我们要感谢他们对这个项目的贡献的下列人员：Kakali萨卡，博士，Joani克里斯滕森，英国BS，凯特Buretta，英国BS，袁力拔头筹，BS，威廉乐豪，医学博士，约翰娜Grahammer，乔治Furtmüller，医学博士，艾琳拉达医学博士穆罕默德·罗汉医师，卡里姆Sarhane医师，萨米Khalifian，英国BS，毛奇，MD和安杰洛勒托巴罗博士，VCA实验室，整形外科，美国约翰霍普金斯大学医学院，詹尼斯陶布，医学博士，马克·菲舍尔，医学博士，皮肤科及病理，美国约翰霍普金斯大学医学院，苏埃勒，微创外科培训中心，约翰霍普金斯大学医学院和程鸿霖医师长庚纪念医院林口，台湾的部门。 资金来源：再生医学的武装部队学院（国防部W81XWH-08-2-0032）

在这个视频发布，所有动物的程序按照批准的约翰·霍普金斯大学实验动物管理和使用委员会（IACUC）动物的协议进行。 1。术前规划<ol><li>我们利用MHC-MGH定义在我们的翻译研究近交系小型猪（15-20公斤）为异体肢体移植，其中一名男性捐赠者提供了四肢两女受助人（ 图1）。手术前，供体和受体动物由兽医进行全面的临床评估。 </li><li>为了尽量减少麻醉和缺血时间，供体和受体的程序是同时出现在三手术台在一个专用的大动物手术室进行。手术团队以及兽医工作人员之间的团队合作和协调是这个程序的整体成功的关键。手术方案与兽医团队在正式会议上讨论1-2周手术的前一天。 </li><li>隧道式中心静脉导管（7神父双腔导管希克曼）被放置在每个收件人动物的外周或颈内静脉移植前的免疫抑制药浓度的监测和免疫和/或免疫调节药物的静脉注射。这些导管放置在直视下通过静脉切下来的技术和隧道后退出高，对背脖子，并使用不可吸收的单丝缝合线和绕颈粘性绷带缠绕固定。 </li><li>饲养动物的NPO（没有固体食物，但被允许水即兴）至少12小时手术前。 </li><li>在手术当天，镇静动物用氯胺酮20 mg / kg和甲苯噻嗪2毫克/公斤的IM。 </li><li>将适当大小的气管插管，并将其连接到麻醉机和呼吸机（保持在异氟醚0.5-3％） </li><li>放置静脉留置针在耳缘静脉和管理重量的0.9％的生理盐水整个过程。 </li><li>管理先发制人镇痛（丁丙诺啡0.02毫克/千克四）。 </li><li>之前的初始切口捐助国和受援国动物管理预防性抗生素（头孢唑啉20-25毫克/千克四）。 </li><li>使用无菌技术在为受援国和捐助国的所有程序。这包括使用高压消毒或气体消毒器械，口罩，头帽，无菌手术衣和无菌手套。剃切口的区域与剪刀和消毒具有交替的聚维酮碘溶液和酒精。 </li></ol>术中监测： <ol><li>诱导和气管插管后，麻醉维持用氧气吸入混合0.5-3％异氟醚。 </li><li>监测心脏，脉搏血氧饱和度，心电图，呼气末二氧化碳不断直肠温度，并记录每15分钟一班。 </li><li>防止低温与拜尔杂乱暖强制空气毯。 </li></ol><ol><li>将供体动物在手术台上仰卧位，悬垂在无菌的方式。 </li><li>利用近红外激光血管造影，以确定穿支血管的前内侧皮肤。注射吲哚菁绿在约0.06毫克/公斤至末梢血管通路稀释于2-5毫升的生理盐水。大约10秒后，进行近红外录音30秒-2分钟。使用视频从这些记录，以确定穿孔。基于打孔器，标志着皮肤（大约100 平方厘米）的区域进行监控。 </li><li>用手术刀做一个腹股沟皮肤切口在连续性标记为皮肤桨（ 图2）。 </li><li>确定股骨血管和远侧分离它们的suprageniculate分支的水平和近侧至腹股沟韧带的水平。 </li><li>剖析皮肤和皮下组织，同时preservin克皮肤桨的面积。缝合皮肤的边缘，下面的肌肉，避免皮瓣收获期间收缩。 </li><li>执行截骨术只是用一个往复锯（电动随身碟，Synthes公司，西切斯特宾夕法尼亚州）睑板上方。 </li><li>划分的肌肉大腿近端使用电。 </li><li>划分股骨在中间轴的血管蒂留下骨肌皮瓣。皮瓣含有皮肤桨，底层大腿肌肉，膝关节，股骨远端和胫骨近端，其中包括完整的血管骨髓以其独特的免疫功能。 </li><li>用骨蜡，以达到止血的骨髓空间。光滑的骨边缘使用ronjeurs在准备收件人。 </li><li>保持灌注其血管蒂，直到收件人的股血管准备（ 图3）移植。 </li><li>一旦收件人剥离完成后，结扎捐助股血管的近端尽可能一ND注入移植物与组氨酸 - 色氨酸 - 酮戊二酸（HTK，司托，现代城PA）移植前。 注：供体肢移植的接受者，即左施主肢为右侧的接受者和供体右肢的收件人的左侧）的对侧。 </li><li>用100毫克/千克静脉戊巴比妥钠收获第二后肢后立即安乐死捐助猪。死亡是由每设立协议的兽医人员证实。 </li></ol> 4。收件人程序（雌鹿异体肢体移植） <ol><li>最初的捐赠者切口后立即启动收件人诱导和气管插管，让准备接受血管，而捐赠者的肢体正在收获。这消除了不必要的等待时间，并降低了总麻醉时间为捐赠者和接受者。 </li><li>用30°旋转操作侧到异体定位动物仰卧背外侧侧w的风险。 </li><li>使腹股沟切口并暴露股血管;远侧分离它们的suprageniculate分支的水平和近侧至腹股沟韧带的水平。 </li><li>从腹股沟建立皮下囊袋的背外侧腹壁的接枝插块（ 图4）。 </li><li>结扎供体血管和冲洗移植与HTK。 </li><li>在一个倒置的方式进行移植插图：有皮桨面对背外侧，以方便免疫监视在站立姿势，面向尾端，让最终到终端的血管吻合而对船舶的任何张力（股血管的断端图5）。 </li><li>如果可以，静脉吻合用血管耦合装置（SYNOVIS，圣保罗MN）（大小2.5-3.5适用于10-20公斤的动物）。当与常规方法相比本显著降低缺血时间。 </li><li>进行动脉anast使用传统的缝合技术用9-0缝线（SYNOVIS，圣保罗明尼苏达州）omoses </li><li>后血管通畅确认，确保移植的地方用3-0 PDS缝合线（ 图5）。移植物的肌肉被缝合到腹壁的肌肉。移植的肌肉不应该被缝合到收件人大腿肌肉，因为它可以是有害的，当在笼子里的动物移动。 </li><li> Exteriorize皮肤桨上的背外侧边为免疫监视（ 图6），并将其缝合用3-0薇乔皮下缝合线和4-0单丝可吸收间断皮肤缝合线的相邻的皮肤。 </li><li>关闭用3-0薇乔缝合皮下和4-0单丝可吸收皮肤间断缝合腹股沟皮肤层。 </li><li>放置一个50-100微克/小时芬太尼贴剂用于术后镇痛。 </li><li>后肢异体移植后，将动物的钢笔一旦清醒，对自己的呼吸舒适。</li>
</ol>

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作者宣称，他们有没有竞争的财务权益。

从历史上看，异位后肢移植协议包括一个皮肤桨向腹侧腹壁的外化和船只吻合的端 - 侧的方式（2004 Hettiarachty）。然而，在我们修改的方法，一个倒皮瓣insetting和终端到终端的吻合带给肌肤更桨侧向，从而促进免疫监测动物的站立姿势。腓肠动脉的近红外激光血管造影进一步的鉴定和最大灌注区改善皮肤桨的可靠性。 在我们的改装技术，进行端 - 端吻合股血管之间，以减少相...

血管化复合同种异体移植（VCA），如手和脸移植手术是目前临床的现实与世界各地12进行了大量的手和脸移植手术。尽管早期和中间结果是有利的，高度鼓励2，慢性免疫抑制多药的要求继续限制了它的广泛临床应用。在VCA的小鼠模型，包括超显微吻合和nonsuture袖口13技术的进步，3铺平了道路，以更好地了解在VCA同种免疫反应。免疫调节无数的协议已经提出了基于我们更好地了解VCA免疫机制的临床应用，但他们需要在一个大动物模型，这将是合理预测其在人体中7的性能进行验证。基于人类和猪的器官系统之间的生理和免疫学的相似<sup&gt; 6，猪VCA模型可以被认为是可靠和具有成本效益的替代品，以9犬和非人灵长类动物模型1。 本文提供了在我们的MHC定义的猪异位后肢移植模型，它作为基础，为我们的目的是诱导免疫耐受的VCA，从而扩大其临床应用当前和未来的免疫策略所使用的方法的详细介绍。我们利用育成纯合性在猪白细胞抗原位点专门为他们在移植相关研究11利用良好的特点近交系猪。我们提出基于股血管血管化骨肌皮瓣。皮瓣包含完整的血管骨髓在股骨远端和胫骨近端。前内侧大腿皮肤也被包括在接枝和被形象化到受体动物的免疫监视的最免疫原性组分邻背外侧方面F中的VCA。背外侧的定位有利于临床检查在站立和坐姿，并保持移植皮肤比较干净。 Ustener 等人介绍了VCA的第一个大型动物平移模型通过移植前肢桡骨肌皮瓣远交农场的猪只15之一。该小组利用这个模型来演示首次急性排斥反应的VCA其中包括高度免疫原性的皮肤组件可以被延迟，并与临床相关的治疗策略无显著药物特有的并发症和副作用。在这项研究中获得有利的结果随后内置在设计药物疗法为人类移植重建一个基本步骤。虽然这些早期的猪VCA模型是非常适合开发的协议，以防止排斥反应的皮肤，肌肉，骨骼，神经和它们缺乏专门的结构，例如关节cartilag船只e和关节滑膜。后续工作的重点包括数字内侧的动物，因此有必要全长铸安置，以防止移植物移位14。虽然适当的调查拒绝肢体移植的所有主要组件，这种模式的主要限制之一是由于铸造安置移植后卧床困难。因此，异位猪异体肢体移植模型，包括胫骨，腓骨，膝关节，股骨下端，周围的肌肉和皮肤桨，创建了到主要研究VCA的免疫学方面的同时，允许动物自由术后用最少morbidit 8走动。 的良好表征的SLA定义的近交系猪通过戴维H萨克斯博士的开创性工作的发展带动了平移VCA研究的新时代。利用异位后肢移植模型中一个小的抗原不匹配的设置，垫HES 等 10证实肌肉骨骼成分的无限期生存与环孢素治疗的短期课程。皮肤组件生存，但是，只有当相比，没有任何治疗的控制延长。移植物的皮肤成分的损失归因于一种分离和高度有力的免疫应答，特别是在表皮。同样，使用完全不匹配的猪T细胞耗竭，环孢素和诱导耐受只有肌肉骨骼成分与皮肤成分的细胞因子动员的供者外周血单核细胞中的短期课程仍然拒绝5。这种现象，被称为“分裂宽容”，带来了VCA研究模式的转变与更加注重高免疫原性的皮肤组件，这也是广大迄今为止已进行重建移植的一个组成部分上。 在这个修改后的模型，我们利用端 - 端吻合者ligatin克收件人股动脉并旋转它头侧（ 图1）。这不仅降低了局部缺血时通过允许使用常规的连接装置，而且还降低吻合失败的几率。我们并没有观察到以下的股动脉在我们的收件人表明侧支循环是足以提供血管原生腿结扎任何缺血事件。另外，在该改性方法中，外在皮肤组件是基于底层穿支血管动员和被横向定位（ 图1）相反，在传统的模型10腹侧腹股沟位置。这使得移植物方便的可视化免疫监视在站立或动物的坐姿。 因此，可靠的和可重现的大型动物模型是必不可少的探讨耐受诱导策略对VCA的皮肤组分和开发新型Noninvasive免疫监视策略，更好地预测移植物存活的。

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			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>HTK</td>
			<td>Custodial</td>
			<td>N/A</td>
			<td></td>
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		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Electric Pen Drive</td>
			<td>Synthes, Westchester PA </td>
			<td>05.001.011 </td>
			<td>Reciprocating saw</td>
		 </tr>
		 <tr>
			<td>Vascular Coupling device</td>
			<td>Synovis, Newtown PA</td>
			<td>21003B</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (vca) research

血管化复合同种异体移植（VCA），如手和脸移植手术代表了复杂的骨骼肌肉创伤和破坏性的组织损失一个可行的治疗选择。尽管有利和非常令人鼓舞的初，中级实用成果，甩一个VCA的高免疫原性的皮肤组件和慢性多药免疫抑制的潜在不利影响的继续妨碍VCA的广泛临床应用。因此，研究这本小说领域需要关注与VCA独特的免疫功能翻译研究和制定免疫调节和免疫耐受诱导下的VCA，而不需要长期免疫抑制的新型免疫调节策略。 本文介绍一个可靠和可重复的平移大型动物VCA的是被包括在MHC定义的猪后腿异位异体肢体移植的骨肌皮瓣模型。 BRIEFLY，良好的血管化皮桨确定了利用近红外激光血管造影大腿前内侧区。底层肌肉，膝关节，股骨远端和胫骨近端的收获上的股骨血管蒂。这种移植可以认为既是一个VCA和血管骨髓移植以其独特的免疫特权功能。移植物移植到腹部皮下口袋受体动物用形象化的背外侧区的免疫监测皮肤组件。 三种手术团队在充分协调的方式同时工作，以减少麻醉和缺血时间，从而提高了这种模式的效率，并减少实验协议潜在的混杂因素。该模型作为基础，为旨在减少和可能省去在VCA慢性多药物的免疫抑制将来的治疗策略。

二十四SLA定义的猪异位后肢移植是用我们的技术改良与78分钟（：62-94分钟范围），平均缺血时间进行。移植物插入和背外侧皮肤桨的定位是毫无困难地在所有动物中实现。近红外激光血管造影显示出优异的移植灌注在所有收件人。最初的12静脉吻合均采用传统的缝合技术进行，而最后12静脉吻合均使用血管耦合装置进行。一个动物与传统缝合技术需要再吻合时，静脉血栓后立即过程识别。无并发?...

Watch this Scientific Journal Video about A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research at JoVE.com

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

血管化复合Allotransplantations（VCA）已成为临床现实。然而，VCA的广泛临床应用是由慢性多药免疫抑制的限制。作者提出一个可靠和可重复的大动物模型来翻译新型免疫调节策略，可以最大限度地减少或消除潜在的免疫抑制的VCA的需要。

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 ...

a-modified-heterotopic-swine-hind-limb-transplant-model-for

Research

JoVE Journal

Medicine

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

12.4K 次观看.  Johns Hopkins University School of Medicine. 血管化复合Allotransplantations（VCA）已成为临床现实。然而，VCA的广泛临床应用是由慢性多药免疫抑制的限制。作者提出一个可靠和可重复的大动物模型来翻译新型免疫调节策略，可以最大限度地减少或消除潜在的免疫抑制的VCA的需要。 

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

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

对于异位小鼠心脏移植技术的一种改进方法

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

科研

医学

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

小鼠异位心脏移植技术

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

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 ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

应用改良袖套技术的小鼠胰腺移植模型

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

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 ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

血管化异位脾移植小鼠模型在脾细胞生物学和移植免疫研究中的应用

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

采取下一步：神经整形正交后肢移植模型，以最大限度地提高大鼠的功能恢复

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...