非体外循环冠状动脉旁路移植术治疗慢性心肌缺血的外科猪模型
Summary
该协议提出了一种外科大动物模型, 慢性, 单血管缺血, 导致区域异常, 但不会造成梗塞, 称为冬眠心肌。在慢性缺血的建立后, 动物用非泵浦的利马-小伙子冠状动脉旁路移植术治疗 revascularize 缺血性组织。
Abstract
慢性心肌缺血损害心功能, 但不导致梗塞, 称为冬眠心肌 (HM)。冠状动脉疾病 (CAD) 患者的一个大的临床子集有, 除了造成功能受损, 使他们在心律失常和未来心脏事件更高的风险。这种情况的标准治疗是血管重建, 但这已被证明是一个不完善的治疗。大多数临床前心脏研究的重点是梗死模型的心脏缺血, 留下这个子集的慢性缺血患者大多缺乏服务。为了解决这一研究中的差距, 我们开发了一种具有良好特征和高度可重复性的猪冬眠心肌模型, 因为猪是人类心脏疾病的理想转化模型。除了建立这种独特的疾病模型, 我们已经优化了一个临床相关的治疗模式的冠状动脉旁路手术的猪。这使我们能够准确地研究旁路手术对心脏疾病的影响, 以及调查其他或替代疗法。该模型通过在一只年轻的猪的左前降 (童子) 动脉内植入一个小蟒蛇来手术诱导单血管狭窄。随着猪的生长, 蟒蛇造成了逐渐的狭窄, 导致慢性缺血的区域功能受损, 但保持组织的生存能力。在冬眠心肌表型的建立后, 我们进行非体外循环冠状动脉旁路移植手术, revascularize 缺血性区域, 模仿临床患者的金本位治疗。
Introduction
冠心病 (CHD) 影响了估计在美国的1550万人1 , 是全球死亡的主要原因之一。虽然近年来冠心病的死亡率下降了, 但病人和保健系统的发病率和负担仍然很高1。严重 CAD 的初级治疗是血管重建, 改善生存和减少心绞痛2,3,4。然而, 心脏功能往往仍然压抑, 特别是在增加的工作量和可能进展到心力衰竭5,6。冠状动脉旁路手术 (CABG) 治疗慢性缺血的临床试验表明, 生存和症状有改善, 但弹射分数仅显示 8-10% 7,8的适度改善。我们的创新性和良好特征的猪慢性心肌缺血模型是一个渐进性血管狭窄的临床 CAD 模型。我们已经表明减少的心肌收缩力导致的血液流量的逐步减少9。心肌不梗塞, 可以保持在这种情况下可行。恢复是可能的, 虽然结果是可变的, 即使及时的血管重建。长期缺血性心肌仍然可行的特点是减少血流量和功能休息与保留收缩储备已被称为 HM 和治疗需要冠状动脉搭桥术。虽然 HM 重建术应恢复收缩功能, 实验和临床观察表明, 恢复不完整8,10。
HM 的特点是存在的可行的, 但功能失调的心肌存在减少区域血流11。尽管在休息时收缩力和新陈代谢活动受损, 但在强心剂刺激下, 他能够表现出功能性和新陈代谢的储备12。在大多数的冠心病患者中都有怀疑, 包括广泛的疾病。在这项协议中, 我们展示了我们建立的猪模型, 它绕过左内乳动脉 (利马) 到童子动脉, 模仿临床情况。猪提供了一个很好的模型的心脏疾病超过其他大型动物, 因为他们没有心外膜桥接络。这允许小男孩的狭窄导致区域缺血13。
在这里, 我们描述了通过在童子动脉中创建单血管狭窄来诱导猪冬眠心肌的手术方法。当慢性缺血已经建立 (8 周后植入的小伙子蟒蛇), 我们描述了重建的方法, 在我们的猪模型的临床治疗: 非体外循环冠状动脉旁路移植。这些手术方法不仅可用于研究慢性心肌缺血的临床相关模型, 还可以探讨旁路手术对心脏缺血的影响, 以及试验电位交替或辅助治疗心肌缺血的疗效。
Protocol
所有动物研究都获得了明尼阿波利斯 VA 医疗中心和明尼苏达大学的机构动物护理和使用委员会的批准。遵循目前的国家卫生研究院的使用和护理实验动物指南。 1. 冬眠手术 动物制剂 要创建冬眠心肌模型, 使用5周的老, 女性, 约克郡猪, 它的重量在8-10 公斤的范围内以9.2 公斤作为平均重量。 对于手术前的健康监测, 在手术前3天内对动物进行称量。 <li…
Representative Results
在最初的冬眠手术后, 临床成像技术, 如血管造影或心脏 MRI (图 1A) 可以观察到大于70% 的狭窄。手术后8周, 回声或心脏 MRI 对区域功能的分析显示在增加的工作负载下功能减少 (图 2)。这可以通过刺激心脏与多巴酚丁胺 (5 毫克/千克/分钟), 并测量周向应变和区域壁厚。PET 成像显示, 与同一心脏的非童子领地相比, 冬眠?…
Discussion
在这里, 我们表明, 我们的猪模型, 准确地模仿病人的临床经验, 单血管疾病和保留左心室功能。在血管重建之前, 单血管的动物在全球功能上表现出最小的损伤, 以弹射分数来衡量, 但区域壁增厚明显减少。在血管重建术后, 三月的 CMR 成像显示保留的生存能力和移植通畅性, 但持续的区域功能障碍, 如低剂量多巴酚丁胺压力测试的收缩储备的估计指出。
初始休眠操作有几个关键?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国 (美国) #I01 BX000760 (肯尼迪) 的 VA 功绩审查的支持。退伍军人事务部 BLR & D。这项工作的内容并不代表美国政府退伍军人事务部的意见。
Materials
| Bair Hugger | 3M | Model 505 | Patient Warming system |
| SR Buprenorphine 10 mg/mL | Abbott Labs | NADA 141-434 | Post operative Analgesic |
| Surgical Spring Clip | Applied Medical | A1801 | Clamp end of LIMA after takedown |
| Arterial Line Kit | Arrow | ASK-04510-HF | Femoral catheter for blood pressure monitoring |
| 1000mL 0.9% Sodium chloride | Baxter | 2B1324X | IV replacement fluid |
| 250 mL 0.9% saline | Baxter | UE1322D | Replacement IV Fluid |
| 500mL 0.9% Sodium chloride | Baxter | 2B1323Q | Drug delivery, Provide mist for Blower Mister |
| Flo-thru 1.0 | Baxter | FT-12100 | used to anastomos LIMA to L |
| Flo-thru 1.25 | Baxter | FT-12125 | |
| Flo-thru 1.5 | Baxter | FT-12150 | |
| Flo-thru 2.0 | Baxter | FT-12200 | |
| Cloroprep | Becton Dickenson | 260815 | Surgical skin prep |
| Meloxicam | Boehringer Ingelheim Vetmedica, Inc. | 0010-6013-01 | NSAID for analgesia |
| Hypafix | BSN Medical | 4210 | Secure wound dressing and IV catheters |
| IV Tubing for Blower Mister | Carefusion | 42493E | Adapts to IV Fluids for Blower/Mister |
| Bovie Cautery hand piece | Covidien | E2516 | Hemostasis |
| Chest Tube | Covidien | 8888561043 | Evacuates air from chest cavity |
| Monopolar Cautery | Covidien | Valleylab FT10 | Hemostasis |
| Telpha pad | Covidien | 2132 | Sterile wound dressing |
| 4-0 Tevdek II Strands | Deknatel | 7-922 | Suture to secure constrictor around LAD |
| Propofol | Diprivan | 269-29 | Induction agent |
| long blade for laryngoscope | DRE | 12521 | Allows for visualization of trachea for intubation |
| ECG Pads | DRE | 1496 | Monitor heart rhythm |
| laryngoscope | DRE | 12515 | |
| Anesthesia Machine + ventilator | DRE Drager- Fabius Tiro | DRE0603FT | Deliver Oxygen and inhalant to patient |
| 5 Ethibond | Ethicon | MG46G | Suture |
| 0 Vicryl | Ethicon | J208H | Suture |
| 2-0 Vicryl | Ethicon | J317H | Suture |
| 3-0 Vicryl | Ethicon | VCP824G | Suture |
| 7-0 Prolene | Ethicon | M8702 | Suture |
| Dermabond | Ethicon | DNX12 | Skin adhesive |
| Ligaclips | Ethicon | MSC20 | Surgical Staples for LIMA takedown |
| Sterile Saline 20 mL | Fisher Scientific | 20T700220 | Flush for IV catheters |
| Telazol 100 mg/mL | Fort Dodge | 01L60030 | Pre operative Sedative |
| Triple Antibiotic Ointment | Johnson & Johnson | 23734 | Topical over wound |
| 6.0 mm ID endotracheal tube | Mallinckrodt | 86049 | Establish airway for Revasc,MRI and Termination |
| 1" medical tape | Medline | MMM15271Z | Secure wound dressing and IV catheters |
| 4.0 mm ID endotracheal tube | Medline | DYND43040 | Establish airway for Hibernation |
| 4.5 mm ID endotracheal tube | Medline | DYND43045 | Establish airway for Hibernation |
| 5.0 mm ID endotracheal tube | Medline | DYND43050 | Establish airway for Hibernation |
| 6.5 mm ID endotracheal tube | Medline | DYND43065 | Establish airway for Revasc,MRI and Termination |
| 7.0 mm ID endotracheal tube | Medline | DYND43070 | Establish airway for Revasc,MRI and Termination |
| Bair Hugger Blanket - Large size, underbody | Medline | AUG55501 | Patient Warming system |
| Basic pack | Medline | DYNJP1000 | Sterile drapes and table cover |
| Bone Wax | Medline | ETHW31G | Hemostasis of cut bone |
| Suction tubing | Medline | DYND50223 | |
| Suction Container | Medline | DYNDCL03000 | |
| 1 mL Syringe | Medtronic/Covidien | 1188100777 | Administer injectable agents |
| 12 mL Syringe | Medtronic/Covidien | 8881512878 | Administer injectable agents |
| 20 mL Syringe | Medtronic/Covidien | 8881520657 | Administer injectable agents |
| 3 mL Syinge | Medtronic/Covidien | 1180300555 | Administer injectable agents |
| 6 mL Syringe | Medtronic/Covidien | 1180600777 | Administer injectable agents |
| 60 mL Syringe | Medtronic/Covidien | 8881560125 | Administer injectable agents |
| Blower Mister Kit | Medtronic/Covidien | 22120 | Clears surgical field for vessel anastomosis |
| Roncuronium | Mylan | 67457-228-05 | Neuromuscular blocking agent |
| # 40 clipper blade | Oster | 078919-016-701 | Remove hair from surgery sites |
| Hair Clipper | Oster | 078566-011-002 | Remove hair from surgery sites |
| Bupivicaine | Pfizer | 00409-1161-01 | Local Anesthetic |
| Cephazolin | Pfizer | 00409-0805-01 | Antibiotic |
| Heparin | Pfizer | 0409-2720-03 | anticoaggulant |
| Lidocaine 2% | Pfizer | 00409-4277-01 | Local Anesthetic/ antiarrthymic |
| Succinylcholine 20 mg/mL | Pfizer | 00409-6629-02 | Neuromuscular blocking agent |
| Anesthesia Monitor | Phillips Intellivue | MP70 | Supports ventilation with inhalant |
| Artificial Tears | Rugby | 0536-1086-91 | Lubricate eyes to prevent corneal drying |
| Buprenorphine 0.3 mg/mL | Sigma Aldrich | B9275 | Pre operative Analgesic for survivial procedures |
| Isoflurane | Sigma Aldrich | CDS019936 | General Anesthestic- Inhalant |
| 36” Pressure monitoring tubing | Smith’s Medical | MX563 | Connect art. Line to transducer |
| 48” Pressure monitoring tubing | Smith’s Medical | MX564 | Connect art. Line to transducer |
| Jelco 18 ga IV catheter | Smiths medical | 4054 | IV access in Revasc, MRI and Term |
| Jelco 20 ga IV catheter | Smiths medical | 4059 | IV access in the MRI |
| Jelco 22 ga IV catheter | Smiths medical | 4050 | IV access in Hibernation Procedure |
| OPVAC Synergy II | Terumo Cardiovascular System | 401-230 | Heart positioner and Stabilizer |
| Sternal Saw/ Necropsy Saw | Thermo Fisher | 812822 | Used to open chest cavity |
| Delrin Constrictor | U of MN | Custom made | Creates stenosis of LAD |
| Oxygen Tank E cylinder | various | various | Used for Blower Mister if anesthesia machine doesn't have auxiliary flow meter |
| Pressure Transducer | various | Must adapt to anesthesia monitor | Monitor direct arterial pressures |
| Xylazine 100 mg/mL | Vedco | 468RX | Pre operative Sedative/ analgesic |
References
- Mozaffarian, D., et al. Heart Disease and Stroke Statistics-2016 Update A Report From the American Heart Association. Circulation. 133 (4), 350 (2015).
- Di Carli, M. F., et al. Value of metabolic imaging with positron emission tomography for evaluating prognosis in patients with coronary artery disease and left ventricular dysfunction. Am J Cardiol. 73 (8), 527-533 (1994).
- Gerber, B. L., et al. Prognostic value of myocardial viability by delayed-enhanced magnetic resonance in patients with coronary artery disease and low ejection fraction: impact of revascularization therapy. J Am Coll Cardiol. 59 (9), 825-835 (2012).
- McFalls, E., et al. Regional glucose uptake within hypoperfused swine myocardium as measured by positron emission tomography. Am J Physiol-Heart C. 272 (1), 343-349 (1997).
- Velazquez, E. J., et al. Coronary-artery bypass surgery in patients with left ventricular dysfunction. New Engl J Med. 364 (17), 1607-1616 (2011).
- Canty, J. M., Fallavollita, J. A. Hibernating myocardium. J Nucl Cardiol. 12 (1), 104-119 (2005).
- Joyce, D., et al. Revascularization and ventricular restoration in patients with ischemic heart failure: the STICH trial. Curr Opin Cardiol. 18 (6), 454-457 (2003).
- Shah, B. N., Khattar, R. S., Senior, R. The hibernating myocardium: current concepts, diagnostic dilemmas, and clinical challenges in the post-STICH era. Eur Heart J. 34 (18), 1323-1336 (2013).
- Stone, L. L. H., et al. Magnetic resonance imaging assessment of cardiac function in a swine model of hibernating myocardium 3 months following bypass surgery. J Thorac Cardiov Sur. , (2016).
- Kukulski, T., et al. Implication of right ventricular dysfunction on long-term outcome in patients with ischemic cardiomyopathy undergoing coronary artery bypass grafting with or without surgical ventricular reconstruction. J Thorac Cardiov Sur. 149 (5), 1312-1321 (2014).
- Rahimtoola, S. H. The hibernating myocardium. Am Heart J. 117 (1), 211-221 (1989).
- Fallavollita, J. A., Logue, M., Canty, J. M. Stability of hibernating myocardium in pigs with a chronic left anterior descending coronary artery stenosis: absence of progressive fibrosis in the setting of stable reductions in flow, function and coronary flow reserve. J Am Coll Cardiol. 37 (7), 1989-1995 (2001).
- White, F. C., Carroll, S. M., Magnet, A., Bloor, C. M. Coronary collateral development in swine after coronary artery occlusion. Circ Res. 71 (6), 1490-1500 (1992).
- Viscardi, A. V., Hunniford, M., Lawlis, P., Leach, M., Turner, P. V. Development of a Piglet Grimace Scale to Evaluate Piglet Pain Using Facial Expressions Following Castration and Tail Docking: A Pilot Study. Front Vet Sci. 4, 51 (2017).
- Holley, C. T., Long, E. K., Lindsey, M. E., McFalls, E. O., Kelly, R. F. Recovery of hibernating myocardium: what is the role of surgical revascularization. J Cardiac Surg. 30 (2), 224-231 (2015).
- Cooper, H. A., Braunwald, E. Clinical importance of stunned and hibernating myocardium. Coronary Artery Dis. 12 (5), 387-392 (2001).
- McFalls, E. O., et al. Utility of positron emission tomography in predicting improved left ventricular ejection fraction after coronary artery bypass grafting among patients with ischemic cardiomyopathy. Cardiology. 93 (1-2), 105-112 (2000).
- Pasquet, A., et al. Prediction of global left ventricular function after bypass surgery in patients with severe left ventricular dysfunction. Impact of pre-operative myocardial function, perfusion, and metabolism. Eur Heart J. 21 (2), 125-136 (2000).
Cite This Article
Hocum Stone, L., Wright, C., Chappuis, E., Messer, M., Ward, H. B., McFalls, E. O., Kelly, R. F. Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery. J. Vis. Exp. (133), e57229, doi:10.3791/57229 (2018).