Name: a novel microsurgical model for heterotopic, en bloc chest wall, thymus, and heart transplantation in mice
Uploaded: 2016-01-23T14:00:00.000+00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice at JoVE.com

This work was funded by the American Association of Plastic Surgeons 2014 Academic Scholar Award.

所有的手术过程均符合美国约翰霍普金斯大学和农业部的美国农业部和公共卫生服务的要求完成。该协议遵循美国约翰霍普金斯大学动物护理和使用委员会，机构审查委员会批准的准则（协议号M013M490）。最终存活数据被记录为下面描述的外科手术。供体和受体动物接收使用丁丙诺啡先发制人麻醉以0.1mg /千克SC手术前和在受体动物丁丙诺啡之一小时重新施用同样剂量移植后，并根据需要在最初48小时内重新给药手术后。 1.供体移植恢复注意:开始移植40分钟的捐助部分早于接受者的移植，以尽量减少受援国的麻醉时间，便于同时结束时间或略有耳利尔结束时间与收件人的准备。 <ol><li>使用标准的无菌显微器械和无菌手套的程序。我们的实验室使用显微仪器高压灭菌。 </li><li>麻醉用异氟醚感应蒸发器在4％的供体小鼠（男）。使用无创伤机械剪除去从颈椎，胸椎头发，腹部区域。将动物在仰卧位，并通过鼻锥保持异氟烷上1-2％。通过定期评估脚趾捏撤回反射确保整个过程充分麻醉。 </li><li>在此之前的皮肤切口，广泛应用聚维酮碘消毒剂其次是异丙醇用消毒棉签准备工作。 </li><li>开始时先用浅横皮肤切口与整个颈部和腹部的皮肤剪刀。连接两个切口双边沿腋线。 </li><li>采用显微镊解剖颈部两侧识别，结扎和划分颈外静脉用6-0丝线缝合和剪刀。然后使用电划分胸锁乳突肌，露出颈内静脉和颈总动脉，双侧。通过在左路和右路颈总动脉和颈内静脉散装时尚6-0丝线缝合。 注:它们将被捆绑，后来在步骤1.9分。 </li><li>急剧划分带状肌和相关疏松蜂窝组织，位于前部的气管，用剪刀释放颈椎区的其余附件。 </li><li>用双极电和锐性剥离，分胸大肌肌肉和锁骨露出锁骨下血管和结扎（6-0丝线缝合），并划分近端。 </li><li>接下来，轻轻的，把握和撤回动物的阴茎。沿阴茎的背形象化阴茎的背静脉，和消毒区域用异丙醇。使用地下30针，注入30000单位肝素静脉内通过背静脉并允许阴茎反冲回到其原来的位置。可能会发生局部漏肝素溶液到周围组织的。 </li><li>使用周围颈总动脉和颈内静脉的前面放置散装的联系，结扎和划分结构，两侧。 </li><li>接下来用剪刀作一横切口intrabdominal。剔骨肠内以暴露肝下下腔静脉，并注入2毫升冷欧洲柯林斯心麻痹液进入肝下下腔静脉。确保正确注射肝变色和停止心跳的推进到下一步骤之前，可视化。 注:欧洲Collins液是在我们的实验室制备，见表特定的试剂和仪器。 </li><li>用剪刀通过双边DIAPH进入胸腔内空腔ragmatic切口从露出腹部。延伸穿过肋间肌肉和肋头侧的切口。反映胸壁暴露心脏，胸腺和大血管的同时，确保保护沿胸壁胸廓内血管。 </li><li>注入肝上下腔静脉用4ml冷欧洲柯林斯心麻痹液。 </li><li>识别主动脉根部和远侧跟踪到降主动脉。大幅削减降主动脉（保留最大长度）。 </li><li>确定肺动脉主干及分刚好接近其分支点（保留最大长度）。然后，使用2毫升冷欧元柯林斯停搏液，通过放置一个软塑料尖导管进入肺动脉的管腔冲洗肺动脉干和心脏。 </li><li>用6-0丝线缝合，结扎和划分下腔静脉，肺静脉汇合，和双侧上腔静脉辅助分支。然后提升并沿主干支气管和气管小心不要进入呼吸道附件解剖心脏头侧。使用锋利的和双极电解剖胸壁，胸腺和心脏完全从供体小鼠释放它。 </li><li>最后，修整同种异体移植胸壁体外到更小的尺寸，用剪刀沿胸骨和横向肋脉，小心不要破坏胸廓血管（ 图1A）。为了下面的血运重建尽量减少出血，使用沿着osteomusculocutaneous胸骨边界双极电灼。 </li><li>将在10毫升冷（4度摄氏度）的移植欧洲-科林斯的解决方案，如果收款人没有为插入做好准备。然而，如果接收方是准备插页，同种异体移植物转移直到接收者手术视野。 </li></ol> 2.收件人准备注意:要尽量减少收件人麻醉时间，在一个单独的操作站开始接收方准备前的供体移植收获完成约40分钟。 <ol><li>使用单独的一套标准的无菌显微仪器和程序无菌手套。 </li><li>麻醉用异氟醚感应蒸发器在4％的受体小鼠（男或女）。使用无创机械快船移除右颈部和胸部的头发。 </li><li>鼠标放置在仰卧位置和角度的右上肢稍稍下方形成的头部和右上肢之间的110度角。通过鼻锥维持麻醉上1-2％异氟醚。 </li><li>放置石油眼药膏上使用棉签鼠标眼睛。在此之前的皮肤切口，用聚维酮碘防腐剂接着异丙醇广泛准备手术部位。 </li><li>用剪刀，做一个皮肤切口沿右下博尔中线下颌骨DER和infero，横向延伸的切口右胸部。使用钝性分离微血管钳，调动了颈外静脉被周向无血管的软组织和外膜。除以使用电各分公司，并删除使用锐性剥离和电灼对移植的自由空间颌下腺右叶。 </li><li>确保足够的长度的颈外静脉的外翻在一个压脉袋，并结扎外颈静脉用6-0丝线缝合。通过预切聚酰亚胺箍的内腔插入静脉并使用牛头犬微血管钳固定到位容器袖口复杂。然后用剪刀，近侧划分外部颈静脉，外翻在袖口，以及在适当位置10-0尼龙缝合线固定。 （图1B） </li><li>划分右胸锁乳突肌双极电以暴露颈总动脉。周向暴徒ilize颈部区域内头侧向最远侧点的动脉。这是使用钳船只钝性分离，除去软组织和周围的外膜完成。 </li><li>用6-0丝线缝合，结扎并划分颈总动脉。通过预切割的聚酰亚胺袖口的管腔通过动脉和其固定用的牛头犬微血管钳尽量靠近胸部入口越好。除以血管远端，轻轻地扩张用显微扩张血管，埃弗特在袖口，并在适当位置10-0尼龙缝合修复。 （图1B） 注:具体显微扩张器在特定的试剂和仪器表所述。 </li></ol> 3.移植插图<ol><li>保持无菌的标准仪器和无菌手套把收件人颈部区域内的移植在倒置和倾斜位置。 </li><li>下一步，将捐助下降AOR抽动管腔在收件人的动脉袖套构建体和其固定用10-0尼龙缝合线（图1C 和1D）。 </li><li>时尚相同吻合作为供体肺动脉和受体小鼠（图1C 和1D）的外翻颈外静脉袖套构建体之间步骤3.2。 </li><li>首先拆下静脉微血管钳（颈外静脉夹），然后松开动脉夹（颈总动脉夹）。在动脉灌注，检查移植解决任何出血的全部内容。如果出血可视化，重新动脉夹，以减少出血量及减少使用双极电出血的来源。 </li><li>检查移植物，并确保止血。释放并彻底清除动脉微血管夹。观察心脏表现出再灌注的迹象，这将是快速扩容的心脏室立即显现出来，并等待对B吃开始在0.5-1分钟。用温盐水（35摄氏度），以滋润的心脏。 </li><li>悬垂胸壁到解剖位置，以免诱发的吻合任何的扭曲和紧张局势。关闭手术伤口的是用6-0尼龙连续缝线（图1E）的皮肤。 </li></ol> 4.术后护理<ol><li>辖0.3 ml生理盐水腹腔内液体推注，术后立即进行补液。 </li><li>然后经皮下疼痛和感染预防，分别注入丁丙诺啡（0.1mg的/ kg）和恩诺沙星（5毫克/公斤）。 </li><li>放置在热灯下的动物直到从麻醉觉醒，并返回到胸骨斜卧。在恢复过程中，检查颈部以可视化的移植保证足够的移植物灌注的原纤化的心跳。 </li><li>一旦醒来，在卧位，返回鼠标到一个单独的笼子（无其他老鼠的公司）在那里可以领取食物和水自由采食。由于右上肢任何临时次要限制性运动，留在笼子的地板上的明胶的食物来源。 </li><li>观察受体小鼠1小时，术后，然后将其返回到笼子设备，在它能够接收的食物和水随意并进行检查，一天三次的第一个24小时的活性和营养摄取。根据需要为先72小时全天监控小鼠的痛苦和困扰，并重新剂量丁丙诺啡（0.1毫克/千克）的迹象皮下的两倍。检查动物此后每天，每星期体重它们。 </li><li>与兽医工作人员咨询，如果任何小鼠显示的痛苦，痛苦的迹象，或采食量下降。考虑早期安乐死（在我们的协议安乐死技术采用的 CO 2过量为7分钟，接着通过颈椎脱位）。 </li><li>同种异体移植心脏跳动的停止被定义为一个特定端点促使鼠标为sacrificed。 </li></ol>

The authors do not have any conflicts of interest or financial disclosures to declare.

有众多的现象因入同种异体移植，其中包括的免疫学调查，但不限于:急性和慢性排斥反应，直接和间接的抗原呈递，收件人敏化，或混合嵌合体诱导的机制。19动物模型已成为的黄金标准移植免疫学的研究中，和小鼠模型被普遍，由于其成本低，转基因和基因敲除小鼠的可用性，市售的单克隆抗体，相对降低兽医和外壳的要求，并易于复制的实现。迄今为止，多种心脏移植模型已经用于研究固体器官移植。19,22-27同样，过多的小鼠模型已被用于研究血管复合同种异体移植28然而，结合实体器官和血管的复合同种异体移植的研究是有限的，和技术还没有建立在小鼠中。整块胸壁，胸腺，和心脏移植的小鼠这里提出的模型是研究的影响，结合实体器官和血管复合同种异体移植的免疫学机制的可靠和可复制的工具。 为了进一步推进移植领域，通过新的治疗方式延长移植物存活和尽量减少免疫抑制的承诺，必须继续实施。一种这样的方法是通过混合嵌合体（供体造血细胞在受体的局部植入），这可能会导致免疫抑制自由供者特异性耐受的诱导，即使在某些情况下，嵌合体不能持续。29,30骨髓输血/移植搭配实体器官31或血管的复合allotransplanation 32,33需要大量的预处理构成一个显著的挑战。血管的骨髓中，血管化复合移植构建第一部分，可以解决这个问题。血管骨髓移植允许供体骨髓细胞的供体保留微环境中的不间断传送，并且被认为是优于单细胞的骨髓移植耐受性和减少的免疫抑制的要求的诱导。34-36此外，掺入与血管骨髓移植胸腺组织已证明能增加供体来源的T细胞嵌合体，最终打在诱导和维持嵌合体的支撑作用。延长移植物存活和尽量减少免疫抑制的基础概念化的2,9上述策略组合实体器官，胸腺和血管复合同种异体移植小鼠模型。 异位整块胸壁，胸腺和心脏移植是多个的合并其torical动物模型。用小鼠非袖口缝合颈部异位心脏移植已经确立，并认为比异位腹部微血管心脏移植少技术要求高。19事实上，袖口技术已在多个其他动物移植模型中实现37-44异位在大鼠胸骨移植于1999年推出由圣地亚哥等人的研究血管骨髓移植的替代方法。1他们能够表现出长期的外围嵌合体，耐受性和生存以下免疫抑制术后当天停止30. 1 Bozkurt的等人随后开发了一个大鼠模型于2013年合并胸腺和胸壁的骨肌部的充分程度。应当指出，然而，这种模式不同于我们的在多个方面的模型。这包括:（1）他们的模型是没有任何固体器官，（2），使用传统的显微外科技术正在完成大鼠，（3）供体收获期间的胸廓血管结扎，（4）通过一个共同的颈动脉和外颈静脉，和执行一个单方面的，单椎弓根（5 ）移植的同种异体移植到腹股沟区域。2尽管如此，他们的模型是能够证明与单独血管的骨髓移植相比，捐助者出身的胸腺起着不仅为嵌合体隆胸也为嵌合维护显著的作用。2共移植的胸腺和心脏的后续猪模型已经显示在心脏同种异体移植物存活的优良效果。每个动物模型10,11的优点，但缺乏机械体内研究关于结合实体器官，胸腺和血管复合同种异体移植促使我们组设计这个模型。 在经历前ecuting这种新颖的模式表现出一定的经验教训，要求我们的团队，制定修改，以达到更好的动物的生存。这种模式试图与一家运营商，最终延长了手术和麻醉时间超过3-4小时，还延长冷缺血时间。动物不会醒来之后终止程序。的两个团队的方式实施削减总的手术和麻醉时间90分钟。这反映在受体小鼠的60分钟麻醉时间，和0-10分钟同种异体冷缺血时间。期间同种异体移植灌注，鼠标易受出血，这可能限制其在不久的围手术期的生存能力。我们提倡细致的检查移植体外潜在的出血来源，以及斗牛犬微血管钳移植再灌注过程中温和释放。通过将同种异体移植物中的反射位置是更容易识别的特定网站出血。此外，这种接枝插画位置促进血管的最符合人体工学的捻最小化容器扭结的风险。最后，与第一个48小时的恢复，运动的受体小鼠的右上肢范围可能阻碍与问候攀爬笼子，以获得食物和水。因此，我们建议明胶营养源的位置沿笼，方便营养的摄入。通常由3术后第二天，是右上肢内恢复全套动作。 虽然有一些限制这种模式，其中包括需要显微外科技术娴熟，两个同时显微镜可用性和两个团队的方式的要求，它仍然证明是一个成功的方法来执行相关的机理免疫学研究结合实体器官和血管复合同种异体移植。其更广泛的应用程序可能会进一步有助于开发新的与免疫osuppressive协议，急性和慢性排斥反应研究力学，和实施潜力的策略的诱导和维持嵌合，并延长同种异体移植物存活。

Cardiac transplantation is the treatment of choice for end-stage heart failure. Both technical advancements and pharmacological innovations have propelled the field to early graft acceptance rates above 90%.12,13 Despite this, 60-80% 5-year graft survival is at a standstill and chronic rejection, characterized by transplant vasculopathy, remains inevitable.14-16 Furthermore, patients are subjected to multiple surgical procedures and lifelong immunosuppression, which are associated with chest wall deformities and medical sequelae and toxicities, respectively. The need for innovative approaches to extend allograft survival, minimize the immunosuppressive requirements, and offer reconstructive options for anatomical deformities is pressing.
Vascularized composite allotransplantation offers a unique strategy for improving heart transplant outcomes both from an immunological aspect as well as a reconstructive perspective.17 Vascularized composite allografts are also unique in a way that they have an inherent source of donor-derived hematopoietic stem cells which has shown a favorable ability to reduce immunosuppression and induce and sustain mixed chimerism.1-8 Additionally, co-transplantation of the thymus has shown to prolong survival of both, solid organ transplants and vascularized composite allografts.2,9-11 Combining these strategies with heart transplantation offers a novel solution to the aforementioned challenges facing heart transplantation.18
Murine models serve as excellent platforms for mechanistic in vivo investigation because of the availability of antibodies and well-defined inbred and knockout strains.19-21 Although heart transplantation in mice is commonly studied using a heterotopic intraabdominal microsurgical suture transplant model22-25, a heterotopic, cervical, non-suture cuff technique model has shown to be extremely replicable, reliable, and carries fewer rates of thrombosis.19,26,27 The goal of this study is to develop a heterotopic en bloc osteomyocutaneous chest wall, thymus, and heart transplant technique in mice to study the immunological mechanisms of combined solid organ and vascularized composite allotransplantation using a cervical non-suture cuff technique. This cluster allograft is perfused through the anastomosis of the donor descending aorta to the right common carotid artery and the donor pulmonary artery to the right external jugular vein. Preservation of the internal thoracic vessels and associated thymus branches is paramount to perfusing the chest wall (sternum, ribs, muscles, and skin) and thymus.

<table><tbody><tr><td>Euro-Collins Solution</td><td></td><td></td><td>The solution is not commercially purchased but rather prepared in the laboratory. To make a 500 ml solution add the ingredient listed below to a 330 ml of double distilled water. Mix well, and then fill in the rest of the 170 ml of double distilled water into the solution to a final volume of 500 ml.&lt;br/&gt; Ingredients: 1.02 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 3.66 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.56 g KCl, 0.42 g NaHCO&lt;sub&gt;3&lt;/sub&gt;, and 17.52 g of glucose.</td></tr><tr><td>Suture</td><td>Ethilon</td><td>MWI 72667</td><td>6-0 Ethilon


https://www.mwivet.com (MWI - Veterinary Supplies)</td></tr><tr><td>Polyimide Cuff Vein (21G)</td><td>Vention Medical</td><td>141-0043</td><td>http://www.ventionmedical.com/products-and-services/polyimide-tubing/</td></tr><tr><td>Polyimide Cuff Artery (24G)</td><td>Vention Medical</td><td>141-0027</td><td>http://www.ventionmedical.com/products-and-services/polyimide-tubing/</td></tr><tr><td>Soft plastic tip catheter</td><td>Terumo</td><td>SR*OX2419CA</td><td>24G x 3/4&quot;&amp;nbsp;</td></tr><tr><td>Microsurgical dilator</td><td>S&amp;amp;T</td><td>D-5a.1</td><td>Dilator, 11 cm, FH, 0.1 mm AT10d</td></tr></tbody></table>

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.

同系C57BL / 6移植实现长期生存。同种异体移植物（图1）的设计中被证明是成功的，从动物的生存透视，并评价正在进行移植物存活的能力。这是通过覆盖皮肤表明剩余可行，活性持续同种异体移植物的毛发生长，和心跳能够用可视化和手指触诊进行评估。存活数据被表示在图2中进行同源移植小鼠。平均存活时间大于109天。基于存活数据，可以合理推断，移植的同种异体移植物的技术方面的目的是灌注胸壁，胸腺和心脏的全部。此外，同系动物的生存长期的能力进一步支持了这种小鼠模型不仅是可行的，但可以复制​​。这证明了概念整块胸壁，胸腺，和心脏移植验证次Ë小鼠模型来研究结合实体器官和血管复合同种异体移植。 <img alt="图1" src="/files/ftp_upload/53442/53442fig1.jpg" /> 图1.术中的照片。 （一）胸壁，胸腺，和心脏移植的成功回收，整理和可视化体外从后方面。双侧胸廓容器被保留。（B）中的收件人外部颈静脉（箭头）和颈总动脉（箭头）是外翻在准备吻合血管固定过聚酰亚胺袖口。（C）的同种异体移植血管吻合完成。箭头示出了供体肺动脉和受体外颈静脉之间的吻合。箭头显示了捐助降主动脉与受体颈总动脉之间的吻合。星号标识胸腺和反射胸壁被可视化，可欣赏莹胸腺。 （四）较高的放大倍数显示了微血管袖口吻合。 （五）完成移植插图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53442/53442fig1large.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/53442/53442fig2.jpg" /> 图2.整块胸壁，胸腺，和心脏移植物存活的整块胸壁，胸腺，和心脏异体移植在同系C57BL / 6小鼠的Kaplan-Meier生存曲线（N = 3;平均生存时间大于109天）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53442/53442fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice at JoVE.com

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

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 ...

a-novel-microsurgical-model-for-heterotopic-en-bloc-chest-wall-thymus

Nanyang Technological University

Research

JoVE Journal

Medicine

9.2K 次观看.  Johns Hopkins University School of Medicine. 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.

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

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

小鼠异位心脏移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

科研

生物学

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

用改进的袖套法小鼠颈部心脏移植模型

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

医学

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 ...

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 ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

颈椎异位心脏移植小鼠

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

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 ...

免疫与感染

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 ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

利用血管袖带的小鼠异位宫颈心脏移植

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部小鼠心脏移植模型中的血液回路重建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

一种改进的袖带技术用于小鼠颈椎异位心脏移植模型

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...