Name: leukocyte infiltration of cremaster muscle in mice assessed by intravital microscopy
Uploaded: 2020-04-15T13:00:00
Description: Watch this Scientific Journal Video about Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy at JoVE.com

这项研究得到了德国联邦教育和研究部（BMBF）01GL1746E的支持，作为PRIMAL联盟的一部分。作者承认布里塔·赫克曼和西尔维娅·佩泽提供了娴熟的技术援助。

在海德堡的IBF（国际生物梅迪辛里切福辛里希通），动物在受控和特定的无病原体条件下饲养。这里描述的所有程序都得到当地IRB和德国巴登-符腾堡市卡尔斯鲁厄市的批准。
1. 麻醉管理
<ol><li>通过腹内注射125毫克/千克氯胺酮和12.5毫克/千克木拉辛对小鼠进行麻醉。</li>
	<li>将鼠标放在加热垫上的背部固定位置，以保持鼠标的体温 （36.5-38 °C）。使用不可吸收的无菌缝合线 ?...

<ol>
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IVM作为一种研究不同器官不同细胞类型的方法得到了广泛的应用，并被广泛地描述和讨论了19。本研究的主要目的是提供一种有效的方法，在肌肉的肌肉中设置和执行IVM。实践该方法会产生可靠且可重现的结果。因此，规划和标准化是掌握该技术的关键因素。最重要的是，该技术非常依赖于血液动力学和微血管参数，需要密切监测和控制。因此，不同组之间的系统动力学会产生?...

宫内显微镜（IVM）是白细胞生物学领域常用的工具。白细胞招募遵循了由白细胞捕获、滚动和粘附到内皮壁引起的一系列明确事件，最后将白细胞迁移和外移到炎症1的实际部位。每个步骤由各种化学素（如IL-8/CXCL8）、受体（例如LFA-1、Mac-1）和相应的内皮细胞粘附分子（例如ICAM-1、VCAM-1和E-Selectin）进行调节2,和控制。,使用IVM,944、5、6、7、8、95,6,7发现不同调控部位、控制因子和调剂的相互作用，如高级甘化端产物受体（RAGE）、细胞间粘附分子1（ICAM-1）、C-X-C图案配体（CXCL）1/2及其受体CXCR2。8
IVM的方法被描述为许多不同的器官和组织，如肠道10，皮肤11，淋巴结12，胚胎蛋黄袋13等。然而，IVM研究最广泛的方法是在大鼠14中首次描述的克里马斯特模型。虽然这种方法仍然用于大鼠15，但由于不同转基因系的丰度高，目前主要应用于小鼠。我们的团队最近强调了排泄物IVM在炎症性肌肉病领域的潜在作用，如杜琴肌肉萎缩症（DMD），研究肌营养不良的mdx小鼠16。由于其薄交织和易于访问的纤维组成，排泄肌代表理想的候选肌肉，用于使用光或荧光显微镜作为整个坐骑进行研究。白细胞的招募和外征主要发生在毛细管后静脉，这可以很容易地识别在连续肌肉层在排骨肌肉。
与其他体外检测相比，体内成像的优点是其生物环境在活的有机体中。同时，对改变的白细胞招募进行细胞特异性贡献可能需要额外的体外模型，如流室或内皮测定。多种方法的组合将产生最令人信服的数据。科学家应该意识到排泄剂模型的局限性，因为任何手术操作都会导致白细胞贩运和招募的增加。因此，很难用这种方法估计基线征聘。尽管它有着广泛的应用，但 cremaster 的 IVM 可能具有挑战性，而一个新设置可能需要时间和资源才能建立。现在，我们提供了一个简单的协议，这将有助于避免IVM中的一些常见错误。此外，还将讨论限制，并在适用时突出显示免费方法。
排泄物的IVM是炎症和传染性研究领域实施的理想方法。更具体地说，在炎症性疾病的背景下，研究骨骼肌生物学的科学家可能对此非常感兴趣。

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			<td height="20" style="height:20px;width:279px;">Material</td>
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			<td style="width:217px;"></td>
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			<td height="21" style="height:21px;width:279px;">Ketanest S</td>
			<td style="width:204px;">Pfizer Pharma GmbH</td>
			<td style="width:175px;">PZN: 08509909</td>
			<td>anesthesia. Generic / IUPAC Name: ketamine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Xylazine</td>
			<td style="width:204px;">CP-Pharma GmbH</td>
			<td style="width:175px;">Article-nr.: 1205&#160;</td>
			<td>anesthesia. Generic / IUPAC Name: xylazine (as hidrochloride)</td>
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			<td height="21" style="height:21px;width:279px;">Saline Solution</td>
			<td>B. Braun Melsungen&#160;</td>
			<td>PZN 02737756</td>
			<td>surgical preparation. Generic / IUPAC Name: sodium chloride</td>
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			<td height="21" style="height:21px;">Syringe needle Omnican F</td>
			<td>B. Braun Melsungen&#160;</td>
			<td style="width:175px;">REF 9161502</td>
			<td>surgical preparation&#160;</td>
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			<td height="21" style="height:21px;width:279px;">Suture 6/0 USP</td>
			<td style="width:204px;">Resorba</td>
			<td style="width:175px;">REF 4217</td>
			<td>surgical preparation&#160;</td>
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			<td height="21" style="height:21px;width:279px;">Polyethylene tube #10&#160;</td>
			<td>BD GmbH</td>
			<td style="width:175px;">Supplier No. 427401</td>
			<td>surgical preparation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:279px;">Polyethylene tube #90&#160;</td>
			<td>BD GmbH</td>
			<td style="width:175px;">Supplier No. 427421</td>
			<td>surgical preparation&#160;</td>
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			<td height="21" style="height:21px;width:279px;">Rhodamine 6G</td>
			<td style="width:204px;">Sigma-Aldrich Chemie GmbH</td>
			<td style="width:175px;">CAS Number 989-38-8&#160;</td>
			<td>leukocyte staining. Generic / IUPAC Name: ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate</td>
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			<td height="21" style="height:21px;width:279px;">Setup Equipment</td>
			<td style="width:204px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;">Upright microscope&#160;</td>
			<td style="width:204px;">Olympus&#160;</td>
			<td style="width:175px;">BX51W1</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">40-fold objective&#160;</td>
			<td style="width:204px;">Zeiss</td>
			<td style="width:175px;">Achroplan 40 &#215; /0.80 W</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ImSpector software</td>
			<td style="width:204px;">Lavision Biotec GmbH</td>
			<td style="width:175px;">ver. 4.0.469</td>
			<td>software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">ImageJ</td>
			<td style="width:204px;">National Institute of Health, USA</td>
			<td style="width:175px;">ver. 1.51j8</td>
			<td>software</td>
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leukocyte infiltration of cremaster muscle in mice assessed by intravital microscopy

体内显微镜（IVM）广泛用于监测体内白细胞招募级联内的生理和病理生理过程。目前的协议是一种实用且可重复的方法，用于可视化白细胞内皮相互作用，导致白细胞在小鼠完整有机体内的骨骼肌衍生组织中招募白细胞。该模型适用于所有研究领域，重点是颗粒细胞活化及其在疾病中的作用。
我们提供分步协议，以指导该方法，并突出潜在的陷阱和技术困难。该协议涵盖以下几个方面：实验设置和所需材料、小鼠麻醉、排骨肌解剖以及气管和胡萝卜罐、IVM 记录和离线分析。详细解释了粘附白细胞、滚动通量 （RF） 和滚动通量分数 （RFF） 等数据格式，并讨论了适当的应用。结果部分提供了肌营养不良mdx小鼠的代表性结果。
IVM是评估体内白细胞招募的有力工具;然而，例如内皮和白细胞功能，如果内皮和白细胞功能，可能需要与外活体设置（如流室实验）组合。此外，感兴趣的动物的遗传背景可能极大地影响基线的招募，需要个人对所提供的协议进行微调。尽管它有局限性，IVM可以作为一个平台，很容易将体外发现转化为活脊椎动物的有机体。

根据所提供的协议，IVM将产生独特的见解，在骨骼肌中白细胞招募的级联。结果部分将侧重于 IVM 获得的典型结果，并重点介绍可能遇到的潜在问题。
在图1中概述了生命内显微镜的实验设置。准备肌肉和去除结缔组织对于获得具有均匀表面的聚焦显微图像至关重要。执行IVM时，多余的结缔组织最终导致模糊的显微图像（补充视频1）。<strong ...

Watch this Scientific Journal Video about Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy at JoVE.com

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

在这里，我们演示如何对小鼠排毛体肌肉的毛细管后静脉进行体内显微镜检查。通常适用于不同炎症和败血症的模型，特别是那些由化学因子和细胞因子引起的，我们强调其在研究肌肉白细胞渗透的肌肉病的相关性。

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

这种实用且可重复的方法可用于可视化导致完整小鼠内白细胞的内皮细胞相互作用。这项技术也为监测体内白细胞招募级联的生理和病理生理过程提供了强有力的工具。该方法可应用于不同的炎症和化粪池模型。例如，我们最近强调了它的相关性，在研究肌肉病变涉及夸张的肌肉白细胞渗透。我们向考虑使用这项技术的科学家们的建议是，尽可能多地获得实践经验，因为这种方法非常容易受到细微的偏差。演示这个程序的将是西蒙·克拉尼格，一个临床医生和高级科学家从我的实验室。在确认麻醉实验动物对脚趾捏没有反应后，将鼠标固定在37摄氏度加热垫上的背背卧位上，并使用不可吸收的无菌6-0缝合线在门牙周围吹一个简单的环。将缝合线连接端固定到加热垫上，并使用钳子在中线轻轻抬起皮肤。接下来，在颈部和上胸上做一个直径一到两厘米的圆形切口，并使用钳子仔细解剖周围的肌肉、脂肪和结缔组织。在气管下放置缝合线。使用小型手术剪刀将大约 1.3 毫米的横向切口切入气管，并在气管的考特端引入聚乙烯管，以确保上气道的安全。用单个圆形结缝合固定管子，并沿着气管右侧定位胡萝卜动脉。从心肌动脉壁解剖周围组织，并在动脉下传递两块缝合线。将第一根颅状缝合线与肌状动脉的分叉连接，并在不系扎的情况下将第二根缝合线从第一个缝合线定位为约五到八毫米的远方。接下来，将一块30厘米的聚乙烯管连接到一个装满盐水的一毫升注射器针上，并使用7毫米容器夹将胡萝卜动脉紧接到第二缝合线上。在胡萝卜状动脉中进行约0.5毫米的横向切口，将无菌聚乙烯管引入动脉。用第二个缝合线固定管子并拆下容器夹。然后轻轻按下注射器柱塞，将盐水冲洗到容器中。为了准备肌酸，将鼠标转移到定制的塑料显微镜框架，阴囊朝向显微镜阶段，用钳子小心地抓住阴囊最端端。轻轻拉阴囊，去除阴囊皮肤直径约五毫米的圆形部分，使开放组织与盐水水合。使用两个钳子解剖松散的结缔组织，并定位两个睾丸。抓住一个睾丸，轻轻地开始拉出生殖组织，一步一步地取出任何周围的结缔组织。一旦睾丸被外化，将组织的端钉到舞台周围的橡胶圈上，用盐水水合组织。接下来，小心地取出结缔组织，而不损害底层的肌部肌肉。这是至关重要的，删除所有的结缔组织，这将导致模糊的图像，而不会伤害潜在的肌。当所有组织被切除后，做一毫米横向切口，打开肌，然后从组织远端进行纵向切口。肌肉应该以球形方式打开。小心地将肌肉铺在玻璃舞台上，将肌肉固定在橡胶圈上，注意不要触摸或伤害组织的中心区域。然后将剩余的睾丸固定到一侧，以允许访问感兴趣的区域，并用 PSS 水合组织。对于肌肉血管的病毒内可视化，将安装的 cremaster 肌肉放在直立显微镜中并选择 40 倍目标。在 35 至 37 摄氏度的 PSS 连续超融合下执行录制，通过一条小管子，该管子已贴在显微镜的直立目标上，允许将 PSS 与目标一起连续滴到肌肉和组织上。确定毛细后结素。测量直径为 20 到 40 微米的圆环上的微循环。然后记录来自肌肌的微循环的高分辨率图像在30秒期间不断调整焦点，由于肌肉组织的收缩和松弛的轻微变化。过多的结缔组织会导致模糊的微观图像。在这个具有代表性的微观图像中，直径在20至40微米的毛细管后，可以通过两个较小的容器的汇合来识别。粘附和滚动的白细胞可以可视化，而循环的白细胞只能在录制视频的慢动作回放中跟踪。多种参数可能会影响体内的白细胞招募，包括心脏输出、血管直径、中心线速度、壁剪速和循环白细胞数量。请注意，中心线速度受心脏输出、周围血管阻力和血管内体积的影响。例如，通过卡罗提德导管给予的大量罗丹或其他实验物质可提高毛细管后中心线速度，并抑制白细胞捕获和滚动。相反，心脏衰竭、深度麻醉或体温过低可能会减少毛细管后流动。为了确定中心线速度，可以使用自由循环的荧光白细胞，或者白细胞必须用像罗达明这样的荧光试剂染色。请注意，野生型菌株可能显示生理差异。例如，与C57BL/10ScSn动物相比，C57BL/6小鼠表现出明显增强的白细胞滚动、粘附和迁移。规划和标准化阶段对于掌握该技术非常重要，尤其是在为显微镜准备肌科学肌肉时。可进行其他可视化白细胞招募的方法，以产生更多信息，并帮助剖析白细胞和内皮细胞对招募级联的个体贡献。该技术为药理冥想剂和新型免疫调节物质的临床前评价提供了一个平台。

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

Intravital Microscopy of the Inguinal Lymph Node

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...