Name: mouse kidney transplantation: models of allograft rejection
Uploaded: 2014-10-11T15:00:00.000+00:00
Duration: 16 min 15 s
Description: Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

从肾脏研究英国资助，爱丁堡和器官移植的欧洲社会皇家外科学院赞助这项研究。

适当的国家和地方机构的伦理应该在的地方进行动物实验前。具体而言，在英国进行了下列实验下的动物（科学程序）法1986如果两microsurgeons可同时操作的施主外科医生应该执行进行步骤1.1到1.16，然后3.1至3.5，而收件人外科医生进行2.1至2.8 。对于单个操作步骤可以顺序执行。 1，捐赠者准备注:这里介绍的程序是供体C57BL / 6 BM12和收件人年龄8〜16周龄之间有体重大于20克C57BL / 6雄性小鼠。然而这个过程可以在各种小鼠品系可再现地进行。在代表性的结果部分给出的数据是从C57BL / 6，C57BL / 6 BM12和BALB / c小鼠获得的。 <ol><li>行为的程序使用设克无菌手术器械和耗材（灭菌），以努力保持工作区无菌越好。同时与收件人的准备如果两个医生都可以进行捐赠准备。 </li><li>麻醉小鼠用美托咪定（0.5毫克/千克）和盐酸氯胺酮（200毫克/千克）的腹膜内注射（31G针）。这个结果在麻醉平面内保持4小时提供足够的时间用于整个过程被执行。补充麻醉是没有必要的。 </li><li>确认鼠标麻醉（无回应脚趾捏）。 </li><li>剃须鼠标的腹部，并移除任何松散的头发用一条胶带。 </li><li>把鼠标放在其上的无菌披加热垫回来，松松固定用无菌遮蔽胶带四肢。 </li><li>监控整个热烧伤的程序鼠标。如果可能的话使用非电热源。 </li><li>敷上眼部润滑剂和SAnitize腹壁用稀碘溶液。 </li><li>请在腹部进入腹膜腔，并插入一个3厘米宋体腹部拉钩正中切口。 </li><li>适用于温热的盐水，以保持肠道和手术区域湿润，避免内脏不必要的干燥。 </li><li>覆盖鼠标，用无菌巾和移动肠向操作者的左（小鼠的右侧），以暴露主动脉，腔静脉和左肾。 </li><li>应用逐步加大钳子胃和优拉，露出大血管和左肾完全。包受潮无菌拭子（2毫米x 2毫米）到腹部缩回组织远离手术区，如肝叶，精囊，和肠。 </li><li>隔离从周围的外膜，脂肪和左肾上腺在腹膜腔通过使用细尖镊子直截了当地解剖结缔组织左肾。把镊子的封闭技巧，网元之间的区域编辑要被分离并慢慢使钳子的提示开到解剖空间。 </li><li>结扎，然后除以左肾上腺静脉与左性腺静脉9 / O的尼龙隔离左肾静脉。放置缝合靠近肾静脉。 </li><li>结扎和分输尿管与7 / O丝线缝合靠近膀胱离开缝合结束漫长。这些长缝合线端部时，将使用的肾收获和所必需的输尿管吻合术。 </li><li>要动员和优劣势，并全面剖析了主动脉和腔静脉至肾动脉和静脉用细尖镊子直言不讳地剖析淋巴管和脂肪血管的正面和侧面。 </li><li>发现主动脉和腔静脉和慢慢扩散钳子之间的组织平面。慢慢打开细尖镊子的尖端传播以最小创伤的组织。 </li><li>通过传递一个角度细绑在主动脉上，下到肾动脉松散7 / O丝血管周围的背面，并通过绘制缝合尖镊子。这些缝合之前将肾脏检索收紧允许威斯康辛大学解决方案逆行灌注。 </li></ol> 2，收件人准备按捐赠者准备的后续步骤1.2〜1.8。 <ol><li>将肠向操作者的权利（鼠标左键），以暴露主动脉，腔静脉。用无菌生理盐水浸湿棉签肠子。 </li><li>结扎右肾动脉进行正确的性肾切除术，并与7/0丝线缝合静脉在一起，然后分开。 </li><li>结扎右侧输尿管有7 / O的丝线和鸿沟。 </li><li>动员和如在步骤1.14和1.15中所述完全解剖主动脉和下腔静脉的肾动脉和静脉。确保主动脉和腔静脉之间的完全切除。小心保护它运行到前腔静脉和aort的精索内动脉à随着淋巴丛。 </li><li>确定在从腔静脉和静脉脊柱腹膜后运行腰椎血管，结扎带9 / O蚕丝中的连续性，也没有必要分割的血管。 </li><li>确定有足够的空间来放置微血管钳与血管吻合之间的空间。 </li><li>管理通过阴茎背静脉静脉注射肝素（5台）。 </li></ol>三体肾检索拧紧7 / O丝束已经周围放置劣和优越主动脉分离从动脉循环的肾脏。 <ol><li>逆行注入冷0.2 - 0.5毫升威斯康辛大学液用针（31G）进入主动脉。 </li><li>分缝线内主动脉和划分肾静脉在其与腔静脉交界以除去肾和肾动脉和主动脉的长度。分从主动脉而产生腰部容器，如果它们存在，瓦特ithout结扎。 </li><li>发生在冷盐水肾脏在培养皿无菌棉签。 </li><li>安乐死的供体小鼠颈椎脱位。 </li></ol> 4，肾移植 - 肾准备<ol><li>通过纵向直接除以主动脉壁的肾动脉对面创建主动脉修补。确定在补丁任何容器流明需要被连接或避免在执行动脉吻合时。 </li><li>将来自外部10 / O的缝合肾静脉管腔优和第二分别缝合下方内侧。缝线用于划分肾上腺静脉和性腺静脉可用于定向的容器。 </li><li>将在收件人（ 图1.1）的右侧肾脏，确保缝线都处于有利地位，以保证血管吻合，而不变得纠缠在棉签或其他工具的缝线完成。 </li></ol> 5，肾Transplan塔季翁 - 血管吻合术应用微血管夹下方先优后包围腔静脉和主动脉。 <ol><li>请用针（31G）穿刺的腔静脉前墙ventomy。从腔静脉注射约50微升的0.9％NaCl冲洗血液。 </li><li>用细尖镊子由venotomy中打开它们，这样的长度相当于供体肾静脉的直径扩大venotomy。 </li><li>将10 /输出卓越的缝合（这已经是在肾静脉腔）首次在venotomy（ 图1.2）的顶点，并加入了连续缝合吻合口后壁，直到劣顶点到达（ 图1.3）。 </li><li>将10 / O的劣质缝合并用单结（ 图1.4）先绑然后从后墙绑在连续缝合。 </li><li>使用劣质缝线营造正面沃尔玛升吻合（ 图1.5），用连续缝合，并配合上级缝合月底在上级顶点（ 图1.6）。 </li><li>创建一个织脉切开术通过拿起主动脉壁与钳和切割用剪刀椭圆形片（约在织脉切开术应该是主动脉的周长和长度的三倍肾动脉管腔的五分之一）。 </li><li>将10 / O的缝合处优越点的主动脉修补，从外到内（ 图1.7），并通过主动脉在织脉切开术的优越顶点血管外打结。 </li><li>创建动脉吻合了连续缝合起优（ 图1.8）吻合的捐赠者的补丁给收件人主动脉，注意不要通过把缝线太紧（ 图1.9），以收缩吻合。 </li><li>取出劣质血管钳先则优于钳reperfuse肾脏（图1.10和1.11）。输尿管可见蠕动也许观察，如果充足的血液供应，输尿管实现。 </li></ol> 6，肾移植 - 输尿管吻合术<ol><li>划分膀胱任何附件从腹壁。 </li><li>通过两个壁通过从左侧到膀胱的右边的针（21G）。 </li><li>放置直钳子提示在针内腔，并同时通过回通过膀胱，使得镊子正从右到左出膀胱的左侧的传递。 </li><li>利用通过使得所述输尿管通过在左膀胱缺陷然后出右侧的膀胱施主输尿管缝合。 </li><li>缝合输尿管至膀胱的外膜的外膜与周围的入口点三个单间断缝合与9 / O尼龙缝线上的圆的健全针。 </li><li>切输尿管近端结扎，从而揭开日ë输尿管允许尿液流动并允许输尿管端缩回到膀胱的主体。可见生产的尿可被与从周围输尿管血管出血观察沿。 </li><li>关闭右侧气囊缺陷与单个中断9 / O的丝线。 </li></ol> 7，恢复和术后护理重新装上消化道内脏到原始方向的腹部和近似直肌与6 / O的可吸收线缝合关闭腹壁。 <ol><li>近似​​与金属皮肤片段的皮肤。 </li><li>部分逆转了皮下注射atipamazole盐酸（10微升/克）的麻醉。 </li><li>通过皮下注射盐酸丁丙诺啡（0.05毫克/千克）和用于流体支撑注射1毫升皮下的0.9％NaCl中施用镇痛。 </li><li>恢复鼠标在变暖内阁在28℃，24〜48小时。观察小鼠的疾病达实验完成。 </li><li>去除金属皮肤剪辑7 - 11天后操作性。 </li><li>一旦试验完成安乐死的小鼠颈椎脱臼。 </li><li>取出并收集对侧肾和移植的组织学分析。 </li></ol>

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最充分描述的方式来执行的动脉吻合术是使用供体的远端主动脉，与肾动脉中继续，在一个端部到另一侧的方式向收件人主动脉。我们描述了使用主动脉补丁，类似于人类肾脏移植，我们认为是比较方便执行的"Carrell补丁&#39;镜像。虽然在供体和受体手术时间的文献报道是稀疏我们相信，利用端 - 侧供体主动脉的主动脉补丁到收件人主动脉而不是向收件人主动脉是优选的。使用补丁否定，要剖析的...

成功的肾移植肾功能衰竭的治疗在1955年首次被描述同卵双胞胎1之间，从那时起，它已成为患者在世界各地的终末期肾功能衰竭的一种革命性的治疗，提供了长寿命2质量均提高。但是长期移植物的存活已经阻碍了许多导致慢性移植物损伤3病理过程。 在人类的移植肾排斥仍发病率的主要原因，尽管在immunosupporessive方案显著改进。发展肾移植小鼠模型的目的是密切复制人类肾移植4中发现的过程和病理。 Skoskiewicz 等人首先描述肾移植的小鼠模型，1973年5。虽然先进的显微外科技巧是必需的，它是一个有价值的吨OOL有以下几个原因:小鼠基因组中已经得到很好的特点，并有很大的不同的实验方法和可用于小鼠实验技术。 使用肾移植的小鼠模型中的许多团体已经使用的移植肾的生命支持器官，但在其他研究中，并在我们所描述的受体小鼠的天然肾脏的方法1留在原位进行实验4的持续时间。这样做的好处是，鼠标经过一个单一的麻醉和手术从而降低了发病率的鼠标和死亡的第二个步骤的风险。此外，该鼠标不从逐步肾功能衰竭的不利影响受害。 虽然在其他器官，如心脏和皮肤存在异体排斥反应的模型，这并不总是直接相关的肾移植。有证据表明，这些模型引出不同的模式和dy排斥的动力学性能，例如拒绝在心脏同种异体移植物和肾移植的时间过程中的某些菌株的组合6显著不同。我们在BALB / c小鼠的捐助者说明移植肾急性排斥反应的模式到非转基因FVB / NJ小鼠，这种模式表现出与T细胞和巨噬细胞7积聚的细胞介导的损伤。另外，我们也描述了慢性移植物损伤模型，展示间质纤维化和肾小管萎缩，这将导致从移植肾的C57BL / 6 BM12捐助者到C57BL / 6受，因为这些小鼠的特点是一个单一的MHC II类基因位点错-match 8。 移植的多个方面使用肾移植的小鼠模型中，包括急性排斥反应，细胞免疫和体液排斥，局部缺血再灌注损伤，以及试用新治疗剂进行了研究。 我们已修改的手术吨echnique，以减少操作时间，提高了易用性手术。特别是我们所描述的同时供体和受体的准备，并利用连续的主动脉修补吻合简化的血管吻合技术。此视频稿件和提供的关键点在建立这种技术的帮助。

<table><tbody><tr><td colspan="4">Surgical Instruments</td></tr><tr><td>Blunt Dissecting Scissors</td><td>Fine Science Tools</td><td>&amp;nbsp;14072-10</td><td>For skin cutting</td></tr><tr><td>Curved Castoviejo scissors</td><td>Fine Science Tools</td><td>15017-10</td><td>For tissue cutting</td></tr><tr><td>Spring Scissors &amp;ndash; straight</td><td>Fine Science Tools</td><td>15000-08</td><td>For suture cutting</td></tr><tr><td>Toothed forceps 1x2 teeth</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>2 x Fine Tip forceps (Dumont No.5)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Angled Fine Tip forceps (Dumont No. 5/45)</td><td>Fine Science Tools</td><td>11253-25</td><td>For blunt dissecting</td></tr><tr><td>Curved Fine Tip forcep (Dumont No.7)</td><td>Fine Science Tools</td><td>11273-22</td><td>Useful to pass around vessels</td></tr><tr><td>Curved Crile Haemostat</td><td>Fine Science Tools</td><td>1300-04</td><td></td></tr><tr><td>Micro clip applicator with lock</td><td>Fine Science Tools</td><td>18056-14</td><td></td></tr><tr><td>2 x Micro serrefines spring width 2mm, jaw length 4mm</td><td>Fine Science Tools</td><td>18055-04</td><td>Microvascular clamps</td></tr><tr><td>2 x Colibri 3cm wire retractor</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Castroviejo needle holder with lock</td><td>Fine Science Tools</td><td>120660-01</td><td></td></tr><tr><td>Wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7mm wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td>Remove 7 to 10 days after surgery</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>OPMI pico microscope</td><td>Carl Zeiss</td><td>S100</td><td></td></tr><tr><td>Thermal cautery unit with fine tip</td><td>Geiger</td><td>150A</td><td></td></tr><tr><td>Heat electronic pad</td><td>Cozee Cumfort</td><td>n/a</td><td></td></tr><tr><td>Euroklav 23-S</td><td>Melag</td><td>n/a</td><td>Autoclave</td></tr><tr><td colspan="4">Disposable equipment</td></tr><tr><td>7/O Silk braided suture</td><td>Pearsall</td><td>30514</td><td></td></tr><tr><td>10/O Dafilon (polyamide) suture</td><td>B-Braun</td><td>&amp;nbsp;G1118099</td><td></td></tr><tr><td>6/O Vicryl (plygalectin)</td><td>Ethicon</td><td>W9537</td><td></td></tr><tr><td>Regular bevel needle, 1 inch, 21G</td><td>Bection, Dickinson and Company</td><td>305175</td><td>For ureteric anastamosis</td></tr><tr><td>Regular bevel needle, 5/8 inch, 25G</td><td>Bection, Dickinson and Company</td><td>305122</td><td></td></tr><tr><td>Regular bevel needle, 1/2 inch, 30G</td><td>Bection, Dickinson and Company</td><td>304000</td><td></td></tr><tr><td>Insulin needle 1ml, 29G</td><td>Bection, Dickinson and Company</td><td>324827</td><td></td></tr><tr><td>Insulin needle 0.3ml, 30G</td><td>Bection, Dickinson and Company</td><td>324826</td><td></td></tr><tr><td>1 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>300184</td><td></td></tr><tr><td>5 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>302187</td><td></td></tr><tr><td>Wypall paper swabs</td><td>Kimberley-Clark</td><td>L40</td><td>sterilised by autoclave</td></tr><tr><td>Cotton wool buds</td><td>Johnson and Johnson</td><td>n/a</td><td>sterilised by autoclave</td></tr><tr><td>Plain drapes</td><td>Guardian</td><td>CB03</td><td>sterilised by autoclave</td></tr><tr><td>Cell culture dish 60mm x 15mm</td><td>Corning Incorporated</td><td>430166</td><td></td></tr><tr><td>Dispensing Pin</td><td>B-Braun</td><td>DP3500L / 413501</td><td>Used with NaCl 0.9%</td></tr><tr><td>Re-agents and Drugs</td><td></td><td></td><td></td></tr><tr><td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td><td>Allergan Ltd</td><td>21956GB10X</td><td></td></tr><tr><td>(Videne) Povidone-iodine 10%</td><td>Ecolab Ltd</td><td>PL 04509/0041</td><td></td></tr><tr><td>(Vetalar V) Ketamine hydrochloride</td><td>Pfizer Animal Health</td><td>Vm 42058/4165</td><td>100mg/ml solution (dose 200mg/kg)</td></tr><tr><td>(Domitor) Medetomidine hydrochloride&amp;nbsp;</td><td>Orion Pharma</td><td>Vm 06043/4003</td><td>1mg/ml (dose 0.5mg/kg)</td></tr><tr><td>(Vetergesic) Bupernorphine hydrochloride&amp;nbsp;</td><td>Alsto Animal Health</td><td>Vm 00063/4002</td><td>0.3mg/ml (dose 0.05mg/kg)</td></tr><tr><td>(Antisedan) Atipamezole hydrochoride</td><td>Orion Pharma</td><td>Vm 06043/4004</td><td>5mg/ml (dose 2mg/kg)</td></tr><tr><td>University of Wisconsin Solution</td><td>Belzer Bridge to Life</td><td>n/a</td><td>dose approximately 500 microlitres/mouse</td></tr><tr><td>NaCl 0.9%</td><td>Baxter</td><td>FKE1323</td><td></td></tr><tr><td>Heparin Sulphate</td><td>non-proprietary</td><td>n/a</td><td>5000units/ml (dose 5units/mouse)</td></tr></tbody></table>

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.

肾同种异体移植物排斥可以通过methacarn固定，石蜡包埋的组织的移植肾（ 图2）的部分的组织学分析来评估。结果小鼠肾缺血再灌注损伤的同系之间的肾脏移植同基因移植，但4周肾小管已经恢复，并在组织学上相当于本地肾脏。急性排斥反应可以通过C57BL / 6肾移植到BALB / C收件人为蓝本，在1周内有与肾实质弥漫性单核细胞浸润，累及间质，肾小球和肾小管。慢性同种异体移植物损伤?...

Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

Mouse Kidney Transplantation: Models of Allograft Rejection

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 ...

mouse-kidney-transplantation-models-of-allograft-rejection

Research

JoVE Journal

Medicine

20.5K 次观看.  The University of Edinburgh. 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.

视频: Mouse Kidney Transplantation: Models of Allograft Rejection

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

小鼠皮肤移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

生物学

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. 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 using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

免疫与感染

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 ...

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.

一种用于小鼠肾移植的改良手术技术

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

大鼠原位肾移植: 一种新的简化的手术方法

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

一种用于肾同种异体排斥的大鼠原位肾移植模型

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...