原位后肢移植中的鼠标

摘要

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。

摘要

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.

引言

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.

研究方案

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

1. 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。
2. 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。
3. 戴口罩，一次性隔离衣和手套。
4. 剃须手术AREA，特别是后肢和腹股沟，并准备用10％聚维酮 - 碘。
5. 使用无菌区悬垂性，蒸压仪器和高放大倍率的显微镜（40X）。
6. 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。
7. 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。
8. 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。
9. 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。
10. 横切股骨和在用剪刀股骨轴的中间切断。
11. 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。
12. 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）。
13. 广场上的股静脉和动脉1聚酰亚胺袖口，分别为。
14. 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。

2.受体手术

1. 后肢的去除
   1. 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。
   2. 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。
   3. 使用鼠标的眼睛兽医药膏，以防止干燥时的麻醉下。
   4. 剃手术区，特别是后肢和腹股沟和制备用10％聚维酮 - 碘。
   5. 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。
   6. 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。
   7. 一旦血管蒂被解剖，在腹股沟韧带的水平夹紧股血管。
   8. 末节砍船只在腹壁浅动脉水平。
   9. 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。
   10. 横切股骨在用剪刀股骨轴的中间。
   11. 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。
2. 植入
   1. 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。
   2. 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。
   3. Coapt使用可吸收缝合材料（6-0 Polysorb）腹侧和背侧肌肉群。
   4. 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。
   5. 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。
   6. 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。
   7. 关闭使用不可吸收缝线尼龙（6-0 Ethilon）皮肤。
   8. 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。
   9. 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。

结果

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...

讨论

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...

材料

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