Name: optimization of the cuff technique for murine heart transplantation
Uploaded: 2020-06-26T13:00:00
Description: Watch this Scientific Journal Video about Optimization of the Cuff Technique for Murine Heart Transplantation at JoVE.com

这项工作得到了福建省健康教育联合研究项目（WKJ2016-2-20）、中国自然科学基金（81771271和81800664）、中国国家重点研发项目（2018YFA0）的支持。 108304）和福建省中青年教师教育科研项目（JAT170714）、中国湖南省自然科学基金（2019JJ50842）和湖南省湖湘青年人才（2019RS2013）。

动物（BALB/c，C57BL/6，雄性，8-12周）被安置在厦门大学实验动物中心特定的无病原体设施中。C57BL/6 用作接收方，BALB/c 用作捐赠者。所有程序都按照机构动物护理和使用委员会 （IACUC） 的准则执行。
注：操作需要一组显微外科仪器，包括微型剪刀、微型直钳、微弯曲钳和微针架（表和材料，图 1B、C、D、E）。需要一对一次一次?...

<ol>
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小鼠心脏移植模型是移植免疫学研究的重要工具，因为评估和大量基因修饰小鼠的免疫机制的工具和材料是可用的。然而，显微外科技术挑战，如血管缝合和电子化，限制了其广泛使用。本研究对骨髓内心脏移植的某些核心技术挑战进行了调查，取得了良好的效果。协议的一个关键步骤是将内管插入流明中，作为支架，将容器壁戴在袖口上。此优化步骤解决了动脉容器因需要广泛拉伸而引起的技?...

1973年，通过腹部的端到端肿瘤进行小鼠异位心脏移植的建立，是基础移植免疫学研究的一个重要里程碑。该模型为分析缺血再灌注损伤2、免疫排斥,和耐受性3、4的机制提供了重要有效的工具。然而，手术的复杂和耗时的性质，以及潜在的感染可能导致严重的腹腔和炎症反应，导致异位心移植模型的效率低。
1991年5月，陈首先描述了宫颈异位心移植技术。在此模型中，接受者的外血管被移植到移植物的肺动脉上，胡萝卜动脉被移植到上升主动脉上。这种方法的主要优点是便于监测和减少对接受者的创伤。同年，松浦晃一郎描述了一种改进的技术，其中外部血管和胡萝卜动脉的端部被戴在特氟隆袖口上，并用圆周丝结字6固定。一些研究人员还固定袖口在捐赠者心脏的右肺动脉之前插入袖口到接受者的外壶静脉7。迄今为止，袖口技术已广泛应用于各种血管皮囊移植模型，包括肺8、肝9、肾10移植等。
迄今为止，袖口技术存在一些困难。例如，由于额外的弹性，胡萝卜动脉很难在袖口上穿戴，导致组织向后翻转。因此，可能需要额外的练习和显微手术扩张器来完成此步骤。此外，子宫颈血管的准备可能需要长达25分钟。
为了解决这些问题，我们引入了内管技术，该技术基于袖口技术，包括使用内管固定外壶静脉和胡萝卜动脉上的袖口，帮助血管壁的反射。此外，通过简单的培训，接收者的准备时间缩短到15.5分钟。这种技术降低了手术的复杂性，不需要额外的练习或使用血管扩张器。它可以应用于所有移植免疫研究，特别是用于验证第三方免疫耐受性，在此期间，接受者接受两个心脏等值，一个在腹部，另一个在颈部11。我们还建议两位熟练的外科医生合作建立这种模式，一位外科医生准备接受动物，另一名外科医生准备收获和植入供体心脏。这种协作可将操作时间缩短至 25 分钟。利用这个优化的程序，我们建立了合成，异基因12，13，14，15，16，17，18，19，和异种小鼠心脏移植模型20。12,13,14,15,16,17,18,19
内管技术发展的基本原理是缩短建立小鼠心脏移植模型的操作时间，成功率高。与传统缝合和袖口技术相比，宫颈心脏移植模型的优化有助于在短时间内获得高成功率。此外，与使用单一操作者进行的手术相比，合作模式可以进一步缩短供体心脏的暖缺血时间。

<table>
	<tbody>
		<tr>
			<td>Artery cuff</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. diameter: 0.55 mm,length: 1.0 mm</td>
		</tr>
		<tr>
			<td>Artery inner tube</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. Diameter: 0.28mm</td>
		</tr>
		<tr>
			<td>Micro curved forceps</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA3050</td>
			<td>1/8 arc, 0.3-mm tip without a hook</td>
		</tr>
		<tr>
			<td>Micro needle holders</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA2050</td>
			<td>0.2-mm tip</td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA1050</td>
			<td>Straight, blade length: 10 mm</td>
		</tr>
		<tr>
			<td>Micro straight forceps</td>
			<td>Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory</td>
			<td>WA3060</td>
			<td>0.15-mm tip without a hook</td>
		</tr>
		<tr>
			<td>Scanlan Vascu-Statt Bulldog Clamps</td>
			<td>Scanlan International Inc</td>
			<td>1001-531</td>
			<td>Clamping pressure 20&#8211;25 grams</td>
		</tr>
		<tr>
			<td>Vein cuff</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. diameter: 0.9 mm,length: 1.2 mm</td>
		</tr>
		<tr>
			<td>Vein inner tube</td>
			<td>Self-made</td>
			<td></td>
			<td>Polyamide tube. Diameter: 0.6 mm</td>
		</tr>
	</tbody>
</table>

optimization of the cuff technique for murine heart transplantation

穆林心脏移植已经进行了40多年。随着显微外科的进步，某些新技术被用于提高手术效率。在我们的实验室中，我们通过两个主要步骤优化了袖口技术。首先，我们使用内管技术将一个临时内管插入外侧血管和胡萝卜动脉血管，以方便血管在袖口上旋转。其次，我们通过两位经验丰富的外科医生的合作，进行了完整的异位心脏移植。这些修改有效地将操作时间缩短到 25 分钟，成功率达 95%。在此报告中，我们将详细描述这些过程，并提供补充视频。我们相信，这份关于改进的袖口技术的报告将为小鼠异位心移植提供实用的指导，并将增强这种小鼠模型在基础研究上的实用性。

手术时间
培训后，熟练的外科医生可以使用内管技术在35分钟内成功完成手术，其中接受者准备需要大约15.5分钟，供体准备需要10.9分钟，供体心脏麻醉需要4.4分钟。与使用袖口技术进行内管技术和缝合技术（表1）21的手术相比，冷热缺血时间（从供体准备到心脏植入）缩短到15.3分钟。Table 1
我们设计了一个合?...

Watch this Scientific Journal Video about Optimization of the Cuff Technique for Murine Heart Transplantation at JoVE.com

Optimization of the Cuff Technique for Murine Heart Transplantation

我们引入一种内管方法的袖口技术，小鼠宫颈异位心脏移植，以帮助永远在袖口的容器。我们发现，两位经验丰富的外科医生的合作大大缩短了手术时间。

穆林心脏移植袖口技术的优化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

大家好我叫齐忠泉博士，众所周知，袖口技术自1991年以来一直被用作心脏移植。虽然医学外科和新的外科技术都有很大的进步，但手铐技术还是有一定的困难。这是一种揭幕的形式，在那里他们将向您展示一个优异的半技术，它可以显著等于手术效率。主要程序是将一个暂时插入该管子到遮阳板中。校对阅读 ISO 几乎一半。插入管技术。熟练的外科医生可以在35分钟内完成手术，成功率达95%。然后，他们可以使用热外科医生操作模块，这进一步提高了在septimic显示的操作效率，手术时间将缩短到约35分钟。谢谢。这就是将食谱和里程与五巴比妥联系在一起。使用创伤机械剪子去除右后宫颈区域的头发。使用无菌棉尖施用器用碘防腐剂擦拭手术区域，然后用70%乙醇擦拭手术区域。将鼠标放在操作平台上的上位位置。用无菌纱布盖住鼠标。使用眼科剪刀使横向切口从下三分之一的NACMEC线到右肩锁骨关节。用微弯曲钳分离右外侧血管，露出足够的长度。通过电凝切断早午餐。并找到船在迪斯，并使用62 0六缝合线。使用粉末夹将外部血管紧近。然后用微型剪刀将静脉近向连字翻译。用每毫升100单位02 4摄氏度肝素突出清洗血管流明，以去除任何残留的血液。使用微型直钳将外部牙脉通过维纳卡瓦。将静脉内管作为支架插入流明中，并在曲线上用微直钳在血管壁上。使用圆周 8 到 06 缝合固定曲线近端的刺激法索内皮。使用微直钳将静脉内管从静脉血管中取走。用微钳子进行钝解剖，以隔离右胡萝卜状动脉，靠近胸骨的内边缘。使用巴布科克夹紧紧右胡萝卜动脉。使用 6 到 06 缝合线快速定位胡萝卜动脉。并使用微型剪刀将胡萝卜状动脉代理到连结。用每毫升 100 单位 02 4 摄氏度肝素突出洗涤胡萝卜动脉，以去除任何残留的血液。通过动脉曲线的胡萝卜动脉，并使用微直钳将动脉内管插入动脉血管。使用微直钳在曲线上瞄准容器。使用圆周 8 到 06 缝合修复倒置的传真内皮。用微直钳从动脉容器中取走动脉内尖。麻醉及其感染与接受者相同。用眼科剪刀做腹部中线切口，露出腹腔。使用微曲线钳来暴露劣质的维纳卡瓦。然后静脉注射200微升，每毫升100单位02 4摄氏度肝素突出每20克体重通过劣质的静脉注射。用眼科剪刀进行胸腔切除术。切断肋骨通过调色板罗米塔轴光切口。向外剥开前胸壁，露出胸腔。用微钳子切除胸腺。暴露主动脉，然后通过主动脉拱门向冠状动脉突出200微升100单位，每毫升02 4摄氏度。使用微型剪刀在大音的开头横贯上升的母道。用微型剪刀在两个主分支的开头横贯肺动脉。找到优越的维纳卡瓦和劣质的维纳卡瓦大约使用6至02缝合线，并使用微型剪刀将静脉蒸馏物透到连结。使用单个六，到 06 缝合，将肺静脉一起定位，并使用微型剪刀将静脉分支切到连结体上。从周围的软组织去除心脏移植物。将供体心脏倒置到接受者的右颈区域。输入供体心脏的肺动脉，用微直钳将6至06环。将容器流明缠绕在 vena 卡瓦周围，然后将小腿周围的 6 到 06 缝合环拧紧，使容器接头弯曲。对移植物和动脉曲线的主动脉进行大动脉的损伤。释放团状的壶静脉，然后是团状的壶动脉。保持血管连接和扭曲，并确保血流畅通无阻。将传递的心脏移植物放入皮下空间，然后缝合切口。记录时间正常鼻窦节律和保持正常鼻窦节律至少五分钟后夹释放，以监测术后移植功能。单独将接收者放在一条毯子上，直到接受者从麻醉中醒来。综合起来，虽然我们的手术使用已经报道的袖口技术，我们建立了基于此的内在核心技术。通过优化技术，减少手术难度，缩短动脉瘤病时间。我们建议双手术合并座右铭，以进一步提高在示意图所示的手术效率。操作时间将缩短至约25分钟。完全MHC不匹配的BALBC同性心脏在移植到C57BL6接受者小鼠后8天内可以被拒绝。我们研究中使用的Syngeneic心脏移植存活了100多天，除了一个罕见的情况，因为与手术前的正常体重相比，体重减轻了15%。第七天的同位素病理学揭示了急性排斥的典型特征，包括严重的细胞渗透与组织坏死。合成移植接近正常，没有菌细胞坏死或炎症细胞渗透的证据。合成心脏移植100天后，可通过免疫荧光收集和染色的脑膜内膜。在分析中，没有观察到血管壁的厌恶性咆哮、血栓形成或内膜增厚。电显成像显示，光滑的内皮和常规的纵向地壳形成与内皮细胞整齐和紧密地排列，表面没有明显的沉积物。

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Optimization of a Quantitative Micro-neutralization Assay

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Murine Full-thickness Skin Transplantation

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...