这项工作是由陆军，海军，美国国立卫生研究院，空军，VA和卫生事务的支持，支持AFIRM II的努力，在奖号W81XWH-13-2-0053。美国陆军医学研究收购活动，820钱德勒街，德特里克堡MD 21702-5014是颁发和管理采购办公室。意见，解释，结论和建议是那些作者的，不一定是由美国国防部批准。 笔者想在这项研究中感谢杰西卡IZZI，DVM，卡罗琳·加勒特，DVM和朱莉·沃森，DVM的出色兽医支持。

所有实验均按照指南的护理与健康研究所（NIH）的实验室动物的用途进行，由约翰霍普金斯大学动物护理和使用委员会（JHUACUC）获得批准。经批准的ACUC协议MO13M108下进行的具体程序。 1.供体手术<ol><li>对于在手术前各药理配方合适的时间点管理镇痛。按照批准的动物护理和使用协议使用0.1毫克皮下注射丁丙诺啡/公斤体重1小时前皮肤切口。 </li><li>通过连接到4％的异氟醚蒸发室采用沉稳的异氟醚捐助;通过鼻锥保持在2％的镇静和麻醉。执行脚趾捏撤回反射并监控之前的程序的启动麻醉深度。 </li><li>戴口罩，一次性隔离衣和手套。 </li><li>剃须手术AREA，特别是后肢和腹股沟，并准备用10％聚维酮 - 碘。 </li><li>使用无菌区悬垂性，蒸压仪器和高放大倍率的显微镜（40X）。 </li><li>使腹股沟皮肤切口用剪刀近侧到大腿中部区域和周向连接切口划定从小鼠身体的其余部分的后肢。 </li><li>识别和解剖股动脉，静脉和神经。单独使用镊子和微型剪刀三种结构。 </li><li>一旦血管蒂解剖划分容器在使用微剪刀腹股沟韧带的水平。 </li><li>接下来，继续在大腿中部的水平近端划分个体腹（股薄肌和内侧大腿肌肉）和背部肌肉群20到移植使用剪刀供体动物分开。 </li><li>横切股骨和在用剪刀股骨轴的中间切断。 </li><li>异氟醚过量FOL动物实施安乐死颈椎脱位钮。确认心脏跳动和呼吸停止。 </li><li>通过使用安装在注射器33克冲洗针冲洗用肝素化2毫升（30 IE）的冷（4℃）盐水肢体（参见材料数据表）。 </li><li>广场上的股静脉和动脉1聚酰亚胺袖口，分别为。 </li><li>在4℃直至嵌入包裹接枝到湿棉花纱布，置于培养皿中，并存储。 </li></ol> 2.受体手术<ol><li> 后肢的去除 <ol><li>对于在手术前各药理配方合适的时间点管理镇痛。按照批准的动物护理和使用协议使用0.1毫克/公斤丁丙诺啡SC 1小时前，皮肤切口的BW。 </li><li>通过连接到4％的异氟醚蒸发室采用沉稳的异氟醚捐助;通过鼻锥保持在2％的镇静和麻醉。执行脚趾捏撤回反射监察部门之前的过程的开始麻醉小时。 </li><li>使用鼠标的眼睛兽医药膏，以防止干燥时的麻醉下。 </li><li>剃手术区，特别是后肢和腹股沟和制备用10％聚维酮 - 碘。 </li><li>使腹股沟皮肤切口用剪刀近侧到大腿中部区域和周向连接切口划定从小鼠身体的其余部分的后肢。 </li><li>识别和解剖股动脉，静脉和神经，并分开使用镊子和微型剪刀三种结构。 </li><li>一旦血管蒂被解剖，在腹股沟韧带的水平夹紧股血管。 </li><li>末节砍船只在腹壁浅动脉水平。 </li><li>接着，继续向近侧在大腿中部水平来划分各个腹侧（纤细和大腿内侧肌肉）和背的肌肉群20以分离天然后立使用剪刀受体动物MB。 </li><li>横切股骨在用剪刀股骨轴的中间。 </li><li>此前烧灼切断大腿肌肉，防止解剖部位出血，从而收件人失血。 </li></ol></li><li> 植入 <ol><li>通过用温盐水（37℃）冲洗手术区域和前压入0.3毫升温盐水和手术后减少流体损失。 </li><li>放置接枝在反映通过对准收件人和移植物的股骨并用20G的脊椎针头作为一个髓内杆连接它们的天然后肢的精确解剖位置的方法。 </li><li> Coapt使用可吸收缝合材料（6-0 Polysorb）腹侧和背侧肌肉群。 </li><li>连接使用非缝合袖套法股血管;详细地说，拉容器的接收者侧超过预先安装在容器的翻边的格拉夫的结束吨。使用10-0尼龙缝合线，并进行圆周领带到收件人容器固定到袖口。 </li><li>接着松开夹子。在这个阶段，直观地验证袖口旋转和最佳位置以防止错旋转和扭曲的船只。 </li><li>使用电烧灼特别注重肌肉收件人捐助接口和骨端上执行细致的止血。 </li><li>关闭使用不可吸收缝线尼龙（6-0 Ethilon）皮肤。 </li><li>通过允许动物在笼子里恢复加热灯下建立常温条件。继续定期监测之前将其返回到所述壳体设施至少4小时。 </li><li>在3天的剂量为0.1mg / kg的SC每6-8小时提供丁丙诺啡术后镇痛。 </li></ol></li></ol>

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The authors declare that they have no competing financial interest.

血管化复合异体移植，如上肢和面部移植毁灭性的组织缺损，已演变为对患者没有可修正传统的重建手术有效的治疗选择。在重建显微外科学领域的技术进步，以及与强效免疫和实体器官移植免疫调节疗法相当丰富的经验，现在可以在这个独特的患者人群3,21-长期移植存活。然而，对于移植的维护和生存需要长期免疫抑制显著的副作用仍然限制了这些提高生活而不是救命重建模式3,22,23的更广泛的应用。此外，VCAS的对比实体器官移植的成功还取决于接受者神经及时再生到同种异体移植物重新支配运动功能两者的肌肉，以及触摸和温度觉感官元件恢复功能。从免疫学的角度来看，在所有移植医学的，在重建移植总体目标是实现业务宽容，允许接受同种异体移植物，而不需要进行长期免疫抑制维持23的状态。在这方面，小鼠已经发展到代表在体内模型系统中的主要在移植免疫学研究以调查在一个完整的免疫系统实现同种异体抗原特异性耐受的途径。此外，小鼠H2复杂酷似人MHC复合物。因此，近交系和基因分型小鼠品系允许通过采用不同程度同种异体抗原来自同基因的失配到一个完全同种异体菌株组合的相关和非相关的生活和尸体供设置临床情景建模。转基因动物和特定基因敲除动物的可用性。此外，所有通过提供选择性激活或细胞或蛋白成分的耗尽的可能性的作用和对免疫接受和拒绝个别分子途径和调节机制的影响的流入调查。这是通过对在体外的小鼠系统唯一地开发诊断和治疗剂（例如，抗体）的广泛可用性平行和体内研究24。总体而言，这些方面使鼠系统的"金标准"的基本移植免疫学研究。 虽然已有了VCA描述的各种小13,16,18,20,25,26和大型动物模型14,27-29最近由Brandacher等人审查。30只有极少数实际上是适用于机械基础免疫学研究16 ，24,30。由于小鼠股动脉和静脉的小血管直径，它需要先进外科和超英里crosurgical培训和技能来成功执行吻合的原位后肢移植的关键组成部分。虽然解剖结构的细致，认真解剖是在这个模型在此前公布的车型20,31,32严格相关的，袖口技术已较亚毫米级船舶16,25和缝合吻合表示不太陡峭的学习曲线在几个月的夫妇培训与严格的日常实践来实现。根据我们的经验，未经训练的microsurgeon将需要30 -50程序试图获取足够的，可靠的技能来完成这个模型具有较高的成功率。对于经验丰富，训练有素的microsurgeon 15 - 30次尝试应该足以掌握这一基于袖带的鼠标后肢移植model.The减少这里介绍的程序的复杂性是由这种方法需要有限的血管长度，从而剥离的事实进一步体现。我们查出服用Ť在腹股沟韧带，他捐助的船只，并在腹壁浅血管水平提供了足够的长度，尽管普遍的观点，即袖口技术需要大量的额外长度适用接收方船只。例如，一种先前公布的方法描述了在髂外容器和受体血管中的腘容器32的水平的水平的必要性收获供体容器。此外，在压脉袋技术，在执行吻合术要求相比，缝合技术更短的时间，并导致总体减少的操作时间。在有经验的医生，捐助国和受援国的程序可在平均90分钟内完成。这与先前报道的方法，其中延长收件人麻醉时间是成功及围手术期生存的关键因素相比，显著改善;制作一个两外科医生的必要性接近的一个先决条件31-33。此外，最小至没有从吻合进一步有助于出血显著减少失血，从而减少了需要的收件人的液体复苏，如先前在本模型33的其它决定成功说明。最后，袖口技术代表具有一个显著的成本优势的方法相比11-0微缝合材料的高费用。因此，非缝合袖套法表示底层协议的最关键的步骤。在东通过一个优雅的研究等，各种不同的肌及骨肌皮瓣，包括腹股沟血管皮瓣已经描述了基于关闭鼠标股骨头血管蒂32。而这里提出的方法只关注整个小鼠后肢移植上，所提出的原理可以很容易地转换和在不同的其它解剖学瓣设计使用。值得注意的是，虽然以前的作者报告automutilation或移植，因为这并没有在我们的经验中观察到手术后的生存31,32期间显著并发症的自噬。此外，恢复快，饲养和仪容的动物的能力有限减值强调，移植腿的原位插图对其造成只有最小的收件人的发病率。 此视频出版物中描述的模型还引入了明显的优点相比，先前公开的技术的鼠标设置。首先，如先前由我们的组16所报告的，袖套法可以与一个管腔直径小于1毫米船只使用，允许一个后肢移植的原位移植。这种方法提供了用于使用该模型结合在两种免疫以及相关的VCA 16,25神经再生的功能研究的基础。神经再生是至关重要给F重建移植ield，因为这些都是非救生程序的成功主要是由功能性和美学缺陷恢复来确定。 一个骨肌皮瓣从后肢始发的异位插图是提供给外科医生15的额外模型。然而，在这种方法中，手术切除潜在兴趣的结构，如钉子和足垫的无毛皮肤的，限制了这种方法的多功能性与解剖学上未改变的后肢移植的原位移植相比。这是在重建移植特别感兴趣，因为这些结构已在患者造成机械应力到其手移植34被描述为非典型排斥过程的目标。天生特有的一种血管复合组织异体移植是骨成分，如血管化骨中含有可行的骨髓，是一个constantlŸ更新组件和捐赠者的免疫系统前体的来源。虽然这种小心视为可能的来源为移植物抗宿主病（GVHD），这尚未在动物模型35,36以及人类37观察到。多，相反实际上，如图临床前36和临床研究38-40，器官移植的与供体骨髓移植的组合或选择的骨髓来源的细胞产品输血实际上显示了关于免疫抑制的量需要21有利效果;一些协议甚至已经显示操作容差而不需要基于药物的免疫抑制41。 该出版物由所示的模型和方法产生了在同系供体100％长期动物存活-收件人组合（图1）以及证实接枝accepta的良好表征的模式NCE和排斥同种异体应变组合如在图2和3所述。此外，在皮肤中的VCA排斥的可见主目标，如下的排斥与人类VCA相关的4个不同的临床情况可重复图案通过所概述的2007年班夫工作分类VCA 42，使得这种模式特别译含皮肤。因此，对于以上的所有重建移植这个高度可靠的小鼠模型中，引入了基因定义自交系和转基因小鼠品系的一个多功能免疫模型系统，该系统将打开的可能性与临床VCA平移影响诊断和介入研究的可用性。 需要之前着手掌握所描述的技术的基本显微技术可以被视为限制到这种模式，但是，它是在相同的主要障碍任何小鼠显微模型。星期四S，此视频协议的主要目的是提供老练microsurgeon 与体内模型的替代方法微血管吻合在鼠为血管复合同种异体移植。此外，该模型允许一个完整的血管的骨髓成分的移植，以及在神经再生研究使用功能模型。 总之，我们建立了一个新的，多功能的，并且可靠小鼠模型原位后肢移植使用打开有关的VCA的任何方面的门基本机理，以及翻译研究的非缝合袖口技术。

The late nineties heralded the pioneering days of reconstructive transplantation with the first successful hand transplant performed in France in 1998. Since then, the use of VCAs for reconstruction of devastating tissue defects has been successfully employed in a wide spectrum of patients. To date, the world counts 76 recipients of 112 upper extremities as well as 31 faces 1-3. In addition, several other types of VCAs such as abdominal wall 4, larynx 5, trachea 6, vascularized joints 7, and even penis 8 have been performed. Furthermore, the live birth of a baby was recently reported after uterus transplantation 9. This growing world experience is indicative for how reconstructive transplantation has become a valid therapeutic option for patients suffering of significant functional tissue defects not amendable to conventional reconstructive and restorative surgery and treatment. 
While the idea of replacing "like with like" sparked clinical enthusiasm, initial skepticism still prevails with regards to side effects of conventional high-dose immunosuppression required to maintain allografts and their function 10,11. However, as shown by seminal work of Lee et al., these composite grafts are less likely to reject than its individual components, and furthermore, some of the tissue components such as the vascularized bone compartment have fueled optimism as they might exert unique immunological effects onto the balance of immune acceptance and rejection 12. 
Our group pioneered several microsurgical animal models for solid organ transplantation, as well as vascularized composite allotransplantation 13-19. Here we describe a novel surgical procedure using a non-suture cuff technique to perform super micro-vascular anastomosis in an orthotopic mouse hind limb transplantation model. This transplant model provides a useful tool for investigating immune acceptance and rejection mechanisms, as well as the role of individual tissue components, such as the vascularized bone marrow compartment, towards tolerance induction in the immunologically versatile setting of the mouse species. Additionally, the orthotopic placement of the limb opens the possibilities for nerve regeneration and functional outcome studies, which are critically important to the setting of VCA.

<table>
	<tbody>
		<tr>
			<td>Suture, 6-0 Nylon</td>
			<td>MWI</td>
			<td>31849</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 6-0 Polysorb</td>
			<td>MWI</td>
			<td>72667</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 10-0 Nylon</td>
			<td>Aero Surgical</td>
			<td>TK-107038</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 25</td>
			<td>Vention Medical</td>
			<td>141-0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 27</td>
			<td>Vention Medical</td>
			<td>141-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Single)</td>
			<td>Synovis</td>
			<td>00396</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Double)</td>
			<td>Synovis</td>
			<td>00414</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Scissors</td>
			<td>Synovis</td>
			<td>SAS-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Forceps</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Dilators</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8d.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Needledriver</td>
			<td>Synovis</td>
			<td>C-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Clamp Applicator</td>
			<td>Synovis</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Flushing Needle</td>
			<td>Hamilton</td>
			<td>N/A</td>
			<td>10MM, 30&#176;, 33G</td>
		</tr>
		<tr>
			<td>Lactated Ringers Solution</td>
			<td>Fisher Scientific</td>
			<td>NC9968051</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DEA Number required; Obtained from hosptial pharmacy.</td>
		</tr>
		<tr>
			<td>Enrofloxacin; Baytril</td>
			<td>Bayer Health Care</td>
			<td>186599</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Obtained from hosptial pharmacy</td>
		</tr>
	</tbody>
</table>

orthotopic hind limb transplantation in the mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

使用非缝合袖套法在小鼠模型中进行血管复合同种异体移植允许以获得优良的和长期的移植物和动物存活，如图1。此外，它表示在血管复合获得逐渐同种异体移植物排斥的可再现的结果的可靠方法同种异体移植如由图2所示的图像记录。从动物进行排斥进一步强调同种异体移植物排斥在这个小鼠模型中的可再现动力学（图3）获得的组织的H＆E组织学。 <img alt="图1" src="/files/ftp_upload/53483/53483fig1.jpg" /> 图1.移植存活在完全H2相合小鼠品系组合[只Balb / c（H2K D）到C57BL6（H2K B）。虽然所有的同源transpl蚂蚁（N = 10）被接受长期的，未经处理的移植（N = 10）中敏锐地拒绝和平均8 - 9天。根据班夫标准皮肤排斥3级被认为在本研究中完全抑制。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53483/53483fig2.jpg" /> 图2.临床特点拒绝和完全H2-不匹配[的Balb / c（H2K D）到C57BL6（H2K B）]鼠原位后肢移植的动态变化。（ 一）临床 0级，（B）临床1级，（ C）有costimul治疗临床2级，（D）临床3级，和（E）临床4级排斥反应，（F）长期存活的移植（POD 100）通货膨胀封锁（antiCD40单抗+的CTLA4Ig）的方案。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53483/53483fig3.jpg" /> 图3.术后第7天，以及皮肤和肌肉在同基因移植（A）和异基因移植（B）的H＆E染色脚垫皮肤和肌肉在同基因移植（C）和异基因移植术后第7天的H＆E染色（D ）（比例尺:0.1微米） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Orthotopic Hind Limb Transplantation in the Mouse at JoVE.com

Orthotopic Hind Limb Transplantation in the Mouse

这种新颖的模型在小鼠原位后肢移植，申请超微血管吻合的非缝合袖套法，提供了相关的血管化复合同种异体移植（VCA） 体内机械免疫学研究的有力工具。

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

orthotopic-hind-limb-transplantation-in-the-mouse

Nanyang Technological University

Research

JoVE Journal

Medicine

11.6K 次观看.  Johns Hopkins University School of Medicine. 这种新颖的模型在小鼠原位后肢移植，申请超微血管吻合的非缝合袖套法，提供了相关的血管化复合同种异体移植（VCA） 体内机械免疫学研究的有力工具。 

视频: Orthotopic Hind Limb Transplantation in the Mouse

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

大鼠原位小肠移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

大鼠原位肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

平移血管复合同种异体移植（VCA）研究一种改进的异位猪后肢移植模型

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植:移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

正交心脏移植的临床前猪模型

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...