ESLP幼猪模型中的左肺原位移植
1Division of Cardiac Surgery, Department of Surgery, Faculty of Medicine,University of Alberta, 2Mazankowski Alberta Heart Institute, 3Faculty of Medicine and Dentistry,University of Alberta, 4Department of Surgery, Faculty of Medicine,University of Alberta, 5Ray Rajotte Surgical-Medical Research Institute (SMRI),University of Alberta, 6Alberta Transplant Institute, 7Canadian Donation and Transplantation Research Program
Summary
该协议描述了设计用于ESLP研究的原位左肺同种异体移植的幼猪模型。重点是麻醉和手术技术，以及关键步骤和故障排除。
Abstract
肺移植是终末期肺病的金标准治疗方法，全世界每年进行超过 4,600 例肺移植手术。然而，肺移植受到可用供体器官短缺的限制。因此，候补名单的死亡率很高。在一些中心， 非原位 肺灌注 （ESLP） 使供体肺利用率提高了 15%-20%。ESLP 已被应用为评估和修复边缘供体肺的一种方法，并已证明移植扩展标准供体 （ECD） 肺后可接受的短期和长期结果。需要大型动物（体内）移植模型来验证正在进行的 体外 研究结果。人类和猪之间的解剖学和生理学差异带来了重大的技术和麻醉挑战。易于复制的移植模型将允许对当前的ESLP策略进行体内验证，并对旨在改善供体肺功能的各种干预措施进行临床前评估。该方案描述了原位左肺同种异体移植的猪模型。这包括麻醉和手术技术、定制的手术清单、故障排除、修改以及该方法的优点和局限性。
Introduction
肺移植是终末期肺病的首要长期治疗方法。全世界每年进行超过 4,600 例肺移植手术 1.然而，肺移植目前有很大的局限性。首先，器官的必要性继续使可用的捐赠者黯然失色。尽管自 2012 年以来，由于更多移植候选者被列入名单、供体数量增加以及恢复器官使用得到改善的综合影响，肺移植率每年都在上升，但移植候补名单的死亡率并未显着下降2.器官质量问题是另一个主要限制，据报道器官利用率低至 20%–30%3,4,5。最后，肺移植术后结局的趋势不尽如人意，长期移植和患者结局仍落后于其他实体器官移植2.
一种新兴技术，即非原位肺灌注（ESLP），有可能减轻这些局限性。ESLP 已越来越多地被用作评估和修复边缘供体肺的方法，并且已证明移植扩展标准供体 （ECD） 肺6、7、8、9、10 后可接受的短期和长期结果。因此，ESLP 将一些中心的利用率提高了 15%-20%6、7、8、9、10、11。
适当的ESLP研究需要对体外研究结果进行体内验证;然而，关于 ESLP12、13、14、15 的猪肺移植模型的文献有限。此外，现有文献提供的关于约克郡猪肺移植麻醉管理的细节不足，肺移植的血流动力学可能非常不稳定12,13,14,15。建立一个易于重复的模型将允许对当前的ESLP策略进行体内验证，并对各种干预措施进行临床前评估，以减少肺缺血再灌注损伤。本研究的目的是描述用于 ESLP 的原位左肺同种异体移植的猪模型。该方案包括麻醉剂和手术技术的描述、自定义手术清单以及有关故障排除体验和方案修改的详细信息。本文还讨论了左肺猪移植模型的局限性和益处。本手稿没有概述35-50公斤约克郡猪猪肺的检索过程，也没有涵盖ESLP的建立和终止。该协议专门针对受体移植手术。
Protocol
所有程序均按照加拿大动物护理委员会的指导方针和实验动物的护理和使用指南进行。这些协议得到了阿尔伯塔大学机构动物护理委员会的批准。该方案已应用于35-50公斤的雌性幼年约克郡猪。猪是无病原体的食品级标本。它们是从加拿大艾伯塔省埃德蒙顿的猪研究和技术中心购买的（https://srtc.ualberta.ca）。所有参与ESLP程序的人都接受了适当的生物安全培训。 1.术前准备和麻?…
Representative Results
所有结果均在 NPV-ESLP16 12 小时后再灌注 4 小时的背景下进行。在肺外植体期间，有几种临床结果需要预期（图3）。通常，猪在左肺移植成功后将保持血流动力学稳定，但由于对手术的血管舒张反应，可能需要低剂量输注去氧肾上腺素（剂量范围：2-10 mg/h）。心率目标应约为 100-120 次/分，SpO 2 > 90% 时呼吸频率 （RR） 为 8-30%，平均动脉压 （MAP） > 60 m…
Discussion
该协议涉及几个关键的手术步骤，需要进行故障排除以确保移植和肺部评估成功。与成年人的肺相比，幼年猪肺非常脆弱，因此手术外科医生在处理猪肺时必须小心。在进行 12 小时的 ESLP 后尤其如此，因为器官将承担液体量并且容易因过度操作而受伤。任何过大的压力都会导致实验肺不张或创伤，从而影响评估结果。同样，幼猪的血管结构非常脆弱。避免 PA 夹扭转至关重要，因为这会导致组织?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究由大学医院基金会资助。
Materials
| ABL 800 FLEX Blood Gas Analyzer | Radiometer | 989-963 | |
| Adult-Pediatric Electrostatic Filter HME – Small | Covidien | 352/5877 | |
| Allison Lung Retractor | Pilling | 341679 | |
| Arterial Filter | SORIN GROUP | 01706/03 | |
| Backhaus Towel Clamp | Pilling | 454300 | |
| Bovine Serum Albumin | MP biomedicals | 218057791 | |
| Biomedicus Pump | Maquet | BPX-80 | |
| Bronchoscope | |||
| Cable Ties – White 12” | HUASU International | HS4830001 | |
| Calcium Chloride | Fisher Scientific | C69-500G | |
| Cooley Sternal Retractor | Pilling | 341162 | |
| CUSHING Gutschdressing Forceps | Pilling | 466200 | |
| Debakey-Metzenbaum Dissecting | Pilling | 342202 | |
| Scissors | Pilling | 342202 | |
| DeBakey Peripheral Vascular Clamp | Pilling | 353535 | |
| Debakey Straight Vascular Tissue Forceps | Pilling | 351808 | |
| D-glucose | Sigma-Aldrich | G5767-500G | |
| Drop sucker | |||
| Endotracheal Tube 9.0mm CUFD | Mallinckrodt | 9590E | |
| Flow Transducer | BIO-PROBE | TX 40 | |
| Infusion Pump | Baxter | AS50 | |
| Inspire 7 M Hollow Fiber Membrane Oxygenator | SORIN GROUP | K190690 | |
| Intercept Tubing Connector 3/8" x 1/2" | Medtronic | 6013 | |
| Intercept Tubing 1/4" x 1/16" x 8' | Medtronic | 3108 | |
| Intercept Tubing 3/8" x 3/32" x 6' | Medtronic | 3506 | |
| Laryngoscope | N/A | N/A | Custom-made with 10-inch blade |
| Metzenbaum Dissecting Scissors | Pilling | 460420 | |
| Medical Carbon Dioxide Tank | Praxair | 5823115 | |
| Medical Oxygen Tank | Praxair | 2014408 | |
| Medical Nitrogen Tank | Praxair | NI M-K | |
| Mosquito Clamp | Pilling | 181816 | |
| Harken Auricle Clamp | |||
| Organ Chamber | Tevosol | ||
| PlasmaLyte A | Baxter | TB2544 | |
| Poole Suction Tube | Pilling | 162212 | |
| Potassium Phosphate | Fischer Scientific | P285-500G | |
| PERFADEX Plus | XVIVO | 19811 | |
| Satinsky Clamp | Pilling | 354002 | |
| Scale | TANITA | KD4063611 | |
| Silicon Support Membrane | Tevosol | ||
| Sodium Bicarbonate | Sigma-Aldrich | 792519-1KG | |
| Sodium Chloride 0.9% | Baxter | JB1324 | |
| Sorin XTRA Cell Saver | SORIN GROUP | 75221 | |
| Sternal Saw | Stryker | 6207 | |
| Surgical Electrocautery Device | Kls Martin | ME411 | |
| TruWave Pressure Transducer | Edwards | VSYPX272 | |
| Two-Lumen Central Venous Catheter 7fr X2 | Arrowg+ard | CS-12702-E | |
| Vorse Tubing Clamp | Pilling | 351377 | |
| Willauer-Deaver Retractor | Pilling | 341720 | |
| Yankauer Suction Tube | Pilling | 162300 | |
| 0 ETHIBOND Green 1X36" Endo Loop 0 | ETHICON | D8573 | |
| 0 PDS II CP-1 2×27” | ETHICON | Z467H | |
| 1 VICRYL MO-4 1×18” | ETHICON | J702D | |
| 2-0 SILK Black 12" x 18" Strands | ETHICON | SA77G | |
| 4-0 PROLENE Blue TF 1×24” | ETHICON | 8204H | |
| 6-0 PROLENE Blue BV 2×30” | ETHICON | M8776 | |
| 21-Gauge Needle |
Riferimenti
- Chambers, D. C., et al. The international thoracic organ transplant registry of the international society for heart and lung transplantation: Thirty-fifth adult lung and heart-lung transplant report-2018; focus theme: Multiorgan transplantation. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 37 (10), 1169-1183 (2018).
- Valapour, M., et al. OPTN/SRTR 2017 annual data report: Lung. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19, 404-484 (2019).
- Klein, A. S., et al. Organ donation and utilization in the united states, 1999-2008. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 10 (4), 973-986 (2010).
- Kotecha, S., et al. Continued successful evolution of extended criteria donor lungs for transplantation. The Annals of Thoracic Surgery. 104 (5), 1702-1709 (2017).
- Singh, E., et al. Sequence of refusals for donor quality, organ utilization, and survival after lung transplantation. The Journal of Heart and Lung Transplantation. 38 (1), 35-42 (2019).
- Cypel, M., et al. Normothermic ex vivo lung perfusion in clinical lung transplantation. The New England Journal of Medicine. 364 (15), 1431-1440 (2011).
- Wallinder, A., et al. Early results in transplantation of initially rejected donor lungs after ex vivo lung perfusion: A case-control study. European Journal of Cardio-Thoracic Surgery: Official Journal of the European Association for Cardio-Thoracic Surgery. 45 (1), 40-44 (2014).
- Sage, E., et al. Lung transplantation from initially rejected donors after ex vivo lung reconditioning: The french experience. European Journal of Cardio-Thoracic Surgery: Official Journal of the European Association for Cardio-Thoracic Surgery. 46 (5), 794-799 (2014).
- Valenza, F., et al. Extracorporeal lung perfusion and ventilation to improve donor lung function and increase the number of organs available for transplantation. Transplantation Proceedings. 44 (7), 1826-1829 (2012).
- Fildes, J. E., et al. Clinical outcome of patients transplanted with marginal donor lungs via ex vivo lung perfusion compared to standard lung transplantation. Transplantation. 99 (5), 1078-1083 (2015).
- Cypel, M., et al. Experience with the first 50 ex vivo lung perfusions in clinical transplantation. The Journal of Thoracic and Cardiovascular Surgery. 144 (5), 1200-1206 (2012).
- Clark, S. C., et al. A new porcine model of reperfusion injury after lung transplantation. Laboratory Animals. 33, 135-142 (1999).
- Karimi, A., et al. Technical pearls for swine lung transplantation. Journal of Surgical Research. 171, 107-111 (2011).
- Kruger, M., et al. Porcine pulmonary auto-transplantation for ex vivo therapy as a model for new treatment strategies. Interactive CardioVascular and Thoracic Surgery. 23, 358-366 (2016).
- Mariscal, A., et al. Pig lung transplant survival model. Nature Protocols. 13, 1814-1828 (2018).
- Aboelnazar, N. S., et al. Negative pressure ventilation decreases inflammation and lung edema during normothermic ex vivo lung perfusion. The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 37 (4), 520-530 (2018).
Citazione di questo articolo
Forgie, K. A., Fialka, N., Khan, M., Buchko, M., Hatami, S., Himmat, S., Qi, X., Wang, X., Buswell, K., Edgar, R., Domahidi, D., Freed, D. H., Nagendran, J. Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP. J. Vis. Exp. (180), e62979, doi:10.3791/62979 (2022).