The authors thank Peter Bollen, head of the Biomedical Laboratory for lending the equipment needed to record the footage seen through the microscopes.

Animals were housed in the University of Southern Denmark Animal Care Facility as per institutional guidelines. All procedures involving animal subjects have been approved by The Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark.
1. Pre-surgery irradiation
NOTE: Pre-surgery irradiation takes place 7 days before surgery.
<ol>
	<li>Induce anesthesia.
	<ol>
		<li>Place the mouse in an induction box and set the vaporizer to 3% isoflurane wit...

<ol>
	<li>Lawenda, B. D., Mondry, T. E., Johnstone, P. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphedema:+a+primer+on+the+identification+and+management+of+a+chronic+condition+in+oncologic+treatment.">Lymphedema: a primer on the identification and management of a chronic condition in oncologic treatment.</a> CA: A Cancer Journal for Clinicians. 59 (1), 8-24 (2009).</li><li>Greene, A. K., Greene, A. K., Slavin, S. A., Brorson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+morbidity+of+lymphedema.">Epidemiology and morbidity of lymphedema.</a> Lymphedema: Presentation, Diagnosis, and Treatment. , 33-44 (2015).</li><li>Hespe, G. E., Nores, G. G., Huang, J. J., Mehrara, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+lymphedema-Is+there+a+chance+for+medication+treatment?.">Pathophysiology of lymphedema-Is there a chance for medication treatment?.</a> Journal of Surgical Oncology. 115 (1), 96-98 (2017).</li><li>Grada, A. A., Phillips, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphedema:+Pathophysiology+and+clinical+manifestations.">Lymphedema: Pathophysiology and clinical manifestations.</a> Journal of the American Academy of Dermatology. 77 (6), 1009-1020 (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 (3 Suppl), 209S-218S (2016).</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>DiSipio, T., Rye, S., Newman, B., Hayes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+unilateral+arm+lymphoedema+after+breast+cancer:+a+systematic+review+and+meta-analysis.">Incidence of unilateral arm lymphoedema after breast cancer: a systematic review and meta-analysis.</a> The Lancet Oncology. 14 (6), 500-515 (2013).</li><li>Douglass, J., Graves, P., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Care+for+Management+of+Secondary+Lymphedema:+A+Systematic+Review.">Self-Care for Management of Secondary Lymphedema: A Systematic Review.</a> PLoS Neglected Tropical Diseases. 10 (6), e0004740 (2016).</li><li>Shih, Y. C. T., 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=Incidence,+treatment+costs,+and+complications+of+lymphedema+after+breast+cancer+among+women+of+working+age:+a+2-year+follow-up+study.">Incidence, treatment costs, and complications of lymphedema after breast cancer among women of working age: a 2-year follow-up study.</a> Journal of Clinical Oncology. 27 (12), 2007-2014 (2009).</li><li>Ridner, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+psycho-social+impact+of+lymphedema.">The psycho-social impact of lymphedema.</a> Lymphatic Research and Biology. 7 (2), 109-112 (2009).</li><li>Gutknecht, 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=Cost-of-illness+of+patients+with+lymphoedema.">Cost-of-illness of patients with lymphoedema.</a> Journal of the European Academy of Dermatology and Venereology. 31 (11), 1930-1935 (2017).</li><li>Hayes, S., 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=Prevalence+and+prognostic+significance+of+secondary+lymphedema+following+breast+cancer.">Prevalence and prognostic significance of secondary lymphedema following breast cancer.</a> Lymphatic Research and Biology. 9 (3), 135-141 (2011).</li><li>Danese, C. A., Georgalas-Bertakis, M., Morales, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+chronic+postsurgical+lymphedema+in+dogs'+limbs.">A model of chronic postsurgical lymphedema in dogs' limbs.</a> Surgery. 64 (4), 814-820 (1968).</li><li>Das, S. K., Franklin, J. D., O'Brien, B. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+model+of+secondary+lymphedema+in+dogs.">A practical model of secondary lymphedema in dogs.</a> Plastic and Reconstructive Surgery. 68 (3), 422-428 (1981).</li><li>Huang, G. K., Hsin, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+lymphedema+in+rabbit+ear.">An experimental model for lymphedema in rabbit ear.</a> Microsurgery. 4 (4), 236-242 (1983).</li><li>Tobbia, D., 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+development+and+lymphatic+function+following+lymph+node+excision+in+sheep.">Lymphedema development and lymphatic function following lymph node excision in sheep.</a> Journal of Vascular Research. 46 (5), 426-434 (2009).</li><li>Lahteenvuo, 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=Growth+factor+therapy+and+autologous+lymph+node+transfer+in+lymphedema.">Growth factor therapy and autologous lymph node transfer in lymphedema.</a> Circulation. 123 (6), 613-620 (2011).</li><li>Honkonen, 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=Lymph+node+transfer+and+perinodal+lymphatic+growth+factor+treatment+for+lymphedema.">Lymph node transfer and perinodal lymphatic growth factor treatment for lymphedema.</a> Annals of Surgery. 257 (5), 961-967 (2013).</li><li>Wang, G. Y., Zhong, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+experimental+lymphedema+in+rats'+limbs.">A model of experimental lymphedema in rats' limbs.</a> Microsurgery. 6 (4), 204-210 (1985).</li><li>Oashi, K., 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=A+new+model+of+acquired+lymphedema+in+the+mouse+hind+limb:+a+preliminary+report.">A new model of acquired lymphedema in the mouse hind limb: a preliminary report.</a> Annals of Plastic Surgery. 69 (5), 565-568 (2012).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Return+of+lymphatic+function+after+flap+transfer+for+acute+lymphedema.">Return of lymphatic function after flap transfer for acute lymphedema.</a> Annals of Surgery. 229 (3), 421-427 (1999).</li><li>Cheung, L., 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=An+experimental+model+for+the+study+of+lymphedema+and+its+response+to+therapeutic+lymphangiogenesis.">An experimental model for the study of lymphedema and its response to therapeutic lymphangiogenesis.</a> BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy. 20 (6), 363-370 (2006).</li><li>Rutkowski, J. M., Moya, M., Johannes, J., Goldman, J., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+lymphedema+in+the+mouse+tail:+Lymphatic+hyperplasia,+VEGF-C+upregulation,+and+the+protective+role+of+MMP-9.">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>Tammela, T., 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=Therapeutic+differentiation+and+maturation+of+lymphatic+vessels+after+lymph+node+dissection+and+transplantation.">Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation.</a> Nature Medicine. 13 (12), 1458-1466 (2007).</li><li>Frueh, F. S., 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=Animal+models+in+surgical+lymphedema+research--a+systematic+review.">Animal models in surgical lymphedema research--a systematic review.</a> Journal of Surgical Research. 200 (1), 208-220 (2016).</li><li>Jorgensen, M. G., 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=Quantification+of+Chronic+Lymphedema+in+a+Revised+Mouse+Model.">Quantification of Chronic Lymphedema in a Revised Mouse Model.</a> Annals of Plastic Surgery. 81 (5), 594-603 (2018).</li><li>Frueh, F. S., 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=High-resolution+3D+volumetry+versus+conventional+measuring+techniques+for+the+assessment+of+experimental+lymphedema+in+the+mouse+hindlimb.">High-resolution 3D volumetry versus conventional measuring techniques for the assessment of experimental lymphedema in the mouse hindlimb.</a> Scientific Reports. 6, 34673 (2016).</li><li>Biau, D. J., Kerneis, S., Porcher, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistics+in+brief:+the+importance+of+sample+size+in+the+planning+and+interpretation+of+medical+research.">Statistics in brief: the importance of sample size in the planning and interpretation of medical research.</a> Clinical Orthopaedics and Related Research. 466 (9), 2282-2288 (2008).</li><li>Korula, P., Varma, S. K., Sunderrao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+wound+contraction+by+point-to-point+adherent+splintage.">Inhibition of wound contraction by point-to-point adherent splintage.</a> Plastic and Reconstructive Surgery. 95 (4), 725-730 (1995).</li><li>Komatsu, E., 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=Lymph+Drainage+During+Wound+Healing+in+a+Hindlimb+Lymphedema+Mouse+Model.">Lymph Drainage During Wound Healing in a Hindlimb Lymphedema Mouse Model.</a> Lymphatic Research and Biology. 15 (1), 32-38 (2017).</li></ol>

There are a few critical steps in this protocol. Firstly, it is important that the researchers take safety precautions when working with radioactivity. Secondly, during the surgical part of this protocol, it is important to start the procedure once the mouse has been anesthetized and finish it without unnecessary breaks. This is important to avoid an excessively long surgical period for the animal and to prevent that the anesthesia loses effect during surgery. It is recommended to only administer one bolus injection of a...

Lymphedema is characterized by an accumulation of lymph fluid that leads to localized tissue swelling, which mainly occurs due to impaired or disrupted flow of lymph fluid in the lymphatic vessels1. The lymph flow can be impaired or disrupted by infection, obstruction, injury or congenital defects in the lymphatic system2. These etiologies result in accumulation of lymphatic fluid, which leads to a chronic state of inflammation, resulting in subsequent fibrosis, as well as deposition of adipose tissue3. Lymphedema can be categorized as primary or secondary lymphedema. Primary lymphedema is caused by developmental abnormalities or genetic mutation2,4. Secondary lymphedema occurs due to underlying systemic disease, surgery or trauma2,4. Secondary lymphedema is the most common form of lymphedema in the world2. In developed countries, the most common cause of secondary lymphedema is oncological therapy such as adjuvant radiotherapy and lymph node dissection5. Lymphedema is most frequent among breast cancer patients, but can also develop in patients with gynecologic, melanoma, genitourinary or neck cancer6. It has been suggested that out of all women diagnosed with breast cancer, 21% will develop lymphedema7.
Lymphedema can be stressful to the patient both physically and psychologically. Patients with lymphedema have an increased risk of infection5,8,9, poor quality of life and can develop social anxiety and symptoms of depression10. The complications of chronic lymphedema lead to high cost of care and an increased disease burden9,11. Findings have also suggested that lymphedema might be associated with increased risk of death after breast cancer treatment12. Conservative management such as compression of the affected area, manual lymph drainage and general skincare remain the first line approach. There is currently no curative treatment6. Although progress has been made in the field of surgical and medical therapy, there is still room for improvement. More research, providing insight in the pathophysiology and progression of the disease, is needed to enable clinicians to provide better treatment options for the patients5.
Animal models are being used in preclinical research to understand the pathophysiology of diseases and develop potential treatment options. Several different lymphedema animal models have been established in canines13,14, rabbits15, sheep16, pigs17,18 and rodents19,20,21,22,23,24. The rodent model seems to be the most cost-effective model, when investigating the reconstruction of lymphatic function, due to rodents being easily accessible and relatively low-priced25. The majority of the mice models have focused on inducing lymphedema in the tail of the mice21,22,23. The tail model is very reliable but the exact surgical technique for inducing lymphedema varies significantly in previous published material. This results in fluctuations in duration and robustness of the developed lymphedema presented in known litterature25. Different techniques are also being used for inducing lymphedema in the hindlimb model and they also yield varying results, but the hindlimb model might be easier to understand from a translational perspective. Previous lymphedema models have been hampered by spontaneous lymphedema resolution and therefore a reproducible and permanent lymphedema model is needed25. Researchers have previously tried to increase the dose of radiation, to prevent the spontaneous lymphedema resolution, but this has often led to subsequent severe morbidity25.
This model results in statistically significant lymphedema, without causing severe morbidity, by combining microsurgery with radiation. The model has been revised from a previous surgical model by adding a dose of irradiation that induces lymphedema, without causing severe morbidity26. It also offers a great opportunity for microsurgical training. Having access to microsurgical equipment and a microscope is necessary, due to the small anatomical structures of the mice. The surgical procedure can be performed when the user has been taught basic microsurgical techniques, such as suturing with microsurgical instruments. The operators that performed this procedure all watched tutorial videos by Acland on the preconditions of microsurgical skills (1981) and basic microsuture technique (1985). We recommend practicing the surgical procedure 8&#8722;10 times before using it in research. Practicing the procedure ensures that fewer mistakes are made and that the procedure can be performed more efficiently. When mastered, the surgical procedure can be performed in 45 minutes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10-0 Nylon suture</td>
			<td>S&#38;T</td>
			<td>12051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Nylon suture - Dafilon</td>
			<td>B Braun</td>
			<td>C0933112</td>
			<td></td>
		</tr>
		<tr>
			<td>Coagulator - ICC 50</td>
			<td>ERBE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Yibon medical co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps</td>
			<td>Lawton</td>
			<td>09-0190</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic retractors</td>
			<td>Odense University Hospital</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical clipper</td>
			<td>Aesculap</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl 0,315 mg/ml</td>
			<td>Matrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad - PhysioSuite</td>
			<td>Kent Scientific Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 1000mg Attane</td>
			<td>Scan Vet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer - PPV</td>
			<td>Penlon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro jewler forceps</td>
			<td>Lawton</td>
			<td>1405-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Needle holder</td>
			<td>Lawton</td>
			<td>25679-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Lawton</td>
			<td>10128-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tying forceps</td>
			<td>Lawton</td>
			<td>43953-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfine U-40 syringe 0,5ml</td>
			<td>BD</td>
			<td>328821</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance syringe 25g</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance syringe 27g</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam 5 mg/ml (hameln)</td>
			<td>Matrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder - Circle wood</td>
			<td>Lawton</td>
			<td>08-0065</td>
			<td></td>
		</tr>
		<tr>
			<td>Non woven swabs</td>
			<td>Selefa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Opmi pico microscope F170</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patent blue V - 25 mg/ml</td>
			<td>Guerbet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors - Joseph</td>
			<td>BD</td>
			<td>RH1630</td>
			<td></td>
		</tr>
		<tr>
			<td>Siemens INVEON multimodality pre-clinical scanner</td>
			<td>Siemens pre-clinical solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Source of radiation - D3100</td>
			<td>Gulmay</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stata Statistical Software: Release 15</td>
			<td>StataCorp LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temgesic - 0,2 mg</td>
			<td>Indivior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vet eye ointment - viscotears</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a revised method for inducing secondary lymphedema in the hindlimb of mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This procedure has previously been used in three separate experiments. All the experiments were made by different lead investigators who all are co-authors of this article. In all three experiments, great care was taken to adhere to the same procedure as described in this protocol. In all three experiments, secondary lymphedema was induced in one hindlimb while the other hindlimb served as a control. Volumes of the hindlimbs were the primary outcome in all three experiments. <strong class...

Watch this Scientific Journal Video about A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice at JoVE.com

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

The overall goal of this method is to induce consistent secondary lymphedema in the hindlimb of mice without causing severe morbidity. This method can be used to induce secondary lymphedema in the hindlimb of mice. The main advantage of this method is that it resolves insignificant lymphedema lasting at least eight weeks without causing severe morbidity.Someone new to the procedure may struggle with the microsurgical techniques. We therefore recommend microsurgical training and several practice procedures to ensure a successful result, but also to prepare the researcher so that he or she can perform the procedure more efficiently. Set up the source of radiation and anesthetize the mouse as described in the protocol.Place a 1.5 millimeter thick lead pad to ensure that only the area that undergoes surgery gets irradiated. The shown example is of a post-surgery mouse to clarify the area. Administer a dose of 10 gray irradiation.The researchers must take precautions when working with radiation. During this experiment all irradiation was performed in a radiation insulated room. And the source of radiation was only turned on when all personnel had left the room.Weigh the mouse pre-surgery. Examine anesthetic depth by paw or tail pinch test. Shave the leg chosen for the procedure.Set the flow of oxygen to 0.8 Liters per minute. Connect it with a nose cone. Apply ophthalmic ointment.Inject 0.5 milliliters of saline subcutaneously, preferably in the scruff of the mouse to prevent hypovolemia during surgery. Place the mouse on the surgical cloth in supine position. Place the nose cone over the snout.Fixate the end of the hindlimbs gently with tape, to prevent the mouse from shifting during surgery. Lift the skin with smooth forceps and clip a small opening approximately five millimeters proximal to the popliteal fossa. Slide sharp scissors into the opening and clip towards the knee, so that the incision ends just above the knee.Make sure not to puncture the underlying vessels, by lifting the skin with forceps while clipping. Move the mouse to prone position and complete the circumferential incision. Dissect the skin to a couple of millimeters above the angle.Remember to keep the tissue damp with sterile saline during the whole procedure. Retract the skin of the proximal woundage with an elastic retractor. And make sure the sciatic vein is visible as shown.Inject 0.01 milliliters Patent Blue V subcutaneously between the second and third toe. Gently press the paw a couple of times to distribute the Patent Blue V.The distribution of the Patent Blue V can be seen through the microscope. The structures needed to be visualized are, the proximal lymph vessel, PLV, the popliteal lymph node, PLN, the first distal lymph vessel, DLV1, and the second distal lymph vessel, DLV2.Magnify to visualize the PLV and ligate it using a 10-0 nylon suture. Press the paw to ensure no Patent Blue passes proximal to the ligature. Ligate the two distal lymph vessels as shown.Press the paw several times again to make sure that no Patent Blue V passes proximal to the ligature. In this example, it can be seen that one of the lymph vessels bursts due to the ligature hindering the lymph flow. Remove the popliteal lymph node, the lymph node has a smooth pearl-like surface and is colored by the Patent Blue V in contrast to the surrounding fat tissue.Before removing the inguinal fat pad, cauterize this vessel as shown. Then remove the fat pad in one piece. Make sure that there is no active bleeding.This is an example of a located inguinal lymph node. The lymph node located in the inguinal fat pad is rarely colored by the Patent Blue V and can be hard to differentiate from the fat. Removing the fat pad in one piece is the best way to ensure that the lymph node has been removed.Suture the skin edges down to the muscle fascia, with a 6-0 nylon suture using forceps and needle holder, leaving a gap of two to three millimeters, to constrain the superficial lymph flow. This is an example of the finished sutures, in the popliteal fossa, the lateral side of the hindlimb, above the knee, and the medial side of the hindlimb. Administer analgesia and weigh the mouse post-surgery, then place it in a cabinet heated for recovery.Repeat the procedure for pre-surgery irradiation. This graph shows the combined mean hindlimb volume of three separate studies in the eight weeks after surgery. 31 mice were included in total.The x-axis represents weeks after surgery. The y-axis represents hindlimb volume in cubic millimeters. The arrow bars represent the standard deviation.It is to be expected that the swelling from the surgery and the induced lymphedema will decrease during the first four weeks. After the first four weeks we see a relative stable state of lymphedema. The volume of the control hindlimb increases during the eight weeks, thus strictly saying the difference in volume between the lymphedema hindlimb and the control hindlimb, this was unexpected.The cause is speculated to be that the mice used their non-operated hindlimb more than the operated hindlimb in the weeks following surgery. This could lead to hypertrophy of the control hindlimb and maybe explain the increase in volume. For individual figures on the three studies included please read the protocol.This table shows Sidak's multiple comparison test for the combined volume measurements of the three included studies. The calculated P-value shows that there is statistically significant difference between the hindlimbs with induced lymphedema and the control hindlimbs, during all eight weeks of follow up. For individual statistical analysis on the three included projects please read the protocol.This method induces statistically significant lymphedema, lasting at least eight weeks. The procedure can be used to study the petrificiality of lymphedema and to research novel treatment options. After watching this video, you should have gained an understanding of how to induce secondary lymphedema in the hindlimb of mice, using a combination of microsurgery and pre and post-surgery irradiation.Once mastered, the surgical procedure can be performed in 45 minutes.

a-revised-method-for-inducing-secondary-lymphedema-hindlimb

Research

JoVE Journal

Medicine

7.8K Views.  University of Southern Denmark. L'obiettivo generale di questo metodo è quello di indurre un linfedema secondario coerente nell'arto posteriore dei topi senza causare grave morbilità. Questo metodo può essere utilizzato per indurre un linfedema secondario nell'arto posteriore dei topi. Il vantaggio principale di questo metodo è che risolve un linfedema insignificante della durata di almeno otto settimane senza causare grave morbilità.Qualcuno nuovo alla procedura può lottare con le tecniche microchirurgiche. Ra...