组织和采血程序标准化改善闭合性胸猪心肌梗死模型的研究
Summary
在这里, 我们演示了一个标准化的方法, 以规范建立猪急性心肌梗塞模型的抽样程序, 以提高其平移值, 了解心肌缺血/再灌注损伤的病理生理学并测试新的药物候选者。
Abstract
心肌缺血再灌注 (一/R) 损伤对心肌梗死后坏死区的近半数有贡献。到目前为止还没有批准的药物来预防或减少心肌的 I/R 损伤。研究和理解心肌损伤的病理生理机制是发展成功治疗的必要条件。大型动物实验是翻译方法的重要步骤。猪急性心肌梗死模型由我们自己和其他人自行建立和描述。我们的目标是进一步提高模型的价值, 重点放在取样技术, 用于未来的实验。此外, 我们强调小但重要的步骤, 可能影响最终结果的质量。为模拟心肌 I./R 损伤的临床情况, 经皮冠状动脉介入治疗 (PCI) 导管插入麻醉猪左前降冠状动脉 (童子)。°°°This 模型模仿急性心肌梗死和 PCI 治疗的人, 有可能准确地确定的区域在危险, 以及坏死和可行的缺血性组织。该模型用于研究 FXIIa 笼肽抑制剂的作用。该模型也可以修改, 以允许更长的再灌注时间, 以研究晚期心肌梗死的影响。
Introduction
缺血性心脏病, 特别是急性心肌梗死 (MI), 是发达国家死亡的主要原因1。今天, 标准治疗 MI 是经皮冠状动脉介入 (PCI), 气囊导管治疗。影响 PCI 治疗急性心肌梗死患者生活质量和预后的关键因素之一是梗塞面积。大小的减少可能对患者生存和预后有很大的影响2。心肌缺血/再灌注 (I.) 损伤对梗死大小有显著影响, 因此心血管研究的主要目的之一是预防或减少心肌 I./r 损伤3。我/R 损伤的确切机制仍在调查中4。激活血浆叶栅和内皮细胞是 I/R 损伤的标志5。凝血系统的激活明显涉及6、7。最近, FXII 的作用, 作为一个早期的上游肽参与凝血叶的接触相活化, 已显示在 FXII 敲出大鼠脑 I/R 损伤模型8。在猪模型中验证这些结果是临床翻译的重要一步。因此, 我们正在试验一种新的笼 (80 kDa 蛋白酶) FXIIa 抑制剂的背景下心肌 I/R 损伤的试验研究。
动物模型, 模仿急性 MI 和 PCI 治疗的临床情况, 是必要的, 以提高我们对心肌 I./R 损伤的病理生理学的认识, 并测试新的治疗方案。猪是临床心肌 I. 型损伤的良好动物模型。这不仅是因为他们的心脏是非常相似的人的心脏解剖和冠状动脉循环, 但他们也显示类似的病理生理反应心肌缺血和再灌注9,10。其他模型, 如老鼠和老鼠不满足这些标准, 并显示相当大的差异时, 与人的心脏11,12, 而狗例如, 有更多的抵押品冠状动脉血管相比, 人类13。
猪急性心肌梗死模型已广泛应用于心血管研究, 包括心肌 I./R 损伤14,15,16,17。后者是一种炎症状态, 因为它减少了与胸骨或开胸手术有关的炎症反应是必不可少的。采用临床 C 臂血管造影设置的闭合胸模型克服了这一问题。此外, 最重要的一点是, 我们的议定书提供了一个准确的区分缺血性 (危险区域, AAR) 和左心室的非缺血性区域 (没有危险的地区), 以便梗塞大小 (坏死缺血性组织, 吹毛求疵) 可以被准确地确定。本论文的目的是明确界定猪心肌 I./r 损伤模型的可再生方法, 特别是在心肌组织取样方面, 这将有助于更精确地分析 I. r 损伤的分子机制和更清楚地了解新药物治疗的效果。
Protocol
1. 动物: 所有动物根据瑞士国家法律的指南对待。研究由伯尔尼的地方动物实验委员会批准 (允许 no。为 25/16)。 使用两个性别的大白猪 (~ 30 ~ 5 公斤)。将动物盲目分为两组, 一组接受笼 (80 kDa 蛋白酶) 抑制剂的 FXIIa 或治疗的选择, 另一种是不活跃的控制。 2. 外科手术 (图 1) 麻醉和动物的准备: <…
Representative Results
一动物在 FXIIa 抑制剂或控制肽的管理之前过早地死了由于技术错误 (血压突然下落在缺血时间, 在测试物质的加法之前)。一只动物被排除在 FXIIa 抑制剂组, 因为没有观察到缺血/再灌注损伤由于左前降动脉的异常解剖 (小伙子)。左心室的很大一部分, 包括先端, 由该动物的扬抑动脉灌注。最后分析的动物是 n = 2 在笼 FXIIa 肽抑制剂小组 (平均重量 27.5 2.5 公斤) 和 n = 3 接受不活…
Discussion
心肌 I. 型损伤对急性心肌梗死后的最终梗死大小有显著影响, 直接转化为患者的预后3。了解心肌 I./R 损伤的病理生理学是减少或预防其发生的第一步。心肌 I./R 损伤是闭塞血管再灌注后直接发生的急性疾病。I/R 损伤导致活化的先天免疫应答和细胞损伤发生在现场的再灌注和周围组织20。最近的一项研究表明, FXIIa 抑制剂8治疗大鼠脑 I/R 损伤模?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢基督教 Heinis 教授提供 FXIIa 抑制剂和各自的控制。我们还感谢伯尔尼大学生物医学研究系 Olgica Beslac、丹尼尔梅特勒和凯 Nettelbeck 的技术支持。席琳 Guillod 和 Rausch 从诊断, 介入和儿科放射科, 伯尔尼大学医院, Inselspital 提供支持的 x 光设备和技术。该项目由瑞士国家科学基金会320030_156193 项目资助。我们还要感谢伯尔尼大学医院通讯和营销 Reto Haenni 先生, Inselspital 为我们的实验录像录音。
Materials
| ABL 90 Flex, blood gas analyser | Radiometer | – | Blood gas analysis (BGA) |
| ACT Plus | Medtronic | – | Activated clotting time |
| Atropin | Sintetica | – | Atropinum Sulfas, 0,5mg/ml |
| Balance (20-500 Kg) | NAGATA Scale, Tiwan | HTB/HTR | Alternative products can be used |
| Balance (21-4200 g) | Mettler toledo, Switzerland | MS4002SDR | Alternative products can be used |
| Blood collection tubes: EDTA, citrate and serum | S-Monovette, Nuembrecht, Germany | 05.1167.001, 05.1071.001 and 05.1557.001 respectively |
Alternative products can be used |
| BV Pulsera mobile C-arm | Philips | – | Alternative products can be used |
| Centrifuge | Labcare, UK | ALC PK120R | Alternative products can be used |
| Defibrillator Lifepak 12 | Medtronic | – | Alternative products can be used |
| Dextran from Leuconostoc mesenteroides | Sigma-Aldrich, Germany | D3759 | Average M.wt 48000-90000 |
| Digital single lens reflex camera | Sony, Thailand | DSLR-A500/A550 | Alternative products can be used |
| Dissecting forceps | Alternative products can be used | ||
| EMPIRA RX PCI dilatation catheter | Cordis, Johnson&Johnson, USA | 85R15300S | Diameter 3 mm, length 15 mm, Alternative products can be used |
| EMPIRA RX PCI dilatation catheter | Cordis, Johnson&Johnson, USA | 85R15350S | Diameter 3.5 mm, length 15 mm, Alternative products can be used |
| Evans Blue | Sigma-Aldrich, Germany | E2129 | Toxic |
| Fabius GS premium respirator | Dräger, Lübeck, Germany | – | Anesthesia work station, Alternative products can be used |
| Fentanyl | Inselspital ISPI | – | Fentanyl 2500mcg/50ml |
| Formaldehyde | Pathology Institute, Bern University | SI148701 | Alternative products can be used |
| FXIIa inhibitor | Provided by Prof. Christian Heinis' laboratory in EPFL | Novel bicyclic peptide | |
| Galeo, coronary guidewire | Biotronik, Germany | 125497 | Alternative products can be used |
| Guidance catheter | Boston Scientific, Florida, USA | 34356-06 | 6F (100 cm, EB3.75). Alternative products can be used |
| Heparin Sodium | Drossapharm, Basel, Switzerland | – | Liquemin, 25000 U.I./5 ml |
| High end electrosurgery | BOWA, Germany | ARC 400 | Electrical source for blood suction. Alternative products can be used |
| Hydro-Guardmini breathing filter | Intersurgical, Lithuania | 1745000 | Filters |
| Image J | National Institute of Health, USA | 1.47v | Alternative products can be used |
| Inflation device, Atrion QL2530 | Atrion medical product, Alabama, USA | 96402 | Alternative products can be used |
| IntelliVue MP 70 | Philips, Boeblingen, Germany | – | Monitor (ECG, heart rate, blood presure and body temperature). Alternative products can be used |
| KCl | Sintetica SA | – | Potassium chloride 15% |
| Ketamine | Vetoquinol | – | Narketan, 1ml/100mg |
| LR-ACT | Medtronic | 402-01 | ACT special syringes |
| Midazolam | Roche | – | Dormicum, 5mg/ml |
| Monopolar scalpel | Alternative products can be used | ||
| Needle holder | Alternative products can be used | ||
| High resolution camera, PathStand Macro Imaging Stand for Grossing | Spotimaging, USA | 1080 p HD resolution. Alternative products can be used | |
| PBS | In-house preparation | – | Alternative products can be used |
| Peripheral venous cannula, 18 G | Alternative products can be used | ||
| PowerLab 4/35 data acquisition system | Adinstruments, Spechbach, Germany | – | |
| Rotamax120T | Heidolph, Germany | 544-41200-00 | Shaker. Alternative can be used |
| Rüschelit-Super Safety Clear Tube | Teleflex, Dublin, Irland | 112480 | Air way tubing, Alternative products can be used |
| Safe Pico Aspirator Syringes | Radiometer | 956-622 | BGA special syringes |
| Saline | Sintetica Bioren | – | NaCl 0,9%. Alternative products can be used |
| Sevorane 1.5% | AbbVie AG | – | Sevorane 250ml 100% |
| Sheath | Cordis, Johnson&Johnson, USA | 504-607 A | AVANTI + / 7 F, Alternative products can be used |
| Space infusion pump | B.Braun Medical AG, Germany | – | For infusion of fentanyl. Alternative products can be used |
| SPR-350 (Millar catheter) | Adinstruments, Texas, USA | 840-8166 | MIKRO-TIP, 5F, 120 cm |
| Sternotomy saw | Alternative products can be used | ||
| Sutures | ETHICON, Johnson&Johnson, USA | Y3110H | Monocryl 3-0 SH-1 Plus. Alternative products can be used |
| Syringes (20, 10 and 5 mL) | CODAN,Baar, Switzerland | 62.7602, 62.6616, 62.5607 respectively |
Used to inject anesthetic materials intramuscularly or directly into the central venous line. Also to inject heparin or FXIIa or the respective control. Alternative products can be used |
| Thorax spreader | Alternative products can be used | ||
| Tissue-tek | SAKURA, Netherlands | 4583 | O.C.T. compound |
| Vascular forceps | Alternative products can be used | ||
| Xenetix 300 contrast media | Guerbet, Zürich, Switzerland | – | Lobitridol, 300 mg iodide/ml |
| Xylazine | Vetoquinol | – | Xylapan, 20mg/1 mL |
| 2,3,5-Triphenyltetrazolium choride | Sigma-Aldrich, Austria | T8877 | |
| 500 μL tubes (eppendorf) | Trefflab, Switzerland | 96.08185.9.03 | Alternative products can be used |
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Cite This Article
Abdelhafez, M. M., Shaw, J., Wilbs, J., Despont, A., Rieben, R. Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures. J. Vis. Exp. (133), e56856, doi:10.3791/56856 (2018).