小鼠心肌梗死和心肌缺血再灌注损伤的诱导
Summary
在这里，我们描述了一种简单且可重复的方法，该方法可以通过显微操作精确结扎左前降支冠状动脉来诱导小鼠心肌梗死或心肌缺血再灌注损伤。
Abstract
急性心肌梗死是一种常见的心血管疾病，死亡率很高。心肌再灌注损伤可以抵消心脏回流的有益作用，诱发继发性心肌损伤。简单且可重复的心肌梗死和心肌缺血再灌注损伤模型是研究人员的良好工具。在这里，描述了一种通过显微操作精确结扎左前降支冠状动脉 （LAD） 来创建心肌梗死 （MI） 模型和 MIRI 的可定制方法。LAD 的准确且可重复的结扎定位有助于获得一致的心脏损伤结果。ST 段更改有助于识别模型准确性。血清心肌肌钙蛋白T（cTnT）水平用于评估心肌损伤，心脏超声用于评估心肌收缩功能，Evans-Blue/三苯基氯化四氮唑染色用于测量梗死面积。一般来说，该协议减少了手术时间，确保了可控的梗死大小，并提高了小鼠的存活率。
Introduction
急性心肌梗死 （AMI） 是全球常见的心血管疾病，死亡率很高1.技术的进步使AMI患者可以进行早期有效的血运重建。在一些患者进行这些治疗后，可能会发生心肌缺血再灌注损伤 （MIRI）2。因此，了解MI/MIRI的作用机制和改善MI/MIRI具有重要意义。小鼠因其成本低、繁殖时间短且易于进行基因改变而被广泛用作模型3.学者们开发了不同的方法来模拟动物4、5、6、7、8、9 的 MIRI 和 MI。这种策略促进了研究，但采用的不同标准和方法使研究团队对结果的解释变得复杂。
在小鼠中，异丙肾上腺素 10、冷冻损伤11,12 或烧灼13 诱导了心肌梗死。异丙肾上腺素易诱发心肌梗死，但病理生理过程与临床心肌梗死不同。 冷冻损伤诱发的心肌梗死一致性差，引起左冠状动脉前降支（left anterior lower coronary artery，LAD）周围过度心肌损伤，易诱发心律失常。烧灼诱发的心肌梗死与心肌梗死的自然过程有很大不同，烧灼部位的炎症反应更强烈;此外，手术方法存在技术困难。此外，还有一些实验室14 通过介入技术使用球囊阻塞或栓塞或血栓形成方法在小型猪中开发 MI 模型。所有这些方法都可能直接导致冠状动脉闭塞，但需要冠状动脉造影设备，尤其是太薄的小鼠冠状动脉使这些手术不切实际。对于MIRI，不同模型之间的差异非常小，例如是否使用呼吸器/显微操作5,6。
在这里，描述了一种简单可靠的方法可以诱导MI和MIRI模型，该模型改编自先前发表的方法4,5,6,7,8,9,15。该方法可以通过结扎直接阻断LAD来模拟病理生理过程。此外，通过解除结扎，该模型还可以模拟再灌注损伤。在该协议中，解剖显微镜用于LAD可视化。然后，研究人员可以很容易地识别 LAD。随后，LAD的准确结扎导致可重复和可预测的血液闭塞和心室缺血。此外，除了在显微镜下观察到的 LAD 颜色变化外，心电图 （ECG） 变化还可用于确认缺血和再灌注。这种策略导致更短的手术时间，更低的手术并发症风险，以及更少的实验小鼠。还描述了肌钙蛋白-T 试验、心脏超声和氯化三苯基四唑 （TTC） 染色的方法。总体而言，该方案可用于MI / MIR机制的研究以及药物发现。
Protocol
动物研究已获得华中科技大学（中国武汉）动物护理和利用委员会的批准。 注：雄性C57BL / 6J小鼠（8-10周）用作模型。小鼠可以自由获取食物和水，并在特定的无病原体条件下繁殖。房间保持在受控温度（22°C±2°C）和湿度（45%-65%）下。根据该机构制定的指南，将小鼠暴露在同济医学院（中国武汉）动物护理设施的12小时光/暗环境中。使用无菌显微手术器械和手术用品。在整…
Representative Results
实验工作流程如图 1A所示。研究开始后，研究者可以根据实验设计安排时间节点。LAD结扎的持续时间取决于研究目的。对于心肌梗死，研究可以忽略再灌注步骤。心脏超声可在研究的不同阶段进行，因为它是非侵入性的，而 Evans-Blue/TTC 染色只能在小鼠被处死时进行。对于专注于纤维化和心室重塑的研究，观察时间要长得多。 部分实验过程的典型图像如?…
Discussion
近年来，MI和MIRI模型在临床和科学研究中的创建发展迅速20,21。然而，仍有一些问题需要解决，例如作用机制和如何改善MI/MIRI。在这里，描述了用于建立MI和MIRI小鼠模型的修改方案。必须仔细考虑几个关键点。
第一个关键点是气管插管。一些程序6,9 涉及切开颈椎皮肤、组织分离，然后暴…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（82070317,81700390林继斌，8210021880吕冰杰，82000428王博元）和国家重点研发计划（2017YFA0208000给何少林）的支持。
Materials
| 0.9 % sodium chloride solution | Kelun Industry Group,China | – | |
| 4% paraformaldehyde fixing solution | Servicebio,China | G1101 | – |
| 4-0 silk suture | Shanghai Pudong Jinhuan Medical Products,China | C412 | – |
| 8-0 suture | Shanghai Pudong Jinhuan Medical Products,China | H801 | – |
| Buprenorphine | IsoReag,China | IR-11190 | – |
| Camera | Canon,Japan | EOS 80D | – |
| Depilatory cream | Veet,French | – | |
| Elecsys Troponin T hs STAT | Roche,Germany | – | |
| Electrochemical luminescence immunoanalyzer | Roche,Germany | Elecsys 2010 | – |
| Evans blue | Sigma,America | E2129 | – |
| Eye scissors | Shanghai Medical Instruments,China | JC2303 | – |
| Haemostatic forceps | Shanghai Medical Instruments,China | J31020 | – |
| High frequency in vivo imaging systems | Visualsonics,Canada | Vevo2100 | – |
| Ibuprofen | PerFeMiKer,China | CLS-12921 | – |
| Intravenous catheter | Introcan,Germany | 4254090B | – |
| Ketamine | Sigma-Aldrich,America | K2753 | – |
| Medical alcohol | Huichang ,China | – | |
| Microneedle holders | Shanghai Medical Instruments,China | WA2040 | – |
| Microscopic shears | Shanghai Medical Instruments,China | WA1040 | – |
| Microsurgical forceps | Shanghai Medical Instruments,China | WA3020 | – |
| Mouse electrocardiograph | Techman,China | BL-420F | – |
| Needle holders | Shanghai Medical Instruments,China | JC3202 | – |
| operating floor | Chico,China | ZK-HJPT | – |
| PE-10 tube | Huamei,China | – | |
| Pentobarbital | Merck,America | 1030001 | – |
| Rodent Ventilator | Shanghai Alcott Biotech,China | ALC-V8S-P | – |
| Stereo microscope | Aomei Industry,China | SZM0745-STL3-T3 | – |
| Surgical thermostatic heating pad | Globalebio, China | GE0-20W | – |
| Triphenyltetrazolium chloride | Servicebio,China | G1017 | – |
| Xylazine | Huamaike Biochemicals and Life Science Research Prouducts,China | 323004 | – |
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Cite This Article
Lv, B., Zhou, J., He, S., Zheng, Y., Yang, W., Liu, S., Liu, C., Wang, B., Li, D., Lin, J. Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice. J. Vis. Exp. (179), e63257, doi:10.3791/63257 (2022).