of+lymphatic+endothelial+cells+results+in+progressive+lymphedema%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Diphtheria toxin-mediated ablation of lymphatic endothelial cells results in progressive lymphedema.</a> JCI Insight. 1 (15), 84095(2016).</li>
<li>Weiler, M. J., Cribb, M. T., Nepiyushchikh, Z., Nelson, T. S., Dixon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+tail+lymphedema+model+for+observing+lymphatic+pump+failure+during+lymphedema+development%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">A novel mouse tail lymphedema model for observing lymphatic pump failure during lymphedema development.</a> Scientific Reports. 9 (1), 10405(2019).</li>
<li>Avraham, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Th2+differentiation+is+necessary+for+soft+tissue+fibrosis+and+lymphatic+dysfunction+resulting+from+lymphedema%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema.</a> FASEB J. 27 (3), 1114-1126 (2013).</li>
<li>Zampell, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4%28%2B%29+cells+regulate+fibrosis+and+lymphangiogenesis+in+response+to+lymphatic+fluid+stasis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">CD4(+) cells regulate fibrosis and lymphangiogenesis in response to lymphatic fluid stasis.</a> PLoS One. 7 (11), 49940(2012).</li>
<li>Arruda, G., Ariga, S., de Lima, T. M., Souza, H. P., Andrade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+mouse-tail+lymphedema+model%5BTitle%5D)+AND+%22Lymphology%22%5BJournal%5D)">A modified mouse-tail lymphedema model.</a> Lymphology. 53 (1), 29-37 (2020).</li>
<li>Jun, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modified+Mouse+Models+of+Chronic+Secondary+Lymphedema%3A+Tail+and+Hind+Limb+Models%5BTitle%5D)+AND+%22Annals+of+Vascular+Surgery%22%5BJournal%5D)">Modified Mouse Models of Chronic Secondary Lymphedema: Tail and Hind Limb Models.</a> Annals of Vascular Surgery. 43, 288-295 (2017).</li>
<li>Karkkainen, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+model+for+gene+therapy+of+human+hereditary+lymphedema%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A model for gene therapy of human hereditary lymphedema.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (22), 12677-12682 (2001).</li>
<li>Yoon, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGF-C+gene+therapy+augments+postnatal+lymphangiogenesis+and+ameliorates+secondary+lymphedema%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema.</a> Journal of Clinical Investigation. 111 (5), 717-725 (2003).</li>
<li>Gallego-Perez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+tissue+nano-transfection+mediates+non-viral+stroma+reprogramming+and+rescue%5BTitle%5D)+AND+%22Nature+Nanotechnology%22%5BJournal%5D)">Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue.</a> Nature Nanotechnology. 12 (10), 974-979 (2017).</li>
<li>Moore, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanochannel-Based+Poration+Drives+Benign+and+Effective+Nonviral+Gene+Delivery+to+Peripheral+Nerve+Tissue%5BTitle%5D)+AND+%22Advanced+Biosystems%22%5BJournal%5D)">Nanochannel-Based Poration Drives Benign and Effective Nonviral Gene Delivery to Peripheral Nerve Tissue.</a> Advanced Biosystems. , 2000157(2020).</li>
<li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exosome-Mediated+Crosstalk+between+Keratinocytes+and+Macrophages+in+Cutaneous+Wound+Healing%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing.</a> ACS Nano. 14 (10), 12732-12748 (2020).</li>
<li>Roy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenic+tissue+nanotransfection+in+the+management+of+cutaneous+diabetic+polyneuropathy%5BTitle%5D)+AND+%22Nanomedicine%22%5BJournal%5D)">Neurogenic tissue nanotransfection in the management of cutaneous diabetic polyneuropathy.</a> Nanomedicine. 28, 102220(2020).</li>
<li>Sitzia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volume+measurement+in+lymphoedema+treatment%3A+examination+of+formulae%5BTitle%5D)+AND+%22European+Journal+of+Cancer+Care%22%5BJournal%5D)">Volume measurement in lymphoedema treatment: examination of formulae.</a> European Journal of Cancer Care. 4 (1), 11-16 (1995).</li>
<li>Smeltzer, D. M., Stickler, G. B., Schirger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+lymphedema+in+children+and+adolescents%3A+a+follow-up+study+and+review%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Primary lymphedema in children and adolescents: a follow-up study and review.</a> Pediatrics. 76 (2), 206-218 (1985).</li>
<li>Maclellan, R. A., Greene, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphedema%5BTitle%5D)+AND+%22Seminars+in+Pediatric+Surgery%22%5BJournal%5D)">Lymphedema.</a> Seminars in Pediatric Surgery. 23 (4), 191-197 (2014).</li>
<li>Clavin, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta1+is+a+negative+regulator+of+lymphatic+regeneration+during+wound+repair%5BTitle%5D)+AND+%22American+Journal+of+Physiology%3A+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">TGF-beta1 is a negative regulator of lymphatic regeneration during wound repair.</a> American Journal of Physiology: Heart and Circulatory Physiology. 295 (5), 2113-2127 (2008).</li>
<li>Gnyawali, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retooling+Laser+Speckle+Contrast+Analysis+Algorithm+to+Enhance+Non-Invasive+High+Resolution+Laser+Speckle+Functional+Imaging+of+Cutaneous+Microcirculation%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Retooling Laser Speckle Contrast Analysis Algorithm to Enhance Non-Invasive High Resolution Laser Speckle Functional Imaging of Cutaneous Microcirculation.</a> Scientific Reports. 7, 41048(2017).</li>
</ol>

The authors have no competing conflicts of interest.

Lymphedema is categorized as a primary (congenital) or secondary (iatrogenic lymphatic) injury38,39. Secondary lymphedema comprises 99% of cases39. Secondary lymphedema is most commonly caused by infection (filariasis) or post-oncological treatment with lymphadenectomy or radiation4,39. A translational animal model is challenging for secondary lymphedema, as 70% of animals treated ...

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of the accumulation of fluid, adipose, and fibrosis1. Lymphedema affects 250 million people worldwide2,3,4. It is estimated that 20-40% of patients who undergo treatment for solid malignancies, such as breast cancer, melanoma, gynecological/urologic tumors, or sarcomas, develop lymphedema2,4,5. Morbidity from lymphedema includes recurrent infections, pain, and deformity6. There is no cure for this progressive, life-long disease. Current therapies are variaby effective7 and include compression, complete decongestive therapy by physical therapists, excisional procedures, and microsurgical operations, including vascularized lymph node transfer and lymphovenous bypass7,8,9,10,11,12,13,14. The ideal treatment for lymphedema has yet to be discovered.
Studying the mechanism and therapy of lymphedema has been limited. There is an average delayed onset of one year following the lymphatic injury15,16 and most individuals who experience iatrogenic insult with radiation and surgery do not develop lymphedema4,6,17. Although large animal models, including canine, sheep, and pig have been described18,19,20, the mouse tail model has been the most widely applied because of ease, cost, and reproducibilty. Mouse models for investigating lymphedema include a tail model, diptheria-toxin mediated lymphatic ablation, and axillary or popliteal lymph node dissection21,22,23,24,25,26. Most tail models use a focal, full thickness skin excision with lymphatic channel clipping that is performed near the base of the tail22, resulting in tail swelling and histological features similar to human lymphedema24,27,28,29. However, the standard murine tail model typically spontaneous resolves in as few as 20 days and is accompanied by periodic tail necrosis30. The lymphedema mouse tail model extends a sustained lymphedema beyond 15 weeks, demonstrates confirmed arterial and venous patency, and allows functional lymphatic dysfunction assessment.
An murine tail model of lymphedema allows for evaluation of novel therapeutics to treat lymphedema. Gene-based strategies have been used in the mouse model mediated by viral vectors31,32. We also use a novel tissue nanotransfection technology (TNT) for genetic cargo delivery to the lymphedematous mouse tail. TNT facilitates direct, transcutaneous gene delivery using a chip with nanochannels in a rapid focused electric field33,34,35,36. The model includes using TNT2.0 to allow for focal gene delivery of potential gene-based therapeutics to the lymphatic injury site of the mouse tail35.

<table><tbody><tr><td>Tegaderm Film</td><td></td><td>1626W</td><td></td></tr><tr><td>Surgical Microscope</td><td>Leica, Wetzlar, Germany</td><td>MSV266</td><td></td></tr><tr><td>Adherent Dressing (Tegaderm)</td><td>3M, St. Paul, Minn.</td><td>1626W</td><td></td></tr><tr><td>Laser speckle (Pericam PSI System )</td><td>Perimed AB, Stockholm, Sweden)</td><td>PSIZ</td><td></td></tr><tr><td>Near-infrared laser (LUNA)</td><td>Stryker (Formerly Novadaq Technologies, Toronto, Canada)</td><td>LU3000</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratories</td><td>000664</td><td></td></tr><tr><td>Micro-Adson Forceps - 1x2 Teeth</td><td>Fine Science Tools (USA) Inc.</td><td>11019-12</td><td></td></tr><tr><td>V-Hook</td><td>Fine Science Tools (USA) Inc.</td><td>18052-12</td><td></td></tr><tr><td>Scalpel SS NO15</td><td>Fischer Scientific</td><td>29556</td><td></td></tr><tr><td>Disposable Needle 30GX1</td><td>Fischer Scientific</td><td>305128</td><td></td></tr><tr><td>Operating Scissors</td><td>Fischer Scientific</td><td>12-460-796</td><td></td></tr><tr><td>Surgi-Or Jeweler&#039;s Forceps, Sklar 4-1/2 in</td><td>Fischer Scientific</td><td>50-118-4255</td><td></td></tr><tr><td>Spring Scissors - Straight/Sharp-Sharp/8mm Cutting Edge</td><td>Fine Science Tools (USA) Inc.</td><td>15024-10</td><td></td></tr><tr><td>Cardiogreen</td><td>Sigma</td><td>I2633-25MG</td><td></td></tr><tr><td>IsosulfanBlue (Lymphazurin)&amp;nbsp; 50 mg/5ml</td><td>Mylan</td><td>67457-220-05</td><td></td></tr></tbody></table>

a murine tail lymphedema model

A Murine Tail Lymphedema Model

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.

The technique for the mouse tail model for sustained lymphedema is shown in Figure 1. The figure exhibits the relevant anatomy of the mouse tail model. Figure 2 demonstrates the progressive swelling and sustained persistant lymphedema in the mouse tail after lymphedema induction. The mouse tail volume, as calculated by the truncated cone equation, peaks at week 4 and plateaus to week 6 followed by gradual improvement that is sust...

Lymphedema is extremity swelling caused by lymphatic dysfunction. We describe a chronic murine tail model of lymphedema and the novel use of tissue nanotransfection technology (TNT) for genetic cargo delivery to the tail.

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. ...

Research

JoVE Journal

Medicine

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Learn a reproducible murine tail lymphedema model using surgical excision and near-infrared laser angiography. This method enables tissue nanotransfection for genetic cargo delivery and therapeutic development.

A Murine Tail Lymphedema Model

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