穆林尾淋巴水肿模型
Summary
淋巴水肿是由淋巴功能障碍引起的四肢肿胀。我们描述了淋巴水肿的慢性粘液尾部模型，以及组织纳米透射技术 （TNT） 对尾部进行遗传货物输送的新用途。
Abstract
淋巴水肿是由淋巴功能障碍引起的四肢肿胀。受影响的肢体由于积液、脂肪和纤维化而扩大。这种疾病没有治愈的方法。老鼠尾巴模型，使用焦点全厚皮肤切除附近的尾巴底部，导致尾部肿胀，已被用于研究淋巴水肿。然而，该模型可能导致血管组成和随后的尾部坏死和早期尾部肿胀的分辨率，限制其临床可翻译性。慢性阴唇尾淋巴水肿模型诱导持续淋巴水肿超过15周和可靠的灌注到尾巴。传统木质尾淋巴水肿模型的增强包括1）使用手术显微镜精确全厚切除和淋巴剪裁，2）使用高分辨率激光斑点确认术后动脉和静脉灌注，3）使用非多氰氨酸绿色近红外激光淋巴扫描进行功能评估。我们还使用组织纳米传递技术 （TNT） 为新的非病毒性、跨皮性、焦点性遗传货物输送到小鼠尾血管。
Introduction
淋巴水肿是由淋巴功能障碍引起的四肢肿胀。受影响的肢体由于积液、脂肪和纤维化1而扩大。淋巴水肿影响全球2.5亿人2，3，4。据估计，20-40%接受固体恶性肿瘤治疗的患者，如乳腺癌、黑色素瘤、妇科/泌尿肿瘤或肉瘤，会发展成淋巴水肿2、4、5。淋巴水肿的发病率包括复发性感染、疼痛和畸形6。这种渐进的终身疾病是无法治愈的。目前的疗法是变种有效7，包括压缩，完全排泄治疗由物理治疗师，切除程序，和显微手术，包括血管淋巴结转移和淋巴旁路7，8，9，10，11，12，13，14。淋巴水肿的理想治疗方法尚未被发现。
研究淋巴水肿的机理和治疗是有限的。淋巴损伤后平均延迟一年，15、16和大多数经历辐射和手术的诱导侮辱的人不会发展淋巴水肿4，6，17。虽然大型动物模型，包括犬，羊，和猪已经描述了18，19，20，老鼠尾巴模型已被最广泛的应用，因为方便，成本，和繁殖能力。用于研究淋巴水肿的鼠标模型包括尾部模型、白喉毒素介导淋巴消融，以及轴状或弹出性淋巴结解剖 21、22、23、24、25、26。大多数尾部模型使用焦点，全厚皮肤切除与淋巴通道剪裁，这是在尾部底部附近执行22，导致尾部肿胀和组织特征类似于人类淋巴水肿24，27，28，29。然而，标准的木乃伊尾巴模型通常自发解决在短短20天，并伴有周期性尾坏死30。淋巴水肿鼠尾模型将持续淋巴水肿延长超过15周，演示确认的动脉和静脉轻度，并允许功能淋巴功能障碍评估。
淋巴水肿的阴唇尾部模型允许评估治疗淋巴水肿的新型治疗方法。基因策略已被用于由病毒载体31，32调解的老鼠模型。我们还使用一种新的组织纳米传递技术（TNT）将遗传货物运送到淋巴小鼠尾部。TNT利用纳米通道芯片在快速聚焦电场33、34、35、36中促进直接的、皮下基因的传递。该模型包括使用TNT2.0，使潜在的基因治疗焦点基因传递到小鼠尾部的淋巴损伤部位35。
Protocol
该协议遵循该机构动物研究伦理委员会的指导方针。所有动物实验都得到了印第安纳大学医学院动物护理和使用委员会的批准。动物被安置在12小时的光暗循环下，食物和水的脂肪。 1. 小鼠尾淋巴的手术中断 使用八周大的C57BL/6小鼠，性别分布均等。 将鼠标置于全身麻醉下，在诱导室中，3-4%的异黄素在100%的氧气中，然后在手术过程中以1-3%的维持饱和度进行。<…
Representative Results
持续淋巴水肿的鼠标尾部模型技术见 图1。该图展示了鼠标尾部模型的相关解剖。 图2 显示了淋巴水肿诱导后小鼠尾部的渐进肿胀和持续持续淋巴水肿。按截断锥方程计算，鼠标尾部体积在第 4 周达到峰值，在第 6 周达到稳定点，随后逐渐改善，持续到第 15 周。尾部体积可用作结果变量，以评估治疗干预对模型淋巴水肿的影响?…
Discussion
淋巴水肿分为原发性（先天性）或继发性（致畸淋巴）损伤38，39。继发性淋巴水肿占39例病例的99%。继发性淋巴水肿最常由感染（丝虫病）或肿瘤后治疗与淋巴切除术或辐射4，39引起。转化动物模型对继发性淋巴水肿具有挑战性，因为70%接受淋巴切除术和放射治疗的动物不会获得淋巴水肿<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国整形外科医生协会学术奖学金和国防部W81XWH2110135 向AHH提供的赠款资助的支持。美容外科教育和研究基金会向NIH女士U01DK119099、R01NS042617和R01DK125835授予CKS。
Materials
| Surgical Microscope | Leica, Wetzlar, Germany | MSV266 | |
| Adherent Dressing (Tegaderm) | 3M, St. Paul, Minn. | 1626W | |
| Laser speckle (Pericam PSI System ) | Perimed AB, Stockholm, Sweden) | PSIZ | |
| Near-infrared laser (LUNA) | Stryker (Formerly Novadaq Technologies, Toronto, Canada) | LU3000 | |
| C57BL/6 mice | Jackson Laboratories | 000664 | |
| Micro-Adson Forceps – 1×2 Teeth | Fine Science Tools (USA) Inc. | 11019-12 | |
| V-Hook | Fine Science Tools (USA) Inc. | 18052-12 | |
| Scalpel SS NO15 | Fischer Scientific | 29556 | |
| Disposable Needle 30GX1 | Fischer Scientific | 305128 | |
| Operating Scissors | Fischer Scientific | 12-460-796 | |
| Surgi-Or Jeweler's Forceps, Sklar 4-1/2 in | Fischer Scientific | 50-118-4255 | |
| Spring Scissors – Straight/Sharp-Sharp/8mm Cutting Edge | Fine Science Tools (USA) Inc. | 15024-10 | |
| Cardiogreen | Sigma | I2633-25MG | |
| IsosulfanBlue (Lymphazurin) 50 mg/5ml | Mylan | 67457-220-05 |
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Cite This Article
Hassanein, A. H., Sinha, M., Neumann, C. R., Mohan, G., Khan, I., Sen, C. K. A Murine Tail Lymphedema Model. J. Vis. Exp. (168), e61848, doi:10.3791/61848 (2021).