大鼠异位心脏和心肌细胞移植的免疫模型
Summary
我们描述了大鼠异位腹心移植的模型，这意味着对当前策略的修改，从而导致简化的手术方法。此外，我们还描述了一种新的排斥模型，通过注射重要的心肌细胞的耳朵内，允许进一步的移植免疫学分析在大鼠。
Abstract
50多年来，大鼠异位心脏移植一直是各种免疫学研究的常用模型。自1964年第一次描述以来，已经报告了若干修改。在大鼠进行异位心脏移植30年后，我们开发了一种简化的手术方法，无需进一步手术训练或背景，即可轻松教授和进行。
在解剖上升主动脉和肺动脉和上等类腔和肺静脉结扎后，供体心脏被收获，然后注入冰冷盐水溶液补充肝素。在夹紧和切开受体腹血管后，捐赠者提升主动脉和肺动脉分别被麻醉到受体腹部主动脉和劣质的vena卡瓦，使用连续的导联。
根据不同的供体-受体组合，此模型允许分析异体移植的急性或慢性排斥。该模型的免疫意义进一步增强，通过一种新的注射重要心肌细胞的耳内注射方法，以及随后对排空颈淋巴组织的分析， 进一步增强了该模型的免疫意义。
Introduction
异位心脏移植是一种常用的实验模型，用于不同研究移植耐受性、急性和慢性异体移植排斥、缺血再灌注损伤、机器灌注或心脏重塑等。除其他优点外，移植功能可以通过触觉进行非侵入性监测，移植失败不会导致接受者与其他器官（如肾脏或肝脏）相比的重要损伤。
1964年，Abbott等人最初描述了大鼠1的异位腹心移植。后来，在1966年，原子塔等人对阿纳斯托莫的端到端技术作了描述。1969年，奥诺和林赛报告了目前使用的模型的基础。在过去的几十年中，已经发表了一些修改，以创建不同类型的卸载，部分加载或加载左心室心脏移植，包括联合异位心肺移植44，5，6。5,6对于免疫分析，最常见的是非体积加载心脏移植移植。在这种情况下，血流逆行进入供体上升主动脉，随后进入冠状动脉。静脉排水沿冠状窦进入右中庭和心室（图1A-B）。因此，除了来自Thebesian静脉的少量血液外，左心室被排除在血流之外。这也使它成为研究左心室辅助装置治疗7期间的病理生理机制的有用模型。
异位心脏移植已在各种物种中进行，包括小鼠、兔子、猪，甚至被用作,人类8、9、10、119的单体或双心形辅助8装置。10,11大鼠仍然是移植模型的流行实验动物，特别是因为不同大鼠菌株组合的移植存活时间在过去已经明确，大量的免疫试剂是可访问的12，13。12,与老鼠不同，大鼠做手术和获得淋巴组织进行免疫分析更可行12。此外，近年来在大鼠身上引入商业性克隆技术，很可能导致对实验鼠模型14的反复关注。
一般来说，异位心脏移植可以通过进行宫颈或腹部麻醉附着在受体血管上。然而，一些研究表明，由于更好的获得手动触觉或跨体波他心动图，因此可以更精确地检测移植失败15，16，因此，股骨性麻醉有助于改善监测。15,
结果表明，两种阿生虫技术在操作时间、并发症率、结果和移植生存时间上没有差异。显然，必须提及有足够的排空淋巴结，作为宫颈麻醉的好处;但是，需要较长的培训期。相反，腹部麻醉对于免疫学研究来说不那么复杂，同样有价值，特别是当结合一种新疗法的异源性心肌细胞和随后的宫颈淋巴切除术的结果时。两种模型的组合提供了广泛的干预后免疫分析。
以下协议是指对外科医生进行手术，以减少缺血时间。但是，所有实验都可以由一个人执行。图2A-B显示了心脏外植和植入仪器和材料的设置。
Protocol
所有动物经验均按照下萨克森州（德国奥尔登堡州拉维斯）消费者保护和食品安全区域当局当地道德动物审查委员会的准则进行，并经批准，12/0768 和 17/2472。 1. 心脏外植和灌注 注：作为移植捐赠者，使用7-22周年龄的雌性或雄性大鼠。 通过吸入的二氟拉纳对供体大鼠进行麻醉（感应5%，维持3%，O2流量为1升/分钟）。注射5毫克Carprofen下皮每…
Representative Results
过去，不同的免疫问题都根据该模型得到解决，在工作小组中，500多例移植进行了验证，存活率超过95%，13、18、19、20、21、22、23、24。23,2413,18,19,20,21,22,总手术时间（包?…
Discussion
先前描述的大鼠异位心脏移植方法主要基于1969年对小野和林赛的描述。自那时以来，对各种物种进行了若干修改，导致该模型的广泛多样性。结合其中几个修改，并介绍我们自己的经验，在实验室进行了超过30年的异位心脏移植，我们创造了一个可行的手术方法，不需要长时间的训练或手术背景。在下面，我们将讨论此模型的一般限制，并强调协议的关键步骤。此外，我们将强?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢布里塔·特劳特维格、科林娜·勒伯特和英格丽德·梅德的承诺。
Materials
| Anesthesia device (including isoflurane vaporizer) | Summit Anesthesia Solutions | No Catalog Number available | |
| Cannula (27 G) | BD Microlance | 302200 | |
| Carprofen | Pfizer | Rimadyl 50 mg/mL | |
| Cellstar Tubes (15 mL) | GreinerBioOne | 188271 | |
| Cell strainer (40 µm) | BD Falcon | 2271680 | |
| Collagenase Type CLSII | Biochrome | C2-22 | |
| Compresses 5×5 cm | Fuhrmann | 31501 | |
| Compresses 7.5×7.5 cm | Fuhrmann | 31505 | |
| Cotton swabs | Heinz Herenz Medizinalbedarf | 1032128 | |
| Dexpathenol (5 %) | Bayer | "Bepanthen" | |
| DPBS BioWhittaker | Lonza | 17-512F | |
| Forceps | B. Braun | Aesculap BD557R | |
| Forceps | B. Braun | Aesculap BD313R | |
| Forceps | B. Braun | Aesculap BD35 | |
| Heating mat | Gaymar Industries | "T/Pump" | |
| Hemostatic gauze | Ethicon | Tabotamp | |
| Heparin-Natrium 25 000 I.E. | Ratiopharm | No Catalog Number available | |
| Isofluran CP | CP-Pharma | No Catalog Number available | |
| Large-pored sieve (stainless steel) | Forschungswerkstätten Hannover Medical School | No Catalog Number available | |
| Lidocaine | Astra Zeneca | 2 % Xylocain | |
| Metamizol-Natrium | Ratiopharma | Novaminsulfon 500 mg/mL | |
| Micro forceps | B. Braun | Aesculap BD3361 | |
| Micro needle holder | Codman, Johnson & Johnson Medical | Codmann 80-2003 | |
| Micro needle holder | B. Braun | Aesculap BD336R | |
| Micro needle holder | B. Braun | Aesculap FD241R | |
| Micro scissors | B. Braun | Aesculap FD101R | |
| Micro scissors | B. Braun | Aesculap FM471R | |
| Needle holder | B. Braun | Aesculap BM221R | |
| Penicillin/Streptomycin/Glutamine (100x) | PAA | P11-010 | |
| Peripheral venous catheter (18 G) | B. Braun | 4268334B | |
| Peripheral venous catheter (22 G) | B. Braun | 4268091B | |
| Probe pointed scissors | B. Braun | Aesculap BC030R | |
| Retractors | Forschungswerkstätten Hannover Medical School | No Catalog Number available | |
| RPMI culture medium | Lonza | BE12-702F | |
| Saline solution (NaCl 0.9 %) | Baxter | No Catalog Number available | |
| Scissors | B. Braun | Aesculap BC414 | |
| Surgical microscope | Carl-Zeiss | OPMI-MDM | |
| Sutures (anastomoses) | Catgut | Mariderm 8-0 monofil | |
| Sutures (ligature) | Resorba | Silk 5-0 polyfil | |
| Sutures (skin, fascia) | Ethicon | Mersilene 3-0 | |
| Syringe (1 mL) | B. Braun | 9166017V | |
| Syringe (10 mL) | B. Braun | 4606108V | |
| Syringe (20 mL) | B. Braun | 4606205V | |
| Vascular clamp | B. Braun | Aesculap FB708R |
Referências
- Abbott, C. P., et al. A Technique for Heart Transplantation In the Rat. Archives of Surgery. 89 (4), 645-652 (1964).
- Tomita, F. Heart homotransplantation in the rat. Sapporo igaku zasshi. The Sapporo Medical Journal. 30 (4), 165-183 (1966).
- Ono, K., Lindsey, E. S. Improved technique of heart transplantation in rats. The Journal of Thoracic and Cardiovascular Surgery. 57 (2), 225-229 (1969).
- Wen, P., et al. A simple technique for a new working heterotopic heart transplantation model in rats. Transplantation Proceedings. 45 (6), 2522-2526 (2013).
- Benke, K., et al. Heterotopic abdominal rat heart transplantation as a model to investigate volume dependency of myocardial remodeling. Transplantation. 101 (3), 498-505 (2017).
- Kearns, M. J., et al. Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration. Journal of Visualized Experiments. (99), 52418 (2015).
- Ibrahim, M., et al. Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading. The Journal of Surgical Research. 179 (1), e31-e39 (2013).
- Liu, F., Kang, S. M. Heterotopic heart transplantation in mice. Journal of Visualized Experiments. (6), 238 (2007).
- Lu, W., et al. A new simplified volume-loaded heterotopic rabbit heart transplant model with improved techniques and a standard operating procedure. Journal of Thoracic Disease. 7 (4), 653-661 (2015).
- Kitahara, H., et al. Heterotopic transplantation of a decellularized and recellularized whole porcine heart. Interactive Cardiovascular and Thoracic Surgery. 22 (5), 571-579 (2016).
- Kadner, A., et al. Heterotopic heart transplantation: experimental development and clinical experience. European Journal of Cardiothoracic Surgery. 17 (4), 474-481 (2000).
- Zinöcker, S., et al. Immune reconstitution and graft-versus-host reactions in rat models of allogeneic hematopoietic cell transplantation. Frontiers in Immunology. 3 (NOV), 1-12 (2012).
- Klempnauer, J., et al. Genetic control of rat heart allograft rejection: effect of different MHC and non-MHC incompatibilities. Immunogenetics. 30, 81-88 (1989).
- Huang, G., et al. Genetic manipulations in the rat: Progress and prospects. Current Opinion in Nephrology and Hypertension. 20 (4), 391-399 (2011).
- Gordon, C. R., et al. Pulse doppler and M-mode to assess viability of cardiac allografts using heterotopic femoral heart transplantation in rats. Microsurgery. 27 (4), 240-244 (2007).
- Gordon, C. R., et al. A new modified technique for heterotopic femoral heart transplantation in rats. The Journal of Surgical Research. 139 (2), 157-163 (2007).
- Ma, Y., Wang, G. Comparison of 2 heterotopic heart transplant techniques in rats: cervical and abdominal heart. Experimental and Clinical Transplantation. 9 (2), 128-133 (2011).
- Bektas, H., et al. Differential effect of donor-specific blood transfusions after kidney, heart, pancreas, and skin transplantation in major histocompatibility complex-incompatible rats. Transfusion. 37 (2), 226-230 (1997).
- Saiho, K. O., et al. Long-term allograft acceptance induced by single dose anti-leukocyte common antigen (RT7) antibody in the rat. Transplantation. 71 (8), 1124-1131 (2001).
- Bektas, H., et al. Blood transfers infectious immunologic tolerance in MHC-incompatible heart transplantation in rats. Journal of Heart and Lung Transplantation. 24 (5), 614-617 (2005).
- Jäger, M. D., et al. Sirolimus promotes tolerance for donor and recipient antigens after MHC class II disparate bone marrow transplantation in rats. Experimental Hematology. 35 (1), 164-170 (2007).
- Timrott, K., et al. Application of allogeneic bone marrow cells in view of residual alloreactivity: Sirolimus but not cyclosporine evolves tolerogenic properties. PLoS ONE. 10 (4), 1-16 (2015).
- Hadamitzky, M., et al. Memory-updating abrogates extinction of learned immunosuppression. Brain, Behavior, and Immunity. 52, 40-48 (2016).
- Beetz, O., et al. Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats. Plos One. 14 (8), e0220546 (2019).
- Hirschburger, M., et al. Nicotine Attenuates Macrophage Infiltration in Rat Lung Allografts. The Journal of Heart and Lung Transplantation. 28 (5), 493-500 (2009).
- Ruzza, A., et al. Heterotopic heart transplantation in rats: improved anesthetic and surgical technique. Transplantation Proceedings. 42 (9), 3828-3832 (2010).
- Wang, D., et al. A simplified technique for heart transplantation in rats: abdominal vessel branch-sparing and modified venotomy. Microsurgery. 26 (6), 470-472 (2006).
- Wang, C., et al. A modified method for heterotopic mouse heart transplantion. Journal of Visualized Experiments. (88), (2014).
- Al-Amran, F. G., Shahkolahi, M. M. Total arterial anastomosis heterotopic heart transplantation model. Transplantation Proceedings. 45 (2), 625-629 (2013).
- Hoerstrup, S. P., et al. Modified technique for heterotopic rat heart transplantation under cardioplegic arrest. Journal of Investigative Surgery. 13 (2), 73-77 (2000).
- Fry, D. L. Acute vascular endothelial changes associated with increased blood velocity gradients. Circulation Research. 22 (2), 165-197 (1968).
- Ahmadi, A. R., et al. Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach. Journal of Visualized Experiments. (147), (2019).
- Schmid, C., et al. Successful heterotopic heart transplantation in rat. Microsurgery. 15 (4), 279-281 (1994).
- Shan, J., et al. A modified technique for heterotopic heart transplantation in rats. Journal of Surgical Research. 164 (1), 155-161 (2010).
- Moris, D., et al. Mechanisms of liver-induced tolerance. Current Opinion in Organ Transplantation. 22 (1), 71-78 (2017).
- Bickerstaff, A. A., et al. Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity. Journal of Immunology. 167 (9), 4821-4827 (2001).
- Mottram, P. L., et al. Electrocardiographic monitoring of cardiac transplants in mice. Cardiovascular Research. 22 (5), 315-321 (1988).
- Torre-Amione, G., et al. Decreased expression of tumor necrosis factor-α in failing human myocardium after mechanical circulatory support: A potential mechanism for cardiac recovery. Circulation. 100 (11), 1189-1193 (1999).
- Goldstein, D. J., et al. Circulatory resuscitation with left ventricular assist device support reduces interleukins 6 and 8 levels. The Annals of Thoracic Surgery. 63 (4), 971-974 (1997).
- Tang-Quan, K. R., et al. Non-volume-loaded heart provides a more relevant heterotopic transplantation model. Transplant Immunology. 23 (1-2), 65-70 (2010).
- Lakhal-Naouar, I., et al. Transcutaneous immunization using SLA or rLACK skews the immune response towards a Th1 profile but fails to protect BALB/c mice against a Leishmania major challenge. Vaccine. 37 (3), 516-523 (2019).
- Sousa-Batista, A. J., et al. Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-loaded microparticles. International Journal for Parasitology: Drugs and Drug Resistance. , (2019).
Citar este artigo
Weigle, C. A., Lieke, T., Vondran, F. W. R., Timrott, K., Klempnauer, J., Beetz, O. An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats. J. Vis. Exp. (159), e60956, doi:10.3791/60956 (2020).