正交心脏移植的临床前猪模型
1Division of Cardiovascular Surgery, Peter Munk Cardiac Center, Toronto General Hospital,University Health Network, 2Institute of Medical Science,University of Toronto, 3Perfusion and Anesthesia Services, Toronto General Hospital,University Health Network, 4Department of Anesthesia and Pain Management, Toronto General Hospital,University Health Network, 5Department of Anesthesia,University of Toronto, 6Department of Surgery, Faculty of Medicine,University of Toronto
Summary
在这里,我们描述了一个临床前大动物(猪)的正交心脏移植模型,该模型已经牢固建立并用于研究新的心脏保护策略。
Abstract
在第一次成功报告发布五十年后,心脏移植仍然是符合资格的晚期心力衰竭患者的黄金标准治疗。多种小动物心脏移植模型已用于研究新疗法的急性和长期影响。然而,很少在临床试验中进行测试和证明成功。在临床相关的大动物模型中评估新疗法,以便有效可靠地翻译基础研究的发现,这一点至关重要。在这里,我们描述了一个临床前大动物(猪)的正交心脏移植模型,该模型已经牢固建立,并以前用于研究新的心脏保护策略。该程序侧重于急性缺血再灌注损伤,是研究在较小的实验模型(如鼠害模型)中经过测试和验证的新型干预措施的可靠方法。我们证明了它在评估移植后早期的心脏性能以及该模型启用的其他潜在可能性方面的有用性。
Introduction
在第一次成功报告50年后,心脏移植仍然是符合资格的晚期心力衰竭患者的黄金标准治疗。虽然长达4小时的缺血时间是耐受的,但超过6小时的缺血时间与劣质结果2有关。原发性移植功能障碍仍然是移植2、3后早期发病和死亡的主要原因。原发性移植功能障碍的病因是多因素的,包括使用边缘器官、受体肺血管疾病、高急性排斥和在移植时遭受的缺血再灌注损伤3。
多项研究已研究新的方法,供体心脏保存,以减少原发性移植功能障碍的发生率4,5,6,7。评估缺血性灌注损伤或异位心脏移植的小鼠模型中的新技术和治疗方法是常见的做法。此外,小动物模型允许生存模型和长期随访,以调查排斥和心脏异体血管病11,12,13的发展。然而,这些策略大多未能通过初步的临床试验,或从未达到这一阶段。在临床相关的大动物模型中评估新疗法,以便有效可靠地翻译基础研究的发现,这一点至关重要。
使用大型动物模型时,猪心通常被认为是与人类心脏在解剖学上最相似的。因此,它是进行心脏外科研究的理想平台。然而,在使用猪模型时,需要考虑几个重要因素。首先,组织通常被描述为脆弱和易碎,特别是在右中庭和肺动脉,容易撕裂14。此外,猪心脏被认为对操纵敏感,容易出现心律失常,这就是为什么在实验开始时应该定期对每只动物进行抗动脉炎。猪模型和临床心脏移植之间的一个重要解剖区别是猪的左血脉,直接进入冠状动脉。这必须在接受者过程中连接,以避免连续出血。最后,猪模型对缺血非常敏感,但仍适用于心脏移植的急性研究。
本手稿描述了一个临床前大动物(猪)的正交心脏移植模型,该模型已经牢固建立,并用于研究新的心脏保护策略5,6,8 ,9.
Protocol
机构动物护理委员会批准了所有实验规程,动物按照国家研究理事会实验室动物资源研究所编写的《保护和使用实验室动物指南》进行了处理。雄性约克郡猪(40-50公斤)用于进行正交心脏移植(动物大小可能因调查人员的判断力和实验目标而异)。 1. 捐助者程序 麻醉诱导和动物制备: 使用氯胺酮(20毫克/千克)、米达佐兰(0.3毫克/千克)和阿托品(0.04毫克/千克)的肌肉注射?…
Representative Results
这种临床前模型自1994年以来已成功使用5,6,8,9。表1展示了在基线上采取的压力-体积关系和超声心动图参数的代表性结果,以及一组5个实验中3小时移植后的结果。虽然我们看到移植后肌体收缩性下降,但在统计学上并不显著。 图 1显示了从同一个?…
Discussion
本手稿描述了一个大型动物正交心脏移植的临床前模型。各种异位心脏移植的小型动物模型已成功地用于研究新疗法对改善器官保存和减少缺血再灌注损伤的效果11、12、 13.此外,小型动物模型允许生存模型和长期随访,以调查排斥和心脏异体血管病的发展11,12,13。<sup class="xref"…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者没有承认。
Materials
| Amiodarone | Purchased from institutional pharmacy | ||
| Angiocath 20G | BD | 381704 | |
| Calcium Chloride 1g/10ml | Purchased from institutional pharmacy | ||
| Cardioplegia solution | This should be chosen at the investigators discretion. | ||
| Cautery Pencil | Covidien | E2515H | |
| Central Venous Catheter double-lumen | Cook Medical | C-UDLM-501J-LSC | |
| CPB pack | Medtronic | Custom-made cardiopulmonary bypass perfusion circuit. | |
| D5W 5% 250ml | Baxter | JB1064 | |
| DLP Aortic Root Cannula/stabber | Medtronic | 12218 | |
| DLP single-lumen venous cannula (24F or 28F) | This should be chosen at the investigators discretion. | ||
| Dobutamine | Purchased from institutional pharmacy | ||
| Electrode Polyhesive | Covidien | E7507 | |
| EOPA arterial cannula (17F or 21F) | This should be chosen at the investigators discretion. | ||
| Epinephrine | Purchased from institutional pharmacy | ||
| Eppendorf Tubes, 1.5 mL | Sarstedt | 72.690.001 | |
| Gloves, nitrile, medium | Fischer | 27-058-52 | |
| Heparin 1000 IU/ml | Purchased from institutional pharmacy | ||
| Ketalean (Ketamine) inj. 100mg/ml, 50ml/vial | Health Canada | Requires health canada approval | |
| Lidocaine/Xylocaine 1% | Purchased from institutional pharmacy | ||
| Magnesium Sulfate 5g/10ml | Purchased from institutional pharmacy | ||
| Midazolam inj. USP 5mg/ml vial/10ml | Health Canada | Requires Health canada approval | |
| MPS Quest delivery disposable pack | Quest medical | 5001102-AS | |
| NACL 0.9% 1L | Baxter | JB1324 | |
| Organ Bag | CardioMed | 2990 | |
| Pipette Tips, 1 mL | Fisherbrand | 02-707-405 | |
| Propofol 1mg/ml | Purchased from institutional pharmacy | ||
| Rocuronium | Purchased from institutional pharmacy | ||
| Set Admin Prim NF PB W/Checkvalve | Smith Medical | 21-0442-25 | Intravenous infusion pump line. Researchers should choose infusion lines compatible with the infusion pump available at their facilities |
| Set Intro Sheath 8.5FRx 10CM | Arrow | SI-09880 | |
| Sofsilk 0 wax coated | Covidien | S316 | |
| Solumedrol 500mg/5ml | Purchased from institutional pharmacy | ||
| Suction tip | Covidien | 8888501023 | |
| Suction Tubing 1/4" x 120" | Med-Rx | 70-8120 | |
| Suture 5.0 Prolene BB | Ethicon | 8580H | |
| Suture Prolene Blum 4-0 SH 36 | Ethicon | 8521H | |
| Sutures 2.0 Prolene Blu M SH | Ethicon | 8523H | |
| Sutures BB 4.0 Prolene | Ethicon | 8881H | |
| Tracheal Tube, 6.5mm | Mallinckrodt | 86449 | |
Referências
- Lund, L. H., Edwards, L. B., et al. The Registry of the International Society for Heart and Lung Transplantation: Thirty-third Adult Heart Transplantation Report-2016; Focus Theme: Primary Diagnostic Indications for Transplant. The Journal of Heart and Lung Transplantation. 35 (10), 1158-1169 (2016).
- Lund, L. H., Edwards, L. B., et al. The Registry of the International Society for Heart and Lung Transplantation_ Thirty-first Official Adult Heart Transplant Report-2014; Focus Theme_ Retransplantation. The. Journal of Heart and Lung Transplantation. 33 (10), 996-1008 (2014).
- Cosío Carmena, M. D. G., Gómez Bueno, M., et al. Primary graft failure after heart transplantation: characteristics in a contemporary cohort and performance of the RADIAL risk score. The Journal of Heart and Lung Transplantation. 32 (12), 1187-1195 (2013).
- Fedak, P. W. M., Rao, V., et al. Combined endothelial and myocardial protection by endothelin antagonism enhances transplant allograft preservation. The Journal of Thoracic and Cardiovascular Surgery. 129 (2), 407-415 (2005).
- Ramzy, D., Rao, V., et al. Cardiac allograft preservation using donor-shed blood supplemented with L-arginine. The Journal of Heart and Lung Transplantation. 24 (10), 1665-1672 (2005).
- Rao, V., Feindel, C. M., Weisel, R. D., Boylen, P., Cohen, G. Donor blood perfusion improves myocardial recovery after heart transplantation. Journal of Heart and Lung Transplantation. 16 (6), 667-673 (1997).
- Wicomb, W. N., Cooper, D. K., Barnard, C. N. Twenty-four-hour preservation of the pig heart by a portable hypothermic perfusion system. Transplantation. 34 (5), 246-250 (1982).
- Badiwala, M. V., Ramzy, D., et al. Donor pretreatment with hypertonic saline attenuates primary allograft dysfunction: a pilot study in a porcine model. Circulation. 120, 206-214 (2009).
- Ribeiro, R. V. P., Badiwala, M. V., Ramzy, D., Tumiati, L. C., Rao, V. Recipient Hypertonic Saline Infusion Prevents Cardiac Allograft Dysfunction. The Journal of Thoracic and Cardiovascular Surgery. , (2018).
- Townsend, D. Measuring Pressure Volume Loops in the Mouse. Journal of Visualized Experiments. (111), e53810 (2016).
- Ratschiller, T., Deutsch, M. -. A., et al. Heterotopic Cervical Heart Transplantation in Mice. Journal of Visualized Experiments. (102), e52907 (2015).
- Fukunaga, N., Bissoondath, V., Rao, V. Submandibular Gland-preserving Technique for Heterotopic Cervical Heart Transplantation in Mice. Transplantation. 1, (2018).
- Gong, W. Mouse Heterotopic Abdominal Heart Transplant Model. Rodent Transplant Medicine. , 107-118 (2014).
- Robinson, N., Souslian, L., Gallegos, R. P., Rivard, A. L., Dalmasso, A. P., Bianco, R. W. Animal Models for Cardiac Research. Handbook of Cardiac Anatomy, Physiology, and Devices. , 469-491 (2015).
- Bianco, R. W., Gallegos, R. P., Rivard, A. L., Voight, J., Dalmasso, A. P. Animal Models for Cardiac Research. Handbook of Cardiac Anatomy, Physiology, and Devices. , 393-410 (2009).
Citar este artigo
Ribeiro, R. V. P., Alvarez, J. S., Yu, F., Adamson, M. B., Fukunaga, N., Serrick, C., Bissoondath, V., Meineri, M., Badiwala, M. V., Rao, V. A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation. J. Vis. Exp. (146), e59197, doi:10.3791/59197 (2019).