Name: a minimally invasive, accurate, and efficient technique for intrathymic injection in mice
Uploaded: 2022-08-23T14:00:00
Description: Watch this Scientific Journal Video about A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice at JoVE.com

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。

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...

<ol>
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Z., 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=Image-guided+intrathymic+injection+of+multipotent+stem+cells+supports+life-long+T+cell+immunity+and+facilitates+targeted+immunotherapy.">Image-guided intrathymic injection of multipotent stem cells supports life-long T cell immunity and facilitates targeted immunotherapy.</a> Blood. 123 (18), 2797-2805 (2014).</li><li>Tuckett, A. Z., Thornton, R. H., O'Reilly, R. J., vanden Brink, M. R. M., Zakrzewski, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+injection+of+hematopoietic+progenitor+cells+establishes+functional+T+cell+development+in+a+mouse+model+of+severe+combined+immunodeficiency.">Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency.</a> Journal of Hematology &amp; Oncology. 10 (1), 109 (2017).</li><li>Hogan, B. V., Peter, M. B., Shenoy, H. G., Horgan, K., Hughes, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+induced+immunosuppression.">Surgery induced immunosuppression.</a> Surgeon. 9 (1), 38-43 (2011).</li><li>Blair-Handon, R., Mueller, K., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+intrathymic+injections+in+mice.">An alternative method for intrathymic injections in mice.</a> Laboratory Animals. 39 (8), 248-252 (2010).</li><li>Tuckett, A. Z., Zakrzewski, J. L., Li, D., vanden Brink, M. R., Thornton, R. 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J., Seddon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+thymocyte+death+underlies+the+CD4:CD8+T-cell+ratio+in+the+adaptive+immune+system.">Asymmetric thymocyte death underlies the CD4:CD8 T-cell ratio in the adaptive immune system.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 2905-2914 (2013).</li><li>Manna, S., Bhandoola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+injection.">Intrathymic injection.</a> Methods in Molecular Biology. 1323, 203-209 (2016).</li><li>de la Cueva, T., Naranjo, A., de la Cueva, E., Rubio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+intrathymic+injection+in+mice.">Refinement of intrathymic injection in mice.</a> Laboratory Animals. 36 (5), 27-32 (2007).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquasonic 100 Ultrasound Gel</td>
			<td>Parker Laboratories (Fairfield, NJ, USA)</td>
			<td>01-01</td>
			<td>Sterile Ultrasound Transmission Gel</td>
		</tr>
		<tr>
			<td>B6;CAG-luc, -GFP mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>025854</td>
			<td>Bioluminescence cell source</td>
		</tr>
		<tr>
			<td>BD Insulin Syringes with needle</td>
			<td>Becton Dickinson (Franklin Lakes, NJ, USA)</td>
			<td>328431</td>
			<td>Ultra-fine needle - 12.7 mm, 30 G</td>
		</tr>
		<tr>
			<td>C57BL/6 mouse - aged</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>000664</td>
			<td>age 6 months old; aged model</td>
		</tr>
		<tr>
			<td>C57BL/6 mouse - young</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>000664</td>
			<td>age 4-6 weeks; young model</td>
		</tr>
		<tr>
			<td>Chloraprep One-step 0.67 mL</td>
			<td>CareFusion (El Paso, TX, USA)</td>
			<td>260449</td>
			<td>chlorhexidine gluconate applicator</td>
		</tr>
		<tr>
			<td>Curity Cotton Tipped Applicator</td>
			<td>Cardinal Health (Dublin, OH, USA)</td>
			<td>A5000-2</td>
			<td>Sterile, 6&#34;</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Gold Biotechnology (St Louis, MO, USA)</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein (Melville, NY, USA)</td>
			<td>1182097</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina X5</td>
			<td>PerkinElmer (Melville, NY, USA)</td>
			<td>n/a</td>
			<td>In vivo bioluminescence imaging system</td>
		</tr>
		<tr>
			<td>J:NU mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>007850</td>
			<td>Athymic nude model</td>
		</tr>
		<tr>
			<td>Kendall Hypoallergenic Paper Tape</td>
			<td>Cardinal Health (Dublin, OH, USA)</td>
			<td>1914C</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Surgical Nitrile Gloves</td>
			<td>Kimberly-Clark Professional (Irving, TX, USA)</td>
			<td>56892</td>
			<td>Sterile Gloves</td>
		</tr>
		<tr>
			<td>Nair Hair Remover Lotion</td>
			<td>Church and Dwight (Trenton, NJ, USA)</td>
			<td>n/a</td>
			<td>Depilatory agent</td>
		</tr>
		<tr>
			<td>NOD scid gamma (NSG) mouse</td>
			<td>The Jackson Laboratory (Bar Harbor, ME, USA)</td>
			<td>005557</td>
			<td>Immunodeficient model</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS), 1x</td>
			<td>Corning (Corning, NY, USA)</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Med Vet International</td>
			<td>PH-PURALUBE-VET</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Sheathes</td>
			<td>Sheathing Technologies (Morgan Hill, CA, USA)</td>
			<td>10040</td>
			<td>Sterile Ultrasound Probe Covers</td>
		</tr>
		<tr>
			<td>Sure-Seal Induction Chamber</td>
			<td>Braintree Scientific (Braintree, MA, USA)</td>
			<td>EZ-17 85</td>
			<td>Anesthesia induction chamber</td>
		</tr>
		<tr>
			<td>Transducer MX550D</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Vevo 3100 imaging probe (25-55 MHz, Centre Transmit: 40 MHz)</td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% solution in PBS</td>
			<td>MP Biomedicals (Solon, OH, USA)</td>
			<td>91691049</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 3100 Imaging System</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Ultrasound imaging system</td>
		</tr>
		<tr>
			<td>Vevo 3100 Lab Software</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Version 3.2.7 for imaging and analysis</td>
		</tr>
		<tr>
			<td>Vevo Compact Dual Anesthesia System</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Tabletop isoflurane-based anesthesia unit</td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>FUJIFILM VisualSonics (Toronto, ON, Canada)</td>
			<td>n/a</td>
			<td>Procedural platform</td>
		</tr>
	</tbody>
</table>

a minimally invasive, accurate, and efficient technique for intrathymic injection in mice

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。

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。?...

Watch this Scientific Journal Video about A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice at JoVE.com

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

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。

一种微创、准确、高效的小鼠胸腺内注射技术

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...

该方案为传统的手术和盲注方法提供了一种准确、安全和有效的非手术替代方案。它也是老年或免疫缺陷小鼠萎缩胸腺最准确的注射技术。主要优点是压力最小的注射程序，非常安全和精确，而且快速，因此可以在不影响手术压力相关免疫抑制数据的情况下进行大规模实验。保持针头和换能器的对齐，同时推进胸壁需要一些练习。因此，我们建议与经验丰富的介入放射科医生合作进行注射或提供培训。帮助演示该程序的是我实验室的技术员Shaina Anuncio。首先，在小鼠的前胸部区域涂上一层薄薄的脱毛膏，不到一分钟以去除皮毛。用湿纸巾去除奶油和松散的皮毛。通过对后爪捏没有反应来确认适当的麻醉深度。在双眼涂抹眼药膏以防止角膜干燥。将一只小鼠一次仰卧放在小动物超声成像站的加热平台上，鼻锥就位。在后肢和前肢用医用胶带将鼠标固定在载物台上。使用葡萄糖酸氯己定涂抹器对无毛皮的上胸部皮肤进行消毒。激活可用的最高频率线性探头。通常，探头对于被成像的动物的大小具有最高的空间分辨率。通过点击启动屏幕后的相应按钮来激活探头。要优化成像和注射的超声设置，请通过调整屏幕右侧的垂直方向滑块将视野深度调整为目标动物的适当大小。通过沿屏幕底部的水平条滑动按钮来调整灰度增益。然后，将焦点区域调整到胸腺的预期水平。直立时，将约一毫升超声凝胶涂抹在换能器表面，放在超声机支架中或助手手中。要在加热平台旁边准备一个小的无菌区域，请将无菌探针盖、橡皮筋、无菌手套和无菌超声凝胶清空到无菌区域。然后，戴上无菌手套。小心地将无菌探头盖放在超声换能器和凝胶上。为保持无菌，请仅触摸无菌盖。将无菌橡皮筋滑过探头盖以将其固定到位。将两到三毫升无菌超声凝胶放在换能器上。将超声凝胶顶探头垂直放置在小鼠前胸壁的消毒部分进行初始成像，同时保持无菌。如前所述，查看和优化超声图像。要在横向平面上扫描鼠标的前胸部，请垂直握住换能器，并以画笔状或扫荡的动作将其从颈部上下移动到腹部。使心脏以视野为中心，将换能器稍微向颈部扫过。注意上胸部两侧的两个圆形成对的黑色结构。使用超声探头，定位胸腺的最宽部分，这通常是理想的注射目标部位。预测所选位置的水平针轨迹，并注意主要血管在该部位的位置。注射过程中应避免使用这些血管。血管将是低回声脉动结构。如果不确定，请通过点击屏幕上的颜色按钮来激活彩色多普勒模式，以检查血管内的流动。一只手握住换能器，另一只手拿着30号胰岛素针头和10微升注射液。要开始注射过程，请横向移动换能器，使胸腺在超声视野中偏离中心。确保视野的另一侧主要由超声凝胶组成，没有其他内容。将针尖放在换能器下方的凝胶中，然后缓慢移动针头，直到在皮肤表面附近可见。在超声下连续成像针头的同时，将针头插入胸腺，经皮轨迹远离血管。使用十字胸腺水平轨迹将针尖放置在入口部位对侧的胸腺叶中。一旦针尖进入胸腺的所需部分，在使用超声可视化的同时，快速从 30 号注射器中注入内容物。为了在针头插入和注射过程中稳定注射器，请将注射器夹在拇指和食指之间，并用食指控制注射器柱塞。将所有内容物沉积后取出针头。在年轻小鼠中，由于腺体的体积很大，识别胸腺很简单。在老年或免疫缺陷小鼠中更具挑战性。然而，使用现代超声设备仍然非常可行。当针头没有穿过关键结构（例如心脏、主动脉或下腔静脉之一）时，注意到成功注射。在注射过程中，针尖在其目标位置可视化，确认胸腺内沉积。使用荧光素作为荧光素酶转基因小鼠的注射剂确认注射的胸腺内位置。这些立即通过生物发光成像进行评估，在不牺牲动物的情况下确认注射的正确位置。或者，使用离体尸检注射台盼蓝作为注射部位和注射精度的视觉标志物。我们小组一直在将这种技术用于几项大型研究，包括基于注射转基因祖细胞的癌症免疫治疗研究，以及改善免疫缺陷小鼠的胸腺功能。

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

A Method for Labeling Vasculature in Embryonic Mice

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...