Name: a murine tail lymphedema model
Uploaded: 2021-02-10T13:00:00
Description: Watch this Scientific Journal Video about A Murine Tail Lymphedema Model at JoVE.com

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 cham...

<ol>
	<li>Kataru, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+and+secondary+lymphedema:+chicken+or+egg.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphedema+prevalence+and+treatment+benefits+in+cancer:+impact+of+a+therapeutic+intervention+on+health+outcomes+and+costs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filariasis:+diagnosis+and+treatment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+population+burden+of+lymphedema.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer-related+lymphedema--what+are+the+significant+predictors+and+how+they+affect+the+severity+of+lymphedema.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upper-body+morbidity+after+breast+cancer:+incidence+and+evidence+for+evaluation,+prevention,+and+management+within+a+prospective+surveillance+model+of+care.">Upper-body morbidity after breast cancer: incidence and evidence for evaluation, prevention, and management within a prospective surveillance model of care.</a> Cancer. 118, 2237-2249 (2012).</li><li>Carl, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Review+of+the+Surgical+Treatment+of+Extremity+Lymphedema.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+overview+on+the+surgical+management+of+secondary+lymphedema+of+the+upper+and+lower+extremities+related+to+prior+oncologic+therapies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+Inferior+Epigastric+Artery+Vascularized+Lymph+Node+Transfer:+A+Simple+and+Safe+Option+for+Lymphedema.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+perioperative+lymphatic-venous+anastomosis+localization+using+transcutaneous+vein+illumination,+isosulfan+blue,+and+indocyanine+green+lymphangiography.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphedema:+Surgical+and+Medical+Therapy.">Lymphedema: Surgical and Medical Therapy.</a> Plastic and Reconstructive Surgery. 138, 209-218 (2016).</li><li>Gould, D. J., Mehrara, B. J., Neligan, P., Cheng, M. H., Patel, K. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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&#39;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)&#160; 50 mg/5ml</td>
			<td>Mylan</td>
			<td>67457-220-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

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A Murine Tail Lymphedema Model

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. ...

Lymphedema is a chronic condition with no cure. It affects one third of patients who have had an axillary lymph node surgery and radiation in the treatment of breast cancer. Having a valid animal model facilitates understanding the mechanism and development of novel treatment strategies.The mouse tail model of lymphedema is reliable and reproducible with the rapid onset. It exhibits histological changes consistent with human lymphedema. Demonstrating the procedure will be Dr.Ganesh Mohan, a post-doctorate from my laboratory.To begin, position the eight week old sedated mouse sternally and prep the tail with 70%isopropyl alcohol. Use a caliper to measure the tail diameter at five millimeter increments starting 20 millimeters from the base of the tail. Mark a three millimeter circumferential excision on the tail 20 millimeters from the base.Under surgical microscopic magnification, perform a meticulous three millimeter full thickness skin excision leaving all the underlying vasculature intact. Incise the superior circumferential mark first through the dermis followed by a circumferential full thickness incision three millimeters distal to the first incision. Make a perpendicular full thickness vertical incision to connect the two incisions, then use a toothed fine pickup to grasp a leading edge and dissect deep within the avascular plane to the dermis and superficial to the vein adventitia with micro scissors.Inject 0.1 milliliter of 1%isosulfan blue subcutaneously proximal to the tip of the tail. Identify the two lymphatic channels appearing blue due to the injection adjacent to the lateral tail veins. Transect the lymphatics carefully, dissecting a plane between the lateral vein and the lymphatic with straight microsurgical scissors.Pass the tip of one scissor blade between the lymphatic vessel and the lateral vein and close the blades to transect the lymphatic vessel. When finished, dress the tail wound with a sterile adherent clear addressing. Administer indocyanine green 0.1 milliliter subcutaneously into the distal mouse tail near the tip.Dim the room lights then place near infrared laser angiography in buffering setting and perform live imaging. After administration of isosulfan blue into the tail tip, the lymphatics exhibited blue color. The lymphatics were disrupted while adjacent lateral veins were preserved.The progressive swelling and sustained persistent lymphedema in the mouse tail after lymphedema induction is shown here. The mouse tail volume as calculated by a truncated cone equation, peaked at week four and plateaued to week six followed by gradual improvement that was sustained until week 15. High-resolution laser speckle contrast imaging was done to confirm mouse tail perfusion in the lymphedema tail model showing injured lateral veins and intact lateral veins for assessment of tail vasculature patency.Near infrared laser lymphangiography demonstrated preoperative intact lymphatics and no ICG transit beyond surgical site post-operatively, thereby confirming that swelling was caused by lymphatic disfunction. The efficiency of genetic cargo delivery using tissue nanotransfection technology was demonstrated by delivering fluorescein amidite labeled DNA to the murine tail. Using adaptations for the mouse tail lymphedema model and ICG near infrared laser angiography is a clinically translatable and exciting animal model with treatment implications.

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