猪静脉移植病模型的建立与评价
Summary
在该方案中，通过左胸壁上的小切口进行新型猪静脉旁路移植术，而无需体外循环。进行了术后病理学研究，显示内膜增厚。
Abstract
静脉移植疾病 （VGD） 是冠状动脉旁路移植术 （CABG） 失败的主要原因。需要CABG-VGD的大型动物模型来研究疾病机制和制定治疗策略。
为了进行手术，我们通过第三肋间隙进入心腔，仔细解剖乳腺内静脉并将其浸入生理盐水中。然后治疗右主冠状动脉缺血。切开目标血管，放置分流栓，并吻合移植静脉远端。升主动脉部分阻塞，穿孔后吻合移植静脉近端。检查移植静脉是否通畅，并结扎右冠状动脉近端。
在迷你猪中进行冠状动脉旁路移植术，以收获左内乳静脉以用作血管移植物。血清生化检查用于评估手术后动物的生理状态。超声检查显示移植血管近端、中端和远端无梗阻。在手术模型中，在CABG手术后的组织学检查中观察到移植物中的湍流血流，并且在移植物中观察到与内膜增生相关的静脉移植物狭窄。本研究为建立可重复的冠状动脉旁路移植术诱导的VGD模型提供了详细的外科手术。
Introduction
尽管近年来冠心病死亡率显著下降，但美国每年有一半的中年人出现缺血性心脏相关症状，三分之一的老年人死于冠心病1。冠状动脉旁路移植术（CABG）是改善心肌缺血的有效手术方式，更重要的是，它是治疗多血管冠状动脉疾病不可替代的手术方式2。然而，随着时间的推移，血管移植物会出现炎症、内膜增生和进行性动脉粥样硬化，已知会导致静脉移植失败或静脉移植疾病 （VGD）3。在冠状动脉旁路移植术后的患者中，如果发生再狭窄，在某些情况下只能更换患病的血管2。老年患者和增加的合并症使得重做冠状动脉旁路移植术非常具有挑战性。延缓或控制与移植血管相关的病理问题是一个亟待解决的问题。需要CABG-VGD的大型动物模型来研究疾病机制和制定治疗策略。研究人员已经成功地在小鼠4，大鼠5，兔子6和猪7等小型和大型动物中建立了动物VGD模型。与小动物相比，猪等大型动物具有与人类相似的解剖结构和生理特征，寿命更长8，9。因此，大型动物更适合于探索静脉移植疾病的长期病理变化和药物或器械的临床前测试。我们和我们的合作团队已经成功地应用手术技术建立了猪心力衰竭模型，并描述了该模型中的心脏病理变化10。
CABG手术在临床实践中已经标准化，但当它应用于VGD动物模型的建立时，物种之间的差异，动物设备和设施的获取，动物外科手术以及动物饲养和护理是研究人员面临的巨大挑战。与临床实践一样，用于建立VGD动物模型的CABG手术方法包括中线胸骨切开术11 和左外侧开胸术12。中线胸骨切开术更常用13，14。然而，这种方法对人类和动物都有很高的风险。在Thankam等人报告的研究中，用于建模的六头猪中有两头在手术中死亡15。高模型死亡率会增加研究成本并影响结果的准确性。早些时候的一项研究表明，在猪中，左胸壁切口可以建立CABG诱导的VGD11。在这里，本研究旨在描述一种循序渐进的方案，为小型猪中CABG诱导的VGD模型建立可重复的手术，并评估该模型的表型。实验方案由心脏外科和麻醉团队共同设计。手术前根据实验室中其他迷你猪的尸体确定左第三肋间隙的手术入路，并根据中心16使用的方法进行麻醉方法。通过血液生化检查、超声检查和组织学检查来评估动物模型。
Protocol
实验动物的护理和使用程序经广东省实验动物监测研究所机构动物护理和使用委员会批准。所有实验均按照《实验动物护理和使用指南》（2011年第8版，美国国家研究委员会）进行。手术过程如图 1所示。 1.动物术前准备 将10头体重30-35公斤的3个月大的雄性迷你猪随机分为假猪组（n = 5）和VGD组（n = 5）。 使用体重指数（BMI）?…
Representative Results
BMI 和血清生化指标假和VGD组之间的BMI没有显着差异（假与VGD，22.05公斤/厘米2 ±0.46公斤/厘米2 对21.14公斤/厘米2 ±0.39公斤/厘米2， p = 0.46）。血清生化结果列于 表1。4个生化指标显示组间差异有统计学意义，包括天冬氨酸氨基转移酶（AST，假与VGD，25.25 IU/L±1.88 IU/L±31.5 IU/L2.58 IU/L），血清胆红素（假胆红素与VGD，2.5μmol/L±0.47μmol/L?…
Discussion
在这项研究中，我们详细描述了在开发CABG诱导的VGD模型时动物选择，器械准备，外科手术和术后评估的方案。我们在CABG手术前后对静脉移植物进行了超声检查，并在手术后30天对移植物进行了组织学检查。在冠状动脉旁路移植术之前，乳内静脉的血流是正常的，而在乳内静脉的移植物中观察到逆行血流。与假手术组相比，手术组动物的肝肾功能受到一定程度的损害。考虑到冠状动脉移植疾病的发…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢广东省实验动物监测研究所的技术支持、动物护理和样本采集。他们还感谢深圳市迈瑞生物医疗电子有限公司在超声检测方面的技术支持。这项工作得到了中国广东省科技计划和暨南大学中央大学基础科研业务费用项目（2017A020215076、2008A08003和21621409）的支持。
Materials
| Aortic Punch | Medtronic Inc. , America | 3.0mm, 3.5mm, 4.0mm | Used for proximal coronary bridge anastomosis |
| Automatic biochemical analyzer | IDEXX Laboratories, Inc. America | Catalyst One | |
| Cardiac coronary artery bypass grafting instrument kit | LANDANGER, France | ||
| Cardiogram monitor | Shenzhen Mindray Bio-Medical Electronics Co, Ltd | MEC-1000 | |
| Coronary Shunt | AXIUS | OF-1500, OF-2500, OF-3000 | The product temporarily blocks the coronary artery during arteriotomy to reduce the amount of bleeding in the surgical field and provide blood flow to the distal end during anastomosis. The Axius shunt plug is not an implant and should be removed prior to completion of the anastomosis. |
| Defibrillator | MEDIANA | Mediana D500 | |
| Diazepam | Nanguo pharmaceutical Co. LTD, Guangdong, China | H37023039 | Narcotic inducer |
| Disposable manual electric knife | Covidien, America | E2516H | |
| Electric negative pressure suction machine | Shanghai Baojia Medical Instrument Co, Ltd | YX932D | |
| Esmolol | Guangzhou Wanzheng Pharmaceutical Co. LTD | H20055990 | Emergency drugs |
| Ice machine | Local suppliers, Guangzhou, China | ||
| Lidocaine | Chengdu First Pharmaceutical Co. LTD | H51021662 | Emergency drugs |
| Luxtec headlight system | Luxtec, America | AX-1375-BIF | Used for lighting fine parts during operation |
| Medical operation magnifier (glasses) | Germany Lista co, LTD | SuperVu Galilean 3.5× | Used for fine site operation during operation |
| Multi-function high-frequency electrotome | Shanghai Hutong Electronics Co, Ltd | GD350-B | |
| Nitrogen canister | Local suppliers, Guangzhou, China | ||
| Nonabsorbable surgical suture (polypropylene suture) | Johnson & Johnson, America | 6-0, 7-0 | Used to suture blood vessels. |
| Nonabsorbable suture (cotton thread) | Covidien, America | 1-0 | Used for skin and muscle tissue tugging |
| Open heart surgery instrument kit | Shanghai Medical Instrument (Group) Co., LTD | ||
| Propofol injection | Xi 'an Libang Pharmaceutical Co. LTD | H19990282 | Anesthetic sedative |
| Refrigerator | Local suppliers, Guangzhou, China | ||
| Respiratory anesthesia machine for animal | Shenzhen Reward Life Technology Co, Ltd, China | R620-S1 | |
| Semi-occlusion clamp | Xinhua Surgical Instrument Co., Ltd. | ZL1701RB | Temporarily cut off the aortic flow |
| vecuronium bromide | Richter, Hungary | JX20090127 | Muscle relaxant |
| Veterinary ultrasound system | Royal Philips, Netherlands | CX50 | |
| Zoletil | Virbac, France | Zoletil 50 | Animal narcotic |
Riferimenti
- Lloyd-Jones, D., et al. Executive summary: Heart disease and stroke statistics–2010 update: A report from the American Heart Association. Circulation. 121 (7), 948-954 (2010).
- Taggart, D. P. Contemporary coronary artery bypass grafting. Frontiers of Medicine. 8, 395-398 (2014).
- Wolny, R., Mintz, G. S., Pregowski, J., Witkowski, A. Mechanisms, prevention and treatment of saphenous vein graft disease. The American Journal of Cardiology. 154, 41-47 (2021).
- Schachner, T., Laufer, G., Bonatti, J. In vivo (animal) models of vein graft disease. European Journal of Cardio-thoracic Surgery: Official Journal of the European Association for Cardio-thoracic Surgery. 30 (3), 451-463 (2006).
- Suggs, W. D., et al. Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model. Surgery. 126 (2), 443-449 (1999).
- Jiang, Z., et al. A novel vein graft model: Adaptation to differential flow environments. American Journal of Physiology. Heart and Circulatory Physiology. 286 (1), H240-H245 (2004).
- O’Brien, J. E., et al. Early injury to the media after saphenous vein grafting. The Annals of Thoracic Surgery. 65 (5), 1273-1278 (1998).
- Zou, Y., et al. Mouse model of venous bypass graft arteriosclerosis. The American Journal of Pathology. 153 (4), 1301-1310 (1998).
- Klyachkin, M. L., et al. Postoperative reduction of high serum cholesterol concentrations and experimental vein bypass grafts. Effect on the development of intimal hyperplasia and abnormal vasomotor function. The Journal of Thoracic and Cardiovascular Surgery. 108 (3), 556-566 (1994).
- Tan, W., et al. A porcine model of heart failure with preserved ejection fraction induced by chronic pressure overload characterized by cardiac fibrosis and remodeling. Frontiers in Cardiovascular Medicine. 8, 677727 (2021).
- Hocum Stone, ., L, L., et al. Magnetic resonance imaging assessment of cardiac function in a swine model of hibernating myocardium 3 months following bypass surgery. The Journal of Thoracic and Cardiovascular Surgery. 153 (3), 582-590 (2017).
- Gedik, N., et al. Proteomics/phosphoproteomics of left ventricular biopsies from patients with surgical coronary revascularization and pigs with coronary occlusion/reperfusion: Remote ischemic preconditioning. Scientific Reports. 7 (1), 7629 (2017).
- Tsirikos Karapanos, ., N, , et al. The impact of competitive flow on distal coronary flow and on graft flow during coronary artery bypass surgery. Interactive Cardiovascular and Thoracic Surgery. 12 (6), 993-997 (2011).
- Meng, X., et al. Competitive flow arising from varying degrees of coronary artery stenosis affects the blood flow and the production of nitric oxide and endothelin in the internal mammary artery graft. European Journal of Cardio-thoracic Surgery: Official Journal of the. 43 (5), 1022-1027 (2013).
- Thankam, F. G., et al. Association of hypoxia and mitochondrial damage associated molecular patterns in the pathogenesis of vein graft failure: A pilot study. Translational Research: The Journal of Laboratory and Clinical. , 38-52 (2021).
- Li, X., et al. A surgical model of heart failure with preserved ejection fraction in Tibetan minipigs. Journal of Visualized Experiments: JoVE. 180 (180), 63526 (2022).
- Rueda, A. l. c. a. l. &. #. 2. 2. 5. ;., I, , et al. A live porcine model for surgical training in tracheostomy, neck dissection, and total laryngectomy. European Archives of Oto-Rhino-Laryngology: Official Journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS): Affiliated with the German Society for Oto-Rhino-Laryngology – Head and Neck Surgery. 278 (8), 3081-3090 (2021).
- Proudfit, W. L. Prognostic value of coronary arteriography. Cardiovascular Clinics. 12 (2), 1-8 (1981).
- Clark, R. A. Regulation of fibroplasia in cutaneous wound repair. TheAmerican Journal of the Medical Sciences. 306 (1), 42-48 (1993).
- Darby, I., Skalli, O., Gabbiani, G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Laboratory Investigation. 63 (1), 21-29 (1990).
- Sterpetti, A. V., et al. Formation of myointimal hyperplasia and cytokine production in experimental vein grafts. Surgery. 123 (4), 461-469 (1998).
- Shannon, A. H., et al. Porcine model of infrarenal abdominal aortic aneurysm. Journal of Visualized Experiments: JoVE. 153 (153), (2019).
- Langille, B. L., O’Donnell, F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science. 231 (4736), 405-407 (1986).
- Zwolak, R. M., Adams, M. C., Clowes, A. W. Kinetics of vein graft hyperplasia: Association with tangential stress. Journal of Vascular Surgery. 5 (1), 126-136 (1987).
- Kotani, K., et al. A subacute hypoxic model using a pig. Surgery Today. 35 (11), 951-954 (2005).
- Liu, D., et al. Comparison of ketamine-pentobarbital anesthesia and fentanyl-pentobarbital anesthesia for open-heart surgery in minipigs. Lab Animal. 38 (7), 234-240 (2009).
- Geovanini, G. R., Pinna, F. R., Prado, F. A., Tamaki, W. T., Marques, E. Standardization of anesthesia in swine for experimental cardiovascular surgeries. Revista Brasileira de Anestesiologia. 58 (4), 363-370 (2008).
- Alhomary, M., Ramadan, E., Curran, E., Walsh, S. R. Videolaryngoscopy vs. fibreoptic bronchoscopy for awake tracheal intubation: A systematic review and meta-analysis. Anaesthesia. 73 (9), 1151-1161 (2018).
- Parang, P., Arora, R. Coronary vein graft disease: Pathogenesis and prevention. Canadian Journal of Cardiology. 25 (2), e57-e62 (2009).
- Egan, T. D., et al. Fentanyl pharmacokinetics in hemorrhagic shock: a porcine model. Anesthesiology. 91 (1), 156-166 (1999).
Citazione di questo articolo
Li, X., Hu, J., Tan, W., Lin, Z., Zhu, C., Huang, C., Huang, J., Liu, Y., Liao, Q., Lu, H., Zhang, X. Establishment and Evaluation of a Porcine Vein Graft Disease Model. J. Vis. Exp. (185), e63896, doi:10.3791/63896 (2022).