一种微创、准确、高效的小鼠胸腺内注射技术
Summary
本协议描述了为小鼠胸腺内注射建立的介入放射学程序，以避免开放手术的风险并提高盲经皮注射的准确性。
Abstract
小鼠模型中的胸腺内注射是研究胸腺和免疫功能（包括遗传和获得性T细胞疾病）的重要技术。这需要将试剂和/或细胞直接沉积到活小鼠胸腺中的方法。传统的胸腺内注射方法包括胸外科或微创经皮盲注，这两种方法都有明显的局限性。超高频超声成像装置使图像引导经皮注射在小鼠中成为可能，大大提高了经皮注射方法的注射精度，并使注射目标更小成为可能。然而，图像引导注射依赖于集成轨道系统的利用，这使得这是一个严格且耗时的过程。本文介绍了一种独特、安全、高效的小鼠经皮胸腺内注射方法，消除了对轨道系统注射的依赖。该技术依赖于使用高分辨率微超声单元对小鼠胸腺进行无创成像。使用徒手技术，放射科医生可以在超声指导下将针尖直接放入小鼠胸腺中。在成像前清洁和麻醉小鼠。对于擅长超声引导程序的经验丰富的放射科医生来说，所述技术的学习期很短，通常在一个会话内。该方法对小鼠的发病率和死亡率较低，并且比目前的经皮注射机械辅助技术快得多。它使研究者能够有效地对任何大小的胸腺（包括非常小的器官，如老年或免疫缺陷小鼠的胸腺）进行精确可靠的经皮注射，而对动物的压力最小。如果需要，该方法可以注射单个叶，并且由于该程序的省时性质，有助于大规模实验。
Introduction
胸腺在T细胞发育和免疫中起着至关重要的作用。T细胞缺乏症可由胸腺退化，遗传性疾病，感染和癌症治疗等因素引起，导致高死亡率和发病率1，2。小鼠模型在基础和转化免疫学研究中都是必不可少的，几十年来一直用于研究胸腺生物学和T细胞发育，以及为患有胸腺功能障碍和T细胞缺陷的患者开发治疗方法3，4，5。
胸腺研究的核心部分是心腺内注射生物材料，如小鼠模型6，7，8，9，10，11，12中的细胞，基因或蛋白质。传统的胸腺内注射方法使用开胸术，然后在直接可视化下进行胸腔内注射或通过“盲”经皮注射到纵隔。手术方法会显著增加气胸风险等。此外，该手术期间升高的压力会导致免疫抑制，从而可能损害免疫学数据13。经验丰富的研究人员经过一些实践，可以执行盲注射技术，但这种方法不太准确，因此将实验对象限制在胸腺大的年轻小鼠身上。
超声引导的使用已被引入，作为传统胸腺内注射方法的精确和微创替代方案14。但是，当使用集成轨道系统而不是徒手技术时，此过程非常耗时。使用进样支架进行进样需要借助各种附件（如探头支架和支架、X、Y 和 Z 定位系统）对换能器进行仔细的成像优化和定位，以及熟练操作显微操作控制和导轨系统扩展。这里介绍了一种简单的替代技术，即超声引导胸腺注射，由放射科医生使用徒手方法15进行，这是上述方法的快速和准确的微创替代方案。重要的是，目前的方法可以使用任何高分辨率超声成像系统执行，而无需注射安装和集成导轨系统。它对于需要注射大量小鼠11的研究，涉及注射两个胸腺叶的实验，或用于在老年，辐照或免疫功能低下的小鼠中准确注射小胸腺12特别有用。
Protocol
所有程序均按照发现与创新中心的动物护理指南（IACUC协议290）进行。对于本研究，C57BL / 6小鼠（雌性，4-6周龄），C57BL / 6小鼠（雌性，6个月大），J：NU雌性小鼠，NOD scid γ（NSG）雌性小鼠和B6;分别以CAG-luc、-GFP小鼠为幼鼠模型、老年小鼠模型、无胸腺裸体模型、免疫缺陷模型和生物发光细胞源。小鼠是从商业来源获得的（见 材料表）。这个过程通常需要两个人（一个在进行注射时保…
Representative Results
该技术的成功实施依赖于要遵循的几个关键步骤。首先，必须确保胸腺本身的可靠识别。在年轻小鼠中，由于腺体的尺寸很大，这很简单（图3A）。在老年小鼠或免疫缺陷小鼠中，它可能更具挑战性;但是，使用现代超声设备仍然非常可行（图3B，C）。其次，设置针头轨迹至关重要，以便在针尖穿过胸壁层并进入胸腺的过程中连续可视化。?…
Discussion
超声引导徒手注射是一种高度准确的技术，以高效和无菌的方式将研究材料输送到胸腺。在注射部位对皮肤进行初始灭菌后，由于使用了无菌手套、无菌超声探头盖和无菌超声凝胶，因此在手术过程中保持无菌。与盲经皮入路10，17或依靠手术切口直接观察胸腺18，19（这是小鼠胸腺内注射的常用方法）相比，使用徒手…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Raymond H. Thornton在这项技术上的深刻而全面的早期工作。这项研究由美国国家癌症研究所（NCI 1R37CA250661-01A1），儿童白血病研究协会，哈肯萨克子午线医学院和HUMC基金会/解决儿童癌症资助。
Materials
| Aquasonic 100 Ultrasound Gel | Parker Laboratories (Fairfield, NJ, USA) | 01-01 | Sterile Ultrasound Transmission Gel |
| B6;CAG-luc, -GFP mouse | The Jackson Laboratory (Bar Harbor, ME, USA) | 025854 | Bioluminescence cell source |
| BD Insulin Syringes with needle | Becton Dickinson (Franklin Lakes, NJ, USA) | 328431 | Ultra-fine needle – 12.7 mm, 30 G |
| C57BL/6 mouse – aged | The Jackson Laboratory (Bar Harbor, ME, USA) | 000664 | age 6 months old; aged model |
| C57BL/6 mouse – young | The Jackson Laboratory (Bar Harbor, ME, USA) | 000664 | age 4-6 weeks; young model |
| Chloraprep One-step 0.67 mL | CareFusion (El Paso, TX, USA) | 260449 | chlorhexidine gluconate applicator |
| Curity Cotton Tipped Applicator | Cardinal Health (Dublin, OH, USA) | A5000-2 | Sterile, 6" |
| D-Luciferin | Gold Biotechnology (St Louis, MO, USA) | LUCK-1G | |
| Isoflurane | Henry Schein (Melville, NY, USA) | 1182097 | |
| IVIS Lumina X5 | PerkinElmer (Melville, NY, USA) | n/a | In vivo bioluminescence imaging system |
| J:NU mouse | The Jackson Laboratory (Bar Harbor, ME, USA) | 007850 | Athymic nude model |
| Kendall Hypoallergenic Paper Tape | Cardinal Health (Dublin, OH, USA) | 1914C | |
| Kimtech Surgical Nitrile Gloves | Kimberly-Clark Professional (Irving, TX, USA) | 56892 | Sterile Gloves |
| Nair Hair Remover Lotion | Church and Dwight (Trenton, NJ, USA) | n/a | Depilatory agent |
| NOD scid gamma (NSG) mouse | The Jackson Laboratory (Bar Harbor, ME, USA) | 005557 | Immunodeficient model |
| Phosphate-Buffered Saline (PBS), 1x | Corning (Corning, NY, USA) | 21-040-CV | |
| Puralube Vet Ointment | Med Vet International | PH-PURALUBE-VET | Eye ointment |
| Sheathes | Sheathing Technologies (Morgan Hill, CA, USA) | 10040 | Sterile Ultrasound Probe Covers |
| Sure-Seal Induction Chamber | Braintree Scientific (Braintree, MA, USA) | EZ-17 85 | Anesthesia induction chamber |
| Transducer MX550D | FUJIFILM VisualSonics (Toronto, ON, Canada) | n/a | Vevo 3100 imaging probe (25-55 MHz, Centre Transmit: 40 MHz) |
| Trypan Blue, 0.4% solution in PBS | MP Biomedicals (Solon, OH, USA) | 91691049 | |
| Vevo 3100 Imaging System | FUJIFILM VisualSonics (Toronto, ON, Canada) | n/a | Ultrasound imaging system |
| Vevo 3100 Lab Software | FUJIFILM VisualSonics (Toronto, ON, Canada) | n/a | Version 3.2.7 for imaging and analysis |
| Vevo Compact Dual Anesthesia System | FUJIFILM VisualSonics (Toronto, ON, Canada) | n/a | Tabletop isoflurane-based anesthesia unit |
| Vevo Imaging Station | FUJIFILM VisualSonics (Toronto, ON, Canada) | n/a | Procedural platform |
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Cite This Article
McGuire, M. T., Tuckett, A. Z., Myint, F., Zakrzewski, J. L. A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice. J. Vis. Exp. (186), e64309, doi:10.3791/64309 (2022).