左冠状动脉结扎术：心肌梗死的外科小鼠模型
Summary
这里介绍的是小鼠左冠状动脉永久结扎的外科手术。该模型可用于研究心肌梗死后的病理生理学和相关炎症反应。
Abstract
缺血性心脏病和随后的心肌梗塞（MI）是美国和世界各地死亡的主要原因之一。为了探究心肌梗死后的病理生理变化并设计未来的治疗方法，需要心肌梗死的研究模型。小鼠左冠状动脉（LCA）的永久性结扎是研究心肌梗死后心脏功能和心室重塑的流行模型。在这里，我们描述了一种侵入性较小、可靠且可重复的手术鼠 MI 模型，通过永久连接 LCA。我们的手术模式包括易于逆转的全身麻醉、不需要气管切开术的气管插管和开胸术。应进行心电图和肌钙蛋白测量以确保心肌梗死。 心肌梗死后第 28 天的超声心动图可识别心功能和心力衰竭参数。心脏纤维化的程度可以通过Masson的三色染色和心脏MRI进行评估。该心肌梗死模型有助于研究心肌梗死后的病理生理和免疫学改变。
Introduction
心血管疾病是一个重大的公共卫生问题，每年夺走1790万人的生命，占全球死亡率的31%1。最普遍的心血管异常类型是冠心病，心肌梗塞（MI）是冠心病的主要表现之一2。心肌梗死通常是由脆弱斑块破裂引起的冠状动脉血栓闭塞引起的3.由此产生的缺血导致受影响的心肌发生深刻的离子和代谢变化，以及收缩功能的快速下降。心肌梗死导致心肌细胞死亡，可进一步导致心室功能障碍和心力衰竭4。
由于从MI5患者获得的组织稀缺，对患者心肌梗死的研究有限。因此，MI的小鼠模型可用于研究疾病机制以及开发潜在的治疗靶点。目前可用的MI小鼠模型包括不可逆缺血模型（LCA和消融方法）和再灌注模型（缺血/再灌注，I / R）6。小鼠左冠状动脉（LCA）的永久性结扎是最常用的方法，它模仿患者7，8，9中MI的病理生理学和免疫学。永久性心肌梗死也可以通过消融方法诱发，包括电损伤或冷冻损伤。消融方法能够在精确位置10产生均匀大小的梗死。另一方面，瘢痕形成、梗死形态和分子信号传导机制可能因消融方法而异10，11。鼠I/R方法是另一个重要的MI模型，因为它代表了再灌注治疗的临床场景12。I/R 模型存在各种挑战，例如梗死大小可变、难以区分初始损伤的反应和再灌注6。
虽然被广泛使用，但LCA结扎方法与低生存率和术后疼痛有关13。该协议展示了LCA结扎的小鼠手术MI模型，该模型涉及小鼠的准备和插管，LCA结扎，术后护理和MI的验证。 该方法不使用侵入性气管切开术14，而是采用气管插管。通过使用喉镜照亮口咽来对动物进行插管，使手术更容易、更安全、创伤更小15.在整个过程中，小鼠保持在呼吸机支持下并在异氟醚麻醉下。此外，进行超声心动图和Masson三色染色分别评估心肌梗死后的心脏功能和心肌纤维化。总体而言，该方法提供了一种可靠且可重复的MI手术鼠模型，可用于研究MI后的病理生理学和炎症。
Protocol
本研究方案由匹兹堡大学机构动物护理和使用委员会（IACUC）审查和批准。8只（假n = 4和MI n = 4）体重24-30g的1岁雌性C57BL / 6J小鼠用于这些实验。大约100%和至少80%的小鼠分别在前24小时和28天内存活。 1.小鼠的准备和气管插管 将珠子灭菌器（见 材料表）预热至250°C，并将高压灭菌的手术器械放入其中几分钟。 在含有3%异氟醚和1L / min氧气…
Representative Results
图1显示了假（图1A）和MI（图1B）小鼠超声心动图评估期间的代表性主动心电图和呼吸信号。在获取超声心动图数据之前，验证主动心电图和呼吸信号很重要。 图2显示了LCA结扎后28天心脏功能参数的超声心动图测量。图 2 显示了假心脏（图 2A）和 MI（图 2B</s…
Discussion
MI的小鼠模型在心血管研究实验室中越来越受欢迎，本研究描述了一种可重复且临床相关的MI模型。该协议以多种方式改进了LCA连接过程。首先，避免使用可注射的术前麻醉剂，例如甲苯噻嗪/氯胺酮或戊巴比妥钠14，15 。仅使用异氟醚麻醉，这有助于提高动物存活率（术后28天存活率为>80%），最大限度地减少药物引起的并发症，并且与其他药物相比，心?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院拨款（R01HL143967，R01HL142629，R01AG069399和R01DK129339），AHA转型项目奖（19TPA34910142），AHA创新项目奖（19IPLOI34760566）和ALA创新项目奖（IA-629694）（PD）的支持。
Materials
| 22 G catheter needle | Exel INT | 26741 | Thoracentesis |
| 24 G catheter needle | Exel INT | 26746 | Endotracheal intubation |
| 4-0 nylon suture | Covetrus | 29263 | Suturing of muscles and skin |
| 8-0 nylon suture | S&T | 3192 | Ligation of LAD |
| Anesthetic Vaporizers | Vet equip | VE-6047 | Anesthetic support |
| Animal physiology monitor | Fujifilm | VEVO 3100 | Monitor heart rate,respiration rate and body temperature |
| Betadine solution | PBS animal health | 11205 | Antispetic |
| Buprenorphine | Covetrus | 55175 | Analgesic |
| Disecting microscope | OMANO | OM2300S-V7 | Binocular |
| Electric razor | Wahl | 79300-1001M | Shaving |
| Electrode gel | Parker Laboratories | W60698L | Electrically conductive gel |
| Ethanol | Decon Laboratories | 22-032-601 | Disinfectant |
| Forceps | FST | 11065-07 | Stainless Steel |
| Gauze | Curity | CAR-6339-PK | Sterile |
| Heat lamp | Satco | S4998 | Post surgery care |
| Heating pad | Kent scientific | Surgi-M | Temperature control |
| Hot Bead sterilizer | Germinator 500 | 11503 | Sterilization of surgical instrument |
| Isoflurane | Covetrus | 29405 | Anesthesia |
| Masson’s trichrome staining kit | Thermoscientific | 87019 | Measurement of cardiac Fibrosis |
| Micro Needle Holder | FST | 12500-12 | Stainless Steel |
| Micro scissors | FST | 15000-02 | Stainless Steel |
| Ophthalmic ointment | Dechra | Puralube Vet | Sterile occular lubricant |
| Scanning Gel | Parker Laboratories | Aquasonic 100 | Aqueous ultrasound transmission gel |
| Scissors | FST | 14060-11 | Stainless Steel |
| Small Animal Laryngoscope | Penn-Century | Model LS-2-M | Illuminating the oropharynx |
| Small animal ventilator | Harvard apparatus | 557058 | Ventilator support |
| Surgical light | Cole parmer | 41723 | Illuminator Width (in): 7 |
| Vevo 3100 preclinical imaging platform | Fujifilm | VEVO 3100 | Echocardiography |
| VevoLAB software | Fujifilm | VevoLAB 3.2.6 | Echocardiography data analysis |
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Cite This Article
Johny, E., Dutta, P. Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction. J. Vis. Exp. (186), e64387, doi:10.3791/64387 (2022).