Name: a revised method for inducing secondary lymphedema in the hindlimb of mice
Uploaded: 2019-11-02T14:00:00
Description: Watch this Scientific Journal Video about A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice at JoVE.com

作者感谢生物医学实验室主任彼得·博伦提供了记录通过显微镜看到的画面所需的设备。

根据机构准则，动物被安置在南丹麦大学动物照料设施中。涉及动物主体的所有程序都已获得丹麦环境和食品部动物实验监察局的批准。
1. 手术前辐照
注：手术前照射发生在手术前7天。
<ol><li>诱导麻醉。<ol>
<li>将鼠标放入感应箱中，将蒸发器设置为 3% 的无氧，氧气流量为 0.8±1.2 L/min，以诱导吸入麻醉。 注：或者，可以使用注射麻醉剂，但在照射的?...

<ol>
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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>

此协议中有几个关键步骤。首先，研究人员在处理放射性时必须采取安全预防措施。其次，在该协议的手术部分，重要的是开始程序，一旦鼠标已被麻醉，并完成它，没有不必要的休息。这一点很重要，以避免动物的手术时间过长，并防止麻醉在手术过程中失去效果。建议只注射一次麻醉剂，一次完成手术。这也是一个关键步骤， 不要管理太多的专利蓝色 V， 因为过量的专利蓝色 V 将变色周围的...

淋巴水肿的特点是淋巴液的积累，导致局部组织肿胀，这主要是由于淋巴血管1淋巴液的流动受损或中断。淋巴流可能因感染、阻塞、损伤或淋巴系统2的先天性缺陷而受损或中断。这些病因导致淋巴液的积累，导致慢性炎症状态，导致随后的纤维化，以及脂肪组织的沉积3。淋巴水肿可分为原发性或继发性淋巴水肿。原发性淋巴水肿是由发育异常或基因突变2，4引起的。继发性淋巴水肿的发生是由于潜在的全身疾病，手术或创伤2，4。继发性淋巴水肿是世界上最常见的淋巴水肿形式2。在发达国家，继发性淋巴水肿最常见的病因是肿瘤治疗，如辅助放射治疗和淋巴结解剖5。淋巴水肿是乳腺癌患者中最常见的，但也可以在妇科、黑色素瘤、泌尿生殖素或颈部癌症患者中发展。有人建议，在所有被诊断患有乳腺癌的妇女中，21%会发展成淋巴水肿7。
淋巴水肿对患者在身体和心理上都有压力。淋巴水肿患者感染的风险增加5、8、9，生活质量差，可发展社会焦虑和抑郁症症状10。慢性淋巴水肿的并发症导致高昂的护理费用和增加的疾病负担9，11。研究结果还表明，淋巴水肿可能与乳腺癌治疗后死亡风险增加有关。保守管理，如压缩受影响的区域，手动淋巴排水和一般护肤仍然是第一线的方法。目前尚无治疗6。虽然在外科和医疗领域取得了进展，但仍有改进的余地。需要更多的研究，提供洞察的病理生理学和疾病的进展，是需要使临床医生提供更好的治疗选择的患者5。
动物模型正用于临床前研究，以了解疾病的病理生理学，并开发潜在的治疗方案。在13只、14只、兔子15只、绵羊16只、猪17只、18只和啮齿动物19只、20只，建立了几种不同的淋巴水肿动物模型。 21，22，23，24.啮齿动物模型似乎是最具成本效益的模型，当研究淋巴功能的重建，由于啮齿动物容易获得和相对低廉25。大多数小鼠模型都集中在诱导淋巴水肿在小鼠的尾巴21，22，23。尾部模型是非常可靠的，但诱导淋巴水肿的确切手术技术在以前的公开材料中变化很大。这导致在已知的淋巴水肿25中呈现的发达淋巴水肿的持续时间和鲁棒性的波动。在后肢模型中也使用不同的技术诱导淋巴水肿，它们也产生不同的结果，但从翻译的角度来看，后肢模型可能更容易理解。以前的淋巴水肿模型一直受到自发淋巴水肿分辨率的阻碍，因此需要一个可重复的和永久淋巴水肿模型25。研究人员此前曾试图增加辐射剂量，以防止自发淋巴水肿的分辨率，但这往往导致随后的严重发病率。
该模型通过将显微手术与辐射相结合，在不引起严重发病率的情况下，产生具有统计学意义的淋巴水肿。该模型已由以前的手术模型修改，增加了一剂引淋巴水肿的辐照，而不会引起严重的发病率26。它还为显微外科培训提供了绝佳的机会。由于小鼠的解剖结构较小，因此有必要使用显微外科设备和显微镜。当使用者被教导了基本的显微外科技术，如用显微手术器械缝合时，可以进行外科手术。执行此程序的操作员都观看了 Acland 关于显微外科技能（1981 年）和基本显微技术（1985 年）的先决条件的教程视频。我们建议在研究中使用之前，先进行8~10次的外科手术。实施该过程可确保减少错误，并更有效地执行该过程。掌握了后，手术可在45分钟内完成。

<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

动物模型在淋巴水肿研究中至关重要，以便了解疾病的病理生理学，同时探索潜在的治疗方案。这种小鼠模型允许研究人员诱导持续至少8周的显著淋巴水肿。淋巴水肿是诱导使用分段放射治疗和手术消融淋巴。该模型要求小鼠在手术前后获得10格雷（Gy）辐射。模型的手术部分包括三个淋巴血管的结扎和从小鼠后肢提取两个淋巴结。由于小鼠的解剖结构很小，因此获得显微手术工具和显微镜至关重要。该模型的优点是，它导致统计显著淋巴水肿，这为评估不同的治疗方案提供了良好的基础。这也是一个伟大的和容易获得的显微外科培训的选择。此模型的局限性是该过程可能非常耗时，尤其是在未提前练习时。该模型在小鼠中产生客观可量化的淋巴水肿，不会引起严重的发病率，并已在三个单独的项目中进行了测试。

此过程以前已在三个单独的实验中使用。所有的实验都是由不同的首席研究员进行的，他们都是本文的合著者。在所有三个实验中，都非常小心地遵循与本协议中描述的相同的程序。在所有三个实验中，继发性淋巴水肿在一个后肢诱导，而另一个后肢作为控制。后肢的体积是所有三个实验的主要结果。图 1说明了研究设计。
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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

这种动物模型使研究人员能够诱导具有统计学意义的继发性淋巴水肿在小鼠的后肢，持续至少8周。该模型可用于研究淋巴水肿的病理生理学，并研究新的治疗方案。

小鼠下肢诱导继发性淋巴水肿的修订方法

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

这种方法的总体目标是在小鼠的后肢中诱导一致的继发性淋巴水肿，而不会引起严重的发病率。这种方法可用于诱导小鼠后肢的继发性淋巴水肿。这种方法的主要优点是，它解决微不足道的淋巴水肿持续至少八个星期，而不会造成严重的发病率。新到这个程序的人可能与显微外科技术斗争。因此，我们建议进行显微外科训练和几个实践程序，以确保成功的结果，但也准备研究人员，以便他或她可以更有效地执行程序。设置辐射源，并麻醉鼠标，如协议中所述。放置一个 1.5 毫米厚的铅垫，以确保只有进行手术的区域得到辐照。所示示例是手术后鼠标以澄清该区域。管理10次灰色辐照的剂量。研究人员在使用辐射时必须采取预防措施。在这项实验中，所有的辐照是在辐射绝缘室进行的。辐射源只有在所有人员离开房间时才打开。称重小鼠手术前。通过爪子或尾部挤压测试检查麻醉深度。剃掉为手术选择的腿。将氧气流量设置为每分钟 0.8 升。用鼻锥连接它。涂抹眼科软膏。注射0.5毫升的盐水皮下，最好是在小鼠的擦伤，以防止在手术期间低伏血症。将鼠标放在手术布上。将鼻锥放在鼻子上。用胶带轻轻固定后肢的端部，防止小鼠在手术过程中移动。用光滑的钳子抬起皮肤，并夹住一个小开口，约五毫米的近在波普莱特福萨。将锋利的剪刀滑入开口，夹向膝盖，使切口在膝盖上方结束。在剪裁时用钳子抬起皮肤，确保不要刺穿基础血管。将鼠标移到易用位置并完成圆周切口。将皮肤解剖到角度上方几毫米。记住，在整个过程中，用无菌盐水保持组织湿润。用弹性缩回器缩回近位伤口的皮肤。并确保坐骨神经脉是可见的，如图所示。在第二个和第三个地趾之间注射0.01毫升专利蓝色V皮。轻轻按下爪子几次，通过显微镜观察专利蓝色 V 的分布。需要可视化的结构是， 近近淋巴细胞， PLV， 波普莱特淋巴结， PLN， 第一个大元淋巴体， DLV1， 和第二个近头淋巴细胞， DLV2.放大以可视化PLV并使用10-0尼龙缝合线进行平分。按爪子，确保没有专利蓝色通过近向连字。如图所示，将两个大肠淋巴血管拉大。再次按数次爪子，以确保没有专利蓝色 V 通过近向连字。在此示例中，可以看到其中一个淋巴血管因连结阻碍淋巴流动而破裂。去除波光淋巴结，淋巴结有一个光滑的珍珠样表面，并着色的专利蓝色V与周围的脂肪组织对比。在取出内焦脂肪垫之前，如图所示，将这种容器烧灼。然后一块取出脂肪垫。确保没有活动出血。这是位于金内淋巴结的示例。位于内皮脂肪垫中的淋巴结很少被专利蓝色 V 着色，并且很难与脂肪区分开来。将脂肪垫切在一块是确保淋巴结已被移除的最佳方法。缝合皮肤边缘到肌肉筋膜，用6-0尼龙缝合使用钳子和针架，留下两到三毫米的差距，以限制表面的淋巴流动。这是完成缝合的一个例子，在波莱特叶，后肢的侧侧，膝盖以上，和后肢的中侧。管理镇痛和重量小鼠手术后，然后把它放在一个加热的柜子恢复。重复手术前辐照程序。此图显示了手术后八周内三项独立研究的总均后肢体积。总共包括31只老鼠。x 轴表示手术后几周。y 轴表示后光体积（以立方毫米为单位）。箭头条表示标准偏差。预计手术引起的肿胀和诱发的淋巴水肿将在前四周内减少。前四周后，我们看到淋巴水肿的相对稳定状态。在八周内，控制后肢的体积增加，因此严格地说淋巴水肿后肢和控制后肢之间的体积差异，这是出乎意料的。据推测，原因是小鼠在手术后的几周内使用非手术后肢比手术后肢使用更多。这可能导致控制后肢的肥大，并解释体积的增加。有关三项研究的个别数字，请阅读协议。下表显示了 Sidak 对三个包含的研究的组合体积测量的多重比较测试。计算的P值表明，在全部八周的随发中，有诱发淋巴水肿的后肢与对后肢之间有统计学上显著的差异。有关三个项目的单独统计分析，请阅读协议。此方法诱导具有统计显著性的淋巴水肿，持续至少八周。该手术可用于研究淋巴水肿的佩特性，研究新的治疗方案。看完这段视频后，你应该已经了解了如何诱导继发性淋巴水肿在小鼠的后肢，使用微外科和手术前和手术后照射的组合。一旦掌握，外科手术可以在45分钟内完成。

Research

JoVE Journal

Medicine

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

科研

医学

The Trier Social Stress Test Protocol for Inducing Psychological Stress

特里尔社会压力诱发心理应激的测试协议

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

诱导肌内膜增生对战动脉粥样硬化小鼠：两种有效的模型介绍

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

在诱导小鼠心肌梗死的微创手术过程

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

An Improved Method for Rapid Intubation of the Trachea in Mice

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

小鼠肝细胞核球状估计的高通量原位方法

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...