一种改良的小鼠心肌梗死诱导简易方法
Summary
在充分麻醉下，小鼠心脏通过肋间腔外化，并使用大多数实验室中现成的材料结扎左前降支 （LAD） 成功诱导心肌梗死。
Abstract
心肌梗死（MI）是导致死亡的主要原因之一。心肌梗死模型被广泛用于研究心肌梗死后重塑的病理机制和新疗法的评估。与异丙肾上腺素治疗和冷冻损伤相比，冠状动脉结扎术可能更能反映心肌梗死后的缺血反应和慢性重塑。然而，传统的小鼠冠状动脉结扎方法在技术上具有挑战性。目前的研究描述了一种简单有效的过程，用于使用现成的材料在小鼠中诱导MI。在稳定麻醉下切开小鼠胸部皮肤。在胸大肌和胸小肌钝性分离后，心脏立即通过肋间腔外化。左前降支 （LAD） 在距其原点 3 mm 处用 6-0 缝合线结扎。LAD 连接后，用 2,3,5-三苯基氯化四氮唑 （TTC） 染色表明 MI 成功诱导和 MI 后瘢痕大小的时间变化。同时，生存分析结果显示心肌梗死后7天内明显死亡，主要由心脏破裂引起。此外，心肌梗死后超声心动图评估显示成功诱导收缩功能障碍和心室重塑。一旦掌握，就可以在2-3分钟内使用现成的材料在小鼠中建立MI模型。
Introduction
心肌梗死 （MI） 是全球死亡和残疾的重要原因之一 1,2,3,4,5。尽管及时进行了再灌注，但目前缺乏治疗心肌梗死后心脏重塑的有效疗法。相应地，在MI 6,7,8的机理探索和治疗开发方面也做出了相当大的努力。值得注意的是，建立MI模型是实现这些目标的先决条件。
已经提出了几种方法（例如，异丙肾上腺素治疗、冷冻损伤、冠状动脉结扎等）来诱导小动物的心肌梗死模型。异丙肾上腺素治疗是诱导心肌梗死的简单方法，但不能诱发靶区梗死9。冷冻损伤通过冰晶的产生和细胞膜的破坏而不是直接缺血导致心肌坏死10。相比之下，冠状动脉结扎术可以精确控制梗死部位和梗死区域的范围，并忠实地再现梗死后的重塑反应11,12。冠状动脉结扎术通常在插管、机械通气和开胸手术后进行，这在技术上具有挑战性13,14。报道了几种改良的冠状动脉结扎方案（例如，无通气）并增强了心肌梗死的诱导，但缺乏详细的视觉演示15,16,17。这些问题对希望使用MI模型进行研究的团体构成了重大的财务和技术障碍。本报告介绍了一种在小鼠中诱导心肌梗死的方法。目前的方法简单、省时，并且使用大多数实验室中容易找到的手术工具和设备。
Protocol
涉及动物工作的实验是在上海交通大学医学院附属仁济医院实验动物福利伦理委员会（R52021-0506）的所有必要批准下进行的。该研究使用了年龄在8-10周之间的雌性和雄性C57BL / 6J小鼠。 1.简易麻醉设备的准备（可选） 注意： 这是一个可选的术前设置，可以替换为第 2 节中提到的可滴定麻醉。在将此设置调整为动物程序之前，应咨询机构动物伦?…
Representative Results
实验方案和一些关键步骤如图1所示。简化的麻醉设备诱导麻醉。如图2A所示，诱导麻醉是稳定的，如常规呼吸频率（测试小鼠的呼吸频率为90-107次/分钟）所反映的那样。冠状动脉结扎术后，TTC染色分析显示心肌梗死成功诱导，心肌梗死后瘢痕大小发生时间变化（图2B）。同时，存活分析结果显示雄性和雌性C57BL / 6J小鼠在心肌梗死…
Discussion
本报告展示了一种使用现成材料在小鼠中诱导心肌梗死的简单方案，该方案是从 Gao16 报告的方法修改而来的。小鼠心肌梗死模型对于心肌梗死后功能障碍和重塑的机理探索和药物筛选是必不可少的12.在现有的心肌梗死诱导技术中，冠状动脉结扎术是最常用的一种。冠状动脉结扎术忠实地概括了心肌梗死的缺血性质，并导致类似于临床情况的瘢痕愈合和重塑反应…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（81930007,81625002,81800307,81470389,81500221,81770238），上海市杰出学术带头人计划（18XD1402400），上海市科学技术委员会（201409005200），上海浦江人才计划（2020PJD030）和中国博士后科学基金（2020M671161，BX20190216）的资助。
Materials
| 2,3,5-Triphenyltetrazolium chloride | SIGMA | T8877-25G | TTC staining |
| 4-0 silk suture | YUANKANG | 4-0 | Surgical instrument |
| Autoclave | HIRAYAMA | HVE-50 | Sterilization for the solid |
| Buprenorphine | Qinghai Pharmaceutical FACTORY Co., Ltd. | H10940181 | reduce post-operative pain |
| Centrifugation tube | Biological Hope | 1850-K | 15ML |
| Depilatory cream | ZIKER BIOTECHNOLOGY | ZK-L2701 | Depilation agent for laboratory animals |
| Forcep | RWD | F12028 | Surgical instrument |
| Gas filter | ZHAOXIN | SA-493 | Operator protection |
| Isoflurane | RWD | 20071302 | Used for anesthesia |
| Light source | Beijing PDV | LG-150B | Operating lamp |
| Micro-mosquito hemostat | FST | 13011-12 | Surgical instrument |
| Needle | BINXIONG | 42180104 | Surgical instrument |
| Needle and the 6-0 silk suture | JIAHE | SC086 | Surgical instrument |
| Needle holder | ShangHaiJZ | J32030 | Surgical instrument |
| Needle holder | ShangHaiJZ | J32010 | Surgical instrument |
| Povidone-iodine swabs | SingleLady | GB26368-2010 | Skin disinfection |
| Scissors | CNSTRONG | JYJ1030 | Surgical instrument |
| Sterile eye cream | Shenyang Xingqi Pharmaceutical Co., Ltd. | H10940177 | prevent corneal dryness |
| Ultra-high resolution ultrasound imaging system for small animals | VisualSonics | Vevo 2100 | Echocardiographic analysis |
References
- Fu, Y., et al. A simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice. Journal of Visualized Experiments. (134), e57074 (2018).
- Pell, S., Fayerweather, W. E. Trends in the incidence of myocardial infarction and in associated mortality and morbidity in a large employed population. The New England Journal of Medicine. 312 (16), 1005-1011 (1985).
- Ramunddal, T., Gizurarson, S., Lorentzon, M., Omerovic, E. Antiarrhythmic effects of growth hormone–in vivo evidence from small-animal models of acute myocardial infarction and invasive electrophysiology. Journal of Electrocardiology. 41 (2), 144-151 (2008).
- Tabrizchi, R. β-blocker therapy after acute myocardial infarction. Expert Review of Cardiovascular Therapy. 11 (3), 293-296 (2013).
- Virani, S. S., et al. Heart disease and stroke statistics-2020 update: A report from the American Heart Association. Circulation. 141 (9), 139 (2020).
- Cahill, T. J., Choudhury, R. P., Riley, P. R. Heart regeneration and repair after myocardial infarction: Translational opportunities for novel therapeutics. Nature Reviews Drug Discovery. 16 (10), 699-717 (2017).
- Froese, N., et al. Anti-androgenic therapy with finasteride improves cardiac function, attenuates remodeling and reverts pathologic gene-expression after myocardial infarction in mice. Journal of Molecular and Cellular Cardiology. 122, 114-124 (2018).
- Wang, W., et al. Defective branched chain amino acid catabolism contributes to cardiac dysfunction and remodeling following myocardial infarction. American Journal of Physiology-Heart and Circulatory Physiology. 311 (5), 1160-1169 (2016).
- Acikel, M., et al. Protective effects of dantrolene against myocardial injury induced by isoproterenol in rats: Biochemical and histological findings. International Journal of Cardiology. 98 (3), 389-394 (2005).
- vanden Bos, E. J., Mees, B. M. E., de Waard, M. C., de Crom, R., Duncker, D. J. A novel model of cryoinjury-induced myocardial infarction in the mouse: A comparison with coronary artery ligation. American Journal of Physiology-Heart and Circulatory Physiology. 289 (3), 1291-1300 (2005).
- Guo, Y., et al. Demonstration of an early and a late phase of ischemic preconditioning in mice. American Journal of Physiology-Heart and Circulatory Physiology. 275 (4), 1375-1387 (1998).
- Kumar, M., et al. Animal models of myocardial infarction: Mainstay in clinical translation. Regulatory Toxicology and Pharmacology. 76, 221-230 (2016).
- Das, S., MacDonald, K., Chang, H. Y., Mitzner, W. A simple method of mouse lung intubation. Journal of Visualized Experiments. (73), e50318 (2013).
- Johns, T. N., Olson, B. J. Experimental myocardial infarction. I. A method of coronary occlusion in small animals. Annals of Surgery. 140 (5), 675-682 (1954).
- Ahn, D., et al. Induction of myocardial infarcts of a predictable size and location by branch pattern probability-assisted coronary ligation in C57BL/6 mice. American Journal of Physiology. Heart and Circulatory Physiology. 286 (3), 1201-1207 (2004).
- Gao, E., Koch, W. J. A novel and efficient model of coronary artery ligation in the mouse. Methods in Molecular Biology. 1037, 299-311 (2013).
- Most, P., et al. Cardiac S100A1 protein levels determine contractile performance and propensity toward heart failure after myocardial infarction. Circulation. 114 (12), 1258-1268 (2006).
- Christia, P., et al. Systematic characterization of myocardial inflammation, repair, and remodeling in a mouse model of reperfused myocardial infarction. Journal of Histochemistry & Cytochemistry. 61 (8), 555-570 (2013).
- Frantz, S., Bauersachs, J., Ertl, G. Post-infarct remodelling: Contribution of wound healing and inflammation. Cardiovascular Research. 81 (3), 474-481 (2008).
Cite This Article
Jiang, C., Chen, J., Zhao, Y., Gao, D., Wang, H., Pu, J. A Modified Simple Method for Induction of Myocardial Infarction in Mice. J. Vis. Exp. (178), e63042, doi:10.3791/63042 (2021).