一种用于小鼠肾移植的改良手术技术
1Department of Pediatric and Adolescent Medicine, University Hospital Erlangen,Friedrich-Alexander University Erlangen-Nürnberg, 2Department of Pediatric Surgery,Chengdu Women’s and Children’s Central Hospital, 3The Key Laboratory of Hainan Trauma and Disaster Rescue,The first affiliated Hospital of Hainan Medical University, 4Department of Department of Pediatric Kidney, Liver, and Metabolic Diseases,Hannover Medical School, 5Department of Nephrology,Hannover Medical School, 6Department of Nephrology, University Hospital Erlangen,Friedrich-Alexander University Erlangen-Nürnberg, 7Department of Dermatology, University Hospital Erlangen,Friedrich-Alexander University Erlangen-Nürnberg
Summary
该方案提出了一种新的小鼠肾移植手术技术，其重点是改进的动脉吻合术策略。还介绍了一种血管缝合技术，包括一种简单且更安全的输尿管-膀胱吻合术方法。这些修饰缩短了手术时间，提高了小鼠肾移植手术的成功率。
Abstract
小鼠肾移植是一种复杂且具有挑战性的手术过程。很少有出版物演示这一行动的关键步骤。因此，本文介绍了该技术并指出了与此操作相关的手术注意事项。此外，还演示了与传统程序相比的重要修改。首先，切除并制备腹主动脉的贴片，以便将包括输尿管动脉在内的肾动脉的近端分叉与供体肾脏整体一起横切 。 这降低了输尿管坏死的风险，并避免了尿路阻塞的发展。其次，展示了一种新的血管吻合术方法，该方法允许操作员在肾移植再灌注已经开始后灵活地增加或减小吻合口的大小。这避免了血管狭窄和腹腔内出血的发展。第三，显示了一种能够吻合脆弱的供体输尿管和受体膀胱的技术，不会引起创伤。采用该方案可以缩短手术时间并减少对受体膀胱的损伤，从而显着提高受体小鼠的手术成功率。
Introduction
自Sakowitz等人于1973年首次开发肾移植小鼠模型以来1，它已被证明是研究移植缺血性损伤和同种免疫排斥机制以及开发旨在延长同种异体移植存活并可能实现免疫耐受性的新疗法的重要实验工具。然而，手术技术已被证明是复杂且非常苛刻的，有时具有并发症，例如血管吻合口狭窄导致肾前非免疫性肾移植失败2，肾后衰竭引起的缺血和随后移植输尿管坏死，移植输尿管吻合口狭窄和/或受者的尿囊导致尿流出破坏。所有这些都是小鼠肾移植尚未进一步发展的原因，因此未被广泛使用。建立一种有效且长期稳定的小鼠肾移植模型，不伴血管和尿路并发症，对于移植领域的许多研究仍然具有不可替代的意义，重点是肾免疫介导的，但也包括传染病3。此外，与小鼠模型中的其他器官移植如肺、心和肠移植4，5相比，小鼠肾移植模型即使在主要组织相容性抗原差异3，6的情况下也为研究长期生存提供了机会。还表明，在供体 – 受体菌株组合的相同设置中，不同的器官移植（例如心脏或肾脏）具有不同的动力学和同种异体移植物排斥反应的发生3。此外，从肾脏学的角度来看，与简单的皮肤移植实验相比，它是在急性和慢性排斥事件背景下研究实质介导的免疫调节机制的更合适的模型。
根据以前关于小鼠肾移植手术技术的报告3，7，8，9，我们在这里证明了以下可靠的改进，这些改进在过去10年中在我们组内已成功应用10，11，12：首先，输尿管动脉被安全地保存，因为肾动脉被 整体 切除 与腹主动脉的相应部分一起。其次，一种新的，简单而快速的无结血管吻合术技术，其中吻合术的最终缝合线不像传统方法那样与上领带的末端相连，而是保持自由。该技术能够在肾脏再灌注后增加或减少吻合口的大小，以避免血管狭窄和腹腔内出血。第三，使用21 G和30 G注射器针头作为辅助穿刺引导工具，以便将供体输尿管植入受者的膀胱壁，减少对受者膀胱的损伤并促进无狭窄吻合口的形成。
在这份报告中，我们还将传统的，广泛使用的技术与我们实验室建立的改良技术进行了比较，发现肾小管萎缩和肾移植间质组织纤维化的程度没有显着差异。在以前的研究中，我们还将这种新技术与传统方法在局部出血，血栓形成，进行血管吻合术的时间和存活率方面的结果进行了比较。我们发现改善，例如局部血栓形成事件的显着减少（1.1% vs 6.6%），吻合术时间缩短，以及肾脏同源移植物的长期生存率高度可重复（95% vs 84% 使用传统方法）10。
Protocol
所有动物实验均根据欧洲议会关于保护用于科学目的的动物的指令2010/63 / EU的指南进行（动物伦理卡：下萨克森州食品和药物安全部，#33.9-42502-04-11/0492）。使用无菌手术器械和耗材（高压灭菌）进行手术，并尽量保持手术区域无菌。 注意：C57BL / 6J雄性小鼠作为供体和受体（同源移植模型），而Balb / c小鼠作为肾脏同种异体移植受体（研究急性同种异体移植排斥模型<sup class="x…
Representative Results
移植后四周，与天然受体对侧肾脏相比，改良技术和常规技术均显示肾小管萎缩的中度体征14，15 （图1）。肾小管萎缩的程度显示两种不同技术之间没有显着差异。与正常的非移植肾脏相比，Masson Goldner的三色染色14，15 的肾脏移植后12周均匀地显示出间质组织纤维化的明显迹象（<strong c…
Discussion
虽然小鼠的皮肤移植模型对于研究同种免疫排斥事件是简单易行的，但用于更具体地研究心脏16 和肾移植10 后同种免疫相关炎症改变的手术技术已被证明是复杂且非常苛刻的。从移植肾病学家的角度来看，建立一种有效且长期稳定的小鼠肾移植模型对于许多功能和免疫学研究仍然具有不可替代的意义。此外，与其他器官移植相比，小鼠肾移植模型即使存在一定?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢白天天医生团队在画外音方面的帮助，感谢包勉小姐在医学插画方面的帮助。这项工作得到了德国研究基金会（DFG）的部分支持，以促进国际合作（HO2581 / 4-1至AH）和中国国家科学基金（NSFC;#81760291至FJ）。
Materials
| 30G-needles | Braun | 456300 | – |
| acepromazine | CP Pharma | Tranquisol P | – |
| Bepanthen eye ointment | Haus-Apotheke | PZN 01578675 | – |
| Bonn Micro Forceps | FST | 11083-07 | – |
| Box for insulation and oxygen supply device | RUSKINN | INVIV | – |
| C57BL/6J mice | Charles River. Germany | no catalog number | – |
| Carprofen | Zoetis | Rimadyl 50 mg/ml | – |
| CATHETER-FEP 26G | TERUMO | Surflo-W | – |
| Clip Applicator Forceps Style | FST | 18057-14 | – |
| Curved forceps | WPI | 14114-G | – |
| Cutasept skin disinfection | VWR | BODL980365 | – |
| Dehydrator | DIAPATH | Donatello | – |
| electrosurgical pen | Bovie | CHANGE-A-TIP | – |
| Embedding machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | – |
| Ethanol | Sinopharm Group Chemical Reagent Co. LtD | 100092683 | – |
| Frozen platform | Wuhan Junjie Electronics Co., Ltd | JB-L5 | – |
| gauze pads, cotton swabs | Lohmann-Rauscher | 13353 | – |
| Glass slide | Servicebio | G6004 | – |
| HE dye solution set | Servicebio | G1003 | – |
| Heating mat | THERMO MAT PRO 30W | HTP-30 | – |
| hemostatic sponge | CuraSpon | J1276A | – |
| heparine-solution | Haus-Apotheke | PZN 03029820 | – |
| ice box | PETZ | No Catalog Number available | – |
| Imaging system | Nikon | Nikon DS-U3 | – |
| Inhalation anesthesia device | GROPPLER | BKGM 0616 | – |
| isoflurane | CP Pharma | Isofluran CP 1 ml/ml | – |
| ketamine | Zoetis | no catalog numer | – |
| Masson dye solution set | Servicebio | G1006 | – |
| metamizole | WDT | no catalog numer | – |
| Micro scissors | FST | 15000-00,15000-10 | – |
| Micro Serrefine ( Clamp ) Angled / 16 mm | FST | 18055-06 | – |
| Microscope | Leica | LEICAMZ6 | – |
| Microscope light | SCHOTT | KL2500LED | – |
| Neutral gum | SCRC | 10004160 | – |
| Oven | Tianjin Laibo Rui Instrument Equipment Co., Ltd | GFL-230 | – |
| Pathology slicer | Shanghai Leica Instrument Co., Ltd | RM2016 | – |
| Saline solution (NaCl 0.9 %) | Haus-Apotheke | PZN 06178437 | – |
| scissors | Peha Instruments | 991083/4 | – |
| Slides | Servicebio | – | |
| small Petri dish | Sarstedt | 8,33,900 | – |
| straight forceps | WPI | 14113-G | – |
| surgical tape | BSN | 4120 | – |
| Suture Tying Forceps – 10 cm | FST | 18025-10 | – |
| Sutures(10-0) | Medtronic | N2540 | – |
| Sutures(4-0) | ETHILON | V4940H | – |
| Sutures(7-0) | ETHILON | 1647H | – |
| Syringe (0,3 mL) | BD | 324826 | – |
| Syringe (1 mL) | BD | 320801 | – |
| Tissue spreader | Zhejiang Kehua Instrument Co., Ltd | KD-P | – |
| Upright optical microscope | Nikon | Nikon Eclipse E100 | – |
| xylazine | Bayer | Rompun | – |
| Xylene | Sinopharm Group Chemical Reagent Co. LtD | 10023418 | – |
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Citazione di questo articolo
Yin, D., Fu, J., Chen, R., Shushakova, N., Allabauer, I., Wei, X., Schiffer, M., Dudziak, D., Rong, S., Hoerning, A. A Modified Surgical Technique for Kidney Transplantation in Mice. J. Vis. Exp. (185), e63434, doi:10.3791/63434 (2022).