对于异位小鼠心脏移植技术的一种改进方法
Summary
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.
Abstract
老鼠经常被用来作为心脏移植供，受者在移植免疫学研究，由于大范围的转基因小鼠和试剂可用的。的困难，提出由于动物的体积小，参与心脏移植的显微外科的相当大的技术挑战。特别是，移植术后早期高企的技术故障可能导致收件人死亡和术后并发症，如后肢瘫痪或无跳动的心脏。在这里，完整的技术异位小鼠心脏移植证实，涉及收获捐赠的心脏而其随后植入到受体小鼠。供体心脏收获立即就地灌注在与升主动脉和肺动脉的冷肝素生理盐水和横断。接受者操作涉及制备腹主动脉和下腔静脉（IVC），其次是端 – 端吻合术使用一个单一的运行10-0 microsuture和捐助肺动脉与受体下腔静脉的吻合类似的捐助主动脉与受体主动脉。以下操作的动物被注射用0.6 ml生理盐水皮下注射和允许恢复在37℃下加热垫。从227小鼠心脏移植手术的结果总结，成功率在48小时的86.8％。在48小时的13.2％的失败，5（2.2％）出现后肢麻痹，10（4.4％）有因移植缺血性损伤和/或血栓形成非跳动的心脏，15（6.6％），48小时内死亡。
Introduction
器官移植的动物模型可以提供有价值的信息，以提高治疗的临床移植的患者。小鼠模型是研究器官移植排斥或接受免疫机制，由于大范围的转基因小鼠和具体的小鼠不适用于其他动物模型1-3试剂特别有用。与移植小鼠模型的一个挑战是捐助者和受助人的需要相当的技术技能，以达到满意的结果的小尺寸。
一种技术是在大鼠4其后由科里等 5适用于小鼠心脏移植首先描述了心中的异位移植。这项技术涉及的准备之前，供体手术接受者和无灌注供体心脏，这两者都可能损害心脏移植或t的生存他收件人。该过程已被广泛地用作最初描述检查移植排斥和耐受6-8的机制。其他人已经适应了原有的大鼠心脏移植Ono和林赛的心脏移植在小鼠9,10过程。最近，一种技术被发布给小鼠心脏移植是克服了与Corry 等人 11的方法相关联的问题。在这里描述的协议采用了修改，基于莫特拉姆等人 12的方法，该方法包括： 在原位灌注冷肝素生理盐水开胸后立即和事先执行操作供体到受体手术，以减少接受者操作时间。此外，我们使用非创伤性小血管夹，而不是6-0真丝领带。虽然血管夹有采取更多的空间的缺点，他们更容易比的关系来控制，这不应该是太紧或太松并且是那么简单，除去比夹。我们的方法是使用一个单一的连续缝合为容器吻合，虽然在最初的学习阶段的替代方法是使用缝线留在近侧和远侧角，以确保缝合线的均匀性，因此该吻合的通畅。
Protocol
Representative Results
Discussion
小鼠心脏移植是需要相当大的手术技巧掌握一个具有挑战性的显微外科方法。最具挑战性的方面是小直径的血管。此外，有必要限制接收者操作时间和出血。该技术对小鼠心脏移植首先由Corry 等人所述。在1973年，后经莫特拉姆等人 12。我们的修改包括以下几点。首先，供体心脏冷肝素盐水下列开胸主动脉和肺动脉束灌注后不久和横断立即灌注防止热缺血期间收获。其次，从?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由澳大利亚资助计划- 1029205国家健康和医学研究委员会（NHMRC）的支持，由澳大利亚Microsearch基金会，由Myee科德林顿医学研究基金会。
Materials
| Operating microscope | Leica, Heerbrugg, Switzerland | M651 | 10x – 25x magnification |
| Anesthetic machine | Vet Quip Pty Ltd, Sydney, Australia | Vett3 | Capable of delivering a mixture of isoflurane and oxygen in air |
| Operating board | Hardware store or office supplier | Dense cork or synthetic capable of taking pins | |
| Heparinised saline (5 U/ml, 4oC) | Pfizer, USA | FW25 | In 1ml syringe with 23 gauge needle on ice |
| Normal saline (37oC) | AstraZeneca Pty Ltd, Australia | 4538 | In 1ml syringe with 23 gauge needle on warm pad |
| Sutures 10- nylon, 5-0 Vicryl | Ethicon, Inc. NJ, USA | 2870G/J421H | |
| Buprenorphine (0.05mg/kg in 0.1ml saline) | Reckitt Benckiser, Sydney, Australia | In 1ml syringe with 23 gauge needle on ice | |
| Ampcillin (5mg/kg in 0.1 ml saline) | Aspen, Sydney, Australia | In 1ml syringe with 23 gauge needle on ice | |
| Gel Foam | Pharmacia & Upjohn Co. USA | 801289304 | Cut into small pieces |
| High temperature cautery device | Medtronic, USA | 8444000 | |
| Microsurgery instruments: | Shanghai Medical Instruments Co. Ltd., | ||
| micro-needleholders | Shanghai, China | WT2020 | |
| micro scissors | " " " | WT1020 | |
| micro-forceps (straight tip) | " " " | WA3010 | |
| micro-forceps (curved tip) | " " " | WA3020 | |
| micro-mosquito clamps (1 pair) | " " " | W40350 | |
| micro-vessel atraumatic clamps (1 pair) | " " " | W40130/W40150 | |
| Heating Pad/Right Temp | Kent Scientific Corporation, Turrington, CT 06790 | ||
References
- Aramaki, O., et al. Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen. Transplantation. 79, 568-576 (2005).
- Chen, R. H., Bushell, A., Fuggle, S. V., Wood, K. J., Morris, P. J. Expression of granzyme A and perforin in mouse heart transplants immunosuppressed with donor-specific transfusion and anti-CD4 monoclonal antibody. Transplantation. 61, 625-629 (1996).
- Poulin, L. F., et al. Interleukin-9 promotes eosinophilic rejection of mouse heart allografts. Transplantation. 76, 572-577 (2003).
- Ono, K., Lindsey, E. S. Improved technique of heart transplantation in rats. J. Thorac. Cardiovasc. Surg. 57, 225-229 (1969).
- Corry, R. J., Winn, H. J., Russell, P. S. Primarily vascularized allografts of heart in mice. Transplantation. 16, 343-350 (1973).
- Larsen, C. P., et al. CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway. Transplantation. 61, 4-9 (1996).
- Saitovitch, D., Bushell, A., Mabbs, D. W., Morris, P. J., Wood, K. J. Kinetics of induction of transplantation tolerance with a nondepleting anti-Cd4 monoclonal antibody and donor-specific transfusion before transplantation. A critical period of time is required for development of immunological unresponsiveness. Transplantation. 61, 1642-1647 (1996).
- Callaghan, C. J., et al. Regulation of allograft survival by inhibitory FcgammaRIIb signaling. J. Immunol. 189, 5694-5702 (2012).
- Qian, S., et al. Impact of donor MHC Class I or Class II antigen deficiency on first- and second-set rejection of mouse heart or liver allografts. Immunology. 88, 124-129 (1996).
- Wang, C., et al. Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments. Transpl. Immunol. 24, 149-156 (2011).
- Liu, F., Kang, S. M. Heterotopic Heart Transplantation in Mice. J. Vis. Exp. , (2007).
- Mottram, P. L., et al. Electrocardiographic monitoring of cardiac transplants in mice. Cardiovasc. Res. 22, 315-321 (1988).
