Name: 穆林尾淋巴水肿模型 (视频) | JoVE Journal
Uploaded: 2021-02-10T13:00:00.000+00:00
Duration: 4 min 38 s
Description: 了解小鼠尾淋巴水肿模型的建立方法，该模型通过手术切断淋巴管并保留血管完整性，用于研究淋巴功能障碍机制。结合组织纳米转染技术，为开发新型治疗策略提供可靠的临床转化平台。

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5 minutes

本工作获得了美国整形外科医师协会学术奖学金以及美国国防部W81XWH2110135项目的资助&#160;AHH 获得美容外科教育与研究基金会资助；MS 获得美容外科教育与研究基金会资助；CKS 获得美国国立卫生研究院（NIH）U01DK119099、R01NS042617 和 R01DK125835 项目资助。

本方案遵循机构动物研究伦理委员会的指导原则。所有动物实验均经印第安纳大学医学院机构动物护理与使用委员会批准。动物饲养环境保持12小时光照-黑暗循环，并可自由获取食物和水。
1. 小鼠尾部淋巴管的手术破坏
<ol>
	<li>使用八周龄、性别分布均等的C57BL/6小鼠。</li>
	<li>将小鼠置于诱导室中，以100%氧气混合3-4%异氟烷进行全身麻醉诱导，术中维持麻醉时使用1-3%异氟烷。</li>
	<li>皮下注射0.5 mg/kg缓释型（SR）布托啡诺以控制疼痛。 
	注意：术后可额外给予镇痛药物：卡洛芬&#160;每24小时一次，至少持续48小时；布比卡因可在切口形成后或缝合切口前单次使用，滴加于皮肤边缘（作用持续4–6小时）。</li>
	<li>将小鼠仰卧位固定，并用70%异丙醇对尾部进行消毒处理。</li>
	<li>在手术前使用游标卡尺测量尾部直径，从尾根部起每5 mm测量一次，起始点为距尾根20 mm处。这些测量数据将用于通过截锥体公式计算体积37。</li>
	<li>在距尾根20 mm处标记一个3 mm宽的环形切除线。</li>
	<li>在手术显微镜放大下，使用无菌手术刀片（15号）精确切除3 mm全层皮肤，保留其下方所有血管结构完整。首先沿上方环形标记线（距尾根20 mm）切开真皮层，随后在第一条切口远端3 mm处做第二个环形全层切口。
	<ol>
		<li>做一个垂直的全层纵向切口以连接两个环形切口。使用齿状精细镊子夹住一侧切口边缘，用显微剪刀小心地在无血管平面内向深部解剖，直至真皮层，并位于静脉外膜浅层。</li>
	</ol>
	</li>
	<li>在尾尖近端皮下注射0.1 mL 1%伊文思蓝溶液。</li>
	<li>在手术显微镜下识别位于尾侧静脉旁的两条淋巴管。由于注射了伊文思蓝，淋巴管呈蓝色。使用直头显微外科剪刀切断淋巴管。先用剪刀仔细分离侧静脉与淋巴管之间的间隙，然后将一侧剪刀尖端插入淋巴管与侧静脉之间，闭合剪刀以切断淋巴管。</li>
	<li>用无菌&#160;透明粘性敷料包扎尾部伤口。&#160;每日检查术后切口，确保无感染或出血，并持续进行伤口护理两周。&#160;</li>
	<li>将动物单独饲养，以防止尾部再次受伤，并避免动物相互啃咬，从而减少手术并发症的发生。&#160;</li>
</ol>
2. 采用激光散斑对比成像进行尾部血管评估
<ol>
	<li>按照步骤1.2所述方法对小鼠进行麻醉。</li>
	<li>为使用激光散斑对比成像技术观察尾部血管结构，将成像区域宽度设为0.8 cm，高度设为1.8 cm，点密度设为高，帧率设为44张/秒，成像时间设为30秒，彩色照片采集频率设为每10秒1张。</li>
	<li>评估静脉和动脉灌注的通畅性。定性上应观察到血流连续。</li>
</ol>
3. 采用近红外激光血管造影进行功能性淋巴系统评估
<ol>
	<li>按照步骤1.2对动物进行麻醉。</li>
	<li>重悬吲哚菁绿（ICG）（25 mg/10 mL），并在小鼠尾部远端靠近尾尖处皮下注射0.1 mL。</li>
	<li>调暗室内光线。将近红外激光血管造影仪设为缓冲模式，随后进行实时采集。</li>
</ol>
4. 使用TNT技术将核酸货物靶向递送至小鼠尾部
<ol>
	<li>按照步骤1.2所述方法对动物进行麻醉。</li>
	<li>使用外用去角质霜对小鼠尾巴进行表皮去角质处理。</li>
	<li>将小鼠尾巴浸入胶原酶溶液（10 mg/mL）中，在37 °C条件下孵育5分钟。</li>
	<li>将DNA加载至TNT2.0 芯片储液池中35。</li>
	<li>将TNT2.0 硅基芯片装置置于尾巴上目标递送部位，使纳米针与尾巴表面接触。</li>
	<li>将正极电极插入储液池中，将负极电极连接至一支30 G针头，并将针头皮下插入尾巴递送部位。</li>
	<li>施加方波脉冲电刺激（10次，每次10 ms，250 V，10 mA）。</li>
</ol>

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<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>
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</ol>

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淋巴水肿分为原发性（先天性）和继发性（医源性淋巴系统）损伤38,39。继发性淋巴水肿占所有病例的99%39。继发性淋巴水肿最常见的病因是感染（丝虫病）或肿瘤治疗后行淋巴结切除术或放疗所致4,39。建立继发性淋巴水肿的转化动物模型具有挑战性，因为接受淋巴结切除术和放疗的动物中，有70%并未发生淋巴水肿2,16。此外，表型淋巴水肿通常在淋巴系统损伤后延迟出现（约一年后）。小鼠尾部淋巴水肿模型克服了这些障碍，所有接受局灶性尾部淋巴管切除术的小鼠均在术后数天内出现淋巴水肿21,23。在手术显微镜下进行可视化操作，实施全层局灶性皮下切除...

淋巴水肿是由淋巴系统功能障碍引起的肢体肿胀。由于液体、脂肪组织和纤维化物质的积聚，受累肢体出现肿大1。全球约有2.5亿人受到淋巴水肿的影响2,3,4。据估计，在接受实体恶性肿瘤（如乳腺癌、黑色素瘤、妇科/泌尿系统肿瘤或肉瘤）治疗的患者中，有20%-40%会继发淋巴水肿2,4,5。淋巴水肿导致的并发症包括反复感染、疼痛和肢体畸形6。目前尚无治愈这一进行性终身疾病的方法。现有疗法的效果参差不齐7，主要包括加压治疗、物理治疗师实施的综合性消肿治疗、切除性手术以及显微外科手术，后者包括带血管蒂淋巴结移植和淋巴管静脉吻合术7,8,9,10,11,12,13,14。理想的淋巴水肿治疗方法仍有待发现。
淋巴水肿的机制与治疗研究一直受到限制。淋巴损伤后，平均延迟一年才出现症状15,16，而大多数因放疗和手术造成医源性损伤的个体并不会发展为淋巴水肿4,6,17。尽管已有犬、羊和猪等大型动物模型的报道18,19,20，但小鼠尾部模型因其操作简便、成本低且可重复性高，成为应用最广泛的模型。用于研究淋巴水肿的小鼠模型包括尾部模型、白喉毒素介导的淋巴系统消融以及腋窝或腘窝淋巴结切除术21,22,23,24,25,26。大多数尾部模型采用在尾根部附近进行局部全层皮肤切除并夹闭淋巴管的方法22，从而引起尾部肿胀及组织学特征，类似于人类淋巴水肿24,27,28,29。然而，标准的小鼠尾部模型通常在短短20天内自发消退，并伴随周期性尾部坏死30。本淋巴水肿小鼠尾部模型可使淋巴水肿持续超过15周，证实存在动脉和静脉通畅，并可实现对功能性淋巴功能障碍的评估。
一种小鼠尾部淋巴水肿模型可用于评估治疗淋巴水肿的新型疗法。基于基因的治疗策略已通过病毒载体介导的小鼠模型得到应用31,32。我们还采用一种新型的组织纳米转染技术（TNT），将基因载荷递送至患淋巴水肿的小鼠尾部。TNT 利用带有纳米通道的芯片，在快速集中的电场作用下实现直接、经皮的基因递送33,34,35,36。该模型采用 TNT2.0 技术，可将潜在的基因治疗药物靶向递送至小鼠尾部的淋巴系统损伤部位35。

<table><tbody><tr><td>透明敷料薄膜</td><td></td><td>1626W</td><td></td></tr><tr><td>手术显微镜</td><td>Leica, Wetzlar, Germany</td><td>MSV266</td><td></td></tr><tr><td>粘性敷料（Tegaderm）</td><td>3M, St. Paul, Minn.</td><td>1626W</td><td></td></tr><tr><td>激光散斑成像系统（Pericam PSI 系统）</td><td>Perimed AB, Stockholm, Sweden</td><td>PSIZ</td><td></td></tr><tr><td>近红外激光成像系统（LUNA）</td><td>Stryker（原 Novadaq Technologies, Toronto, Canada）</td><td>LU3000</td><td></td></tr><tr><td>C57BL/6 小鼠</td><td>Jackson Laboratories</td><td>000664</td><td></td></tr><tr><td>微型有齿镊（1×2 齿）</td><td>Fine Science Tools (USA) Inc.</td><td>11019-12</td><td></td></tr><tr><td>V 形牵开钩</td><td>Fine Science Tools (USA) Inc.</td><td>18052-12</td><td></td></tr><tr><td>不锈钢手术刀片 NO15</td><td>Fischer Scientific</td><td>29556</td><td></td></tr><tr><td>一次性针头 30G×1</td><td>Fischer Scientific</td><td>305128</td><td></td></tr><tr><td>手术剪</td><td>Fischer Scientific</td><td>12-460-796</td><td></td></tr><tr><td>精细珠宝镊（Surgi-Or，Sklar 4-1/2 英寸）</td><td>Fischer Scientific</td><td>50-118-4255</td><td></td></tr><tr><td>弹簧剪 - 直头/锐-锐/8 mm 刃口</td><td>Fine Science Tools (USA) Inc.</td><td>15024-10</td><td></td></tr><tr><td>吲哚菁绿（Cardiogreen）</td><td>Sigma</td><td>I2633-25MG</td><td></td></tr><tr><td>异硫蓝（IsosulfanBlue，Lymphazurin）&amp;nbsp; 50 mg/5ml</td><td>Mylan</td><td>67457-220-05</td><td></td></tr></tbody></table>

一种小鼠尾部淋巴水肿模型

A Murine Tail Lymphedema Model

淋巴水肿是由淋巴系统功能障碍引起的肢体肿胀。由于液体、脂肪组织和纤维化的积聚，受累肢体出现增大。目前该疾病尚无治愈方法。既往研究淋巴水肿常采用小鼠尾部模型，即在尾根部附近进行局部全层皮肤切除，从而引起尾部肿胀。然而，该模型可能导致血管受损，进而引发尾部坏死及早期肿胀消退，限制了其临床可转化性。慢性小鼠尾部淋巴水肿模型可诱导持续超过15周的淋巴水肿，并保持尾部可靠的血液灌注。相较于传统模型，本模型的改进之处包括：1）在手术显微镜下精确实施全层组织切除并进行淋巴管夹闭；2）采用高分辨率激光散斑成像技术确认术后动脉和静脉的灌注情况；3）利用吲哚菁绿近红外激光淋巴造影术进行功能评估。此外，我们还应用组织纳米转染技术（TNT），实现新型非病毒性、经皮、局部向小鼠尾部血管递送基因物质。

小鼠尾部持续性淋巴水肿模型的技术展示见图1。该图展示了小鼠尾部模型的相关解剖结构。图2显示了淋巴水肿诱导后小鼠尾部逐渐肿胀并维持持续性淋巴水肿的过程。根据截锥体公式计算的小鼠尾部体积在第4周达到峰值，第6周趋于平稳，随后逐渐改善，并持续至第15周。尾部体积可作为评估该模型中淋巴水肿治疗干预效果的结局指标。在图3中，可观察到用于评估尾部血管通畅性的高分辨率激光散斑成像。该方法增强了模型的严谨性，以确保肿胀是由淋巴功能障碍引起，而非静脉损伤所致。由此得出的干预效果可更有信心地转化为淋巴水肿的治疗方法。图4展示了通过近红外激光淋巴造影术进行的功能性淋巴系统评估。这一额外的结局指标可用于评价干预措施对淋巴功能的影响。图5展示了利用组织纳米转染技术（TNT2.0）在...

淋巴水肿是由淋巴系统功能障碍引起的肢体肿胀。本文描述了一种慢性小鼠尾部淋巴水肿模型，以及组织纳米转染技术（TNT）在向尾部递送基因载荷方面的创新应用。

淋巴水肿是由于淋巴系统功能障碍引起的肢体肿胀。受累肢体因液体、脂肪组织积聚以及纤维化而增大。

淋巴水肿是一种无法治愈的慢性疾病，影响约三分之一接受过腋窝淋巴结手术及放疗的乳腺癌患者。建立有效的动物模型有助于阐明其发病机制并开发新的治疗策略。小鼠尾部淋巴水肿模型具有可靠的可重复性，且发病迅速，其组织学改变与人类淋巴水肿一致。本实验操作将由我实验室的博士后Ganesh Mohan博士进行演示。首先，将八周龄的麻醉小鼠置于仰卧位，并用70%异丙醇对尾部进行消毒。使用卡尺从尾根部起每5毫米测量一次尾部直径。在距尾根部20毫米处，标记一个3毫米宽的环形切除区域。在手术显微镜放大下，进行精细的3毫米全层皮肤切除，保留所有下方的血管结构完整。首先沿上方环形标记切开真皮层，随后在距第一切口远端3毫米处行环形全层切口。作一垂直的全层纵行切口以连接上述两个切口，然后使用齿状精细镊子夹住游离缘，用显微剪刀在无血管平面深层进行分离，直至真皮层并位于静脉外膜浅层。在尾尖近端皮下注射 0.1 毫升 1% 伊文思蓝。通过注射后显现的蓝色识别两条位于侧尾静脉旁的淋巴管。使用直显微外科剪刀仔细切断淋巴管，在侧静脉与淋巴管之间分离出一个解剖平面。将一把剪刀的一个刀尖插入淋巴管与侧静脉之间，合拢刀刃以横断淋巴管。操作完成后，用无菌透明粘性敷料包扎尾部伤口。在小鼠尾端近尖端处皮下注射0.1毫升吲哚菁绿。调暗室内光线，然后将近红外激光血管造影仪置于缓冲模式下进行实时成像。向尾尖注射伊文思蓝后，淋巴管呈现蓝色。淋巴管被破坏，而邻近的侧部静脉得以保留。此处展示了小鼠尾部在淋巴水肿诱导后逐渐肿胀并持续存在淋巴水肿的情况。通过截锥体公式计算的小鼠尾部体积在第4周达到峰值，第6周趋于稳定，随后逐渐改善，并持续至第15周。采用高分辨率激光散斑对比成像技术确认小鼠淋巴水肿尾模型中的尾部灌注情况，显示受损的侧尾静脉与完整的侧尾静脉，用于评估尾部血管的通畅性。近红外激光淋巴管造影显示术前淋巴管结构完整，术后无吲哚菁绿（ICG）在手术部位以外区域的流通，从而证实肿胀是由淋巴功能障碍所致。通过将荧光素标记的DNA递送至小鼠尾部，验证了组织纳米转染技术在基因 cargo 递送方面的高效性。结合小鼠尾部淋巴水肿模型的改良方法与ICG近红外激光血管造影技术，是一种具有临床可转化性的、前景广阔的动物模型，对治疗研究具有重要意义。

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科研

JoVE Journal

Medicine

医学

穆林尾淋巴水肿模型

近年来发展的近红外荧光（NIRF）成像技术可能有助于阐明淋巴系统在癌症转移、免疫反应、伤口修复及其他淋巴系统相关疾病中所起的作用。...

小鼠淋巴管系统的非侵入性光学成像

在本视频中，通过尾静脉将黑色素瘤细胞静脉注射到小鼠模型体内。这些黑色素瘤细胞随后经循环系统迁移，适应远端微环境，并在肺等器官中引发转移。...

实验性黑色素瘤转移小鼠模型：研究黑色素瘤细胞肺转移潜能的平台

本视频介绍了一种在体内建立小鼠尾部淋巴水肿的实验技术，该模型表现为间质组织间隙内异常的液体积聚，导致尾部肿胀。该模型可用于研究淋巴水肿的发生机制及治疗方法。...

小鼠尾部淋巴水肿模型：一种在小鼠体内诱导持续性淋巴水肿的实验技术

本视频展示了从小鼠模型中手术分离引流腹股沟淋巴结的操作过程。通过切开腹部区域，可暴露腹股沟淋巴结，并将其手术摘取。所获得的透明腹股沟淋巴结可用于免疫学研究。...

从小鼠模型中采集腹股沟引流淋巴结

该动物模型可诱导小鼠后肢产生具有统计学意义的继发性淋巴水肿，且持续时间至少8周。该模型可用于研究淋巴水肿的病理生理机制，并探索新的治疗方案。...

一种改良的小鼠后肢继发性淋巴水肿诱导方法

介绍了一种通过手术缝合输入淋巴管以阻断淋巴液流动的实验方案。...

通过缝合小鼠传入淋巴管阻断淋巴流

本文介绍了一种使用独特设计的加热/固定装置进行啮齿类动物尾静脉注射的有效且高效的方法。通过简化血管扩张的启动过程和固定操作，该方案可对大量动物进行准确及时的静脉注射，同时将动物的不适降至最低。...

一种用于小鼠真菌性脓毒症模型尾静脉注射的现代保温/固定装置

黑色素瘤是一种极具侵袭性的疾病，会迅速扩散至其他器官。本方案描述了超高频超声成像结合三维重建技术的应用，用于监测转移性黑色素瘤Braf/Pten小鼠模型中腹股沟淋巴结体积的变化。...

通过超高频超声成像分析Braf/Pten基因工程小鼠黑色素瘤模型中的淋巴结体积

本文所提出的实验设计为研究淋巴结（LN）转移过程中抗原特异性CD8+ T细胞提供了一个有效的生殖模型，该模型排除了旁观者CD8+...

肿瘤发生过程中抗原特异性CD8+ T细胞动态变化评估的引流淋巴结转移模型

该方案旨在描述一种基于诱导化疗反应动力学的小鼠急性淋巴细胞白血病复发模型。...

急性淋巴细胞白血病诱导化疗后白血病复发的小鼠模型

了解小鼠尾淋巴水肿模型的建立方法，该模型通过手术切断淋巴管并保留血管完整性，用于研究淋巴功能障碍机制。结合组织纳米转染技术，为开发新型治疗策略提供可靠的临床转化平台。

一种小鼠尾部淋巴水肿模型

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