一种改良的小鼠横主动脉收缩技术
1Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, 2Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiological Disorders, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology
Summary
本协议描述了一种使用自制牵开器进行微创横主动脉收缩（TAC）手术的改进和简化技术。该手术可以在没有呼吸机或显微镜的情况下进行，并引入压力超负荷，最终导致心脏肥大或心力衰竭。
Abstract
横主动脉收缩（TAC）是基于小鼠模型中压力超负荷的形成，在心力衰竭和心脏肥大研究中经常使用的手术。该手术的主要挑战是清晰地看到横主动脉弓并精确地束带目标血管。经典方法进行部分开胸术以暴露横主动脉弓。然而，它是一种开胸模型，会导致相当大的手术创伤，并且在手术过程中需要呼吸机。为了防止不必要的创伤并简化操作程序，通过胸骨的近端比例 接近 主动脉弓，使用包含圈套的小型自制牵开器到达并绑定目标血管。该手术可以在不进入胸膜腔的情况下进行，不需要呼吸机或显微外科手术，这使小鼠具有生理呼吸模式，简化了手术，并显着减少了手术时间。由于侵入性较小，手术时间较短，小鼠可以经历较少的应激反应并迅速恢复。
Introduction
心力衰竭是一种复杂的临床症状，由心室充盈或射血的结构和功能受损引起1。疾病分期主要通过纽约心脏协会根据症状严重程度和身体活动进行功能分类 来 定义2.对于射血分数超过 50% 的患者，结构和/或功能异常应提高利钠肽，以支持射血分数保留 （HFpEF） 诊断心力衰竭2。缺血性心脏病是心力衰竭多种病因的主要原因。因此，心肌梗死模型（如永久性冠状动脉结扎术）常用于研究心脏灌注不足或缺血再灌注损伤后的病理生理学3，4。除急性心肌损伤外，高血压、糖尿病、肥胖症和心肌病家族史等其他危险因素也会导致心力衰竭的发展。患者通过A期（有心力衰竭风险）进入B期（心力衰竭前期）后，会发生结构改变1。例如，高血压患者首先经历适应性左心室肥大，然后通过病理重塑逐渐发展为适应不良的心脏肥大并过渡到心力衰竭5。
作为各种心血管疾病的终末期，慢性心力衰竭已经研究了几十年6。多种小鼠模型已广泛用于心力衰竭研究，包括药物输注（血管紧张素II），代谢紊乱（糖尿病或高热量饮食）和主动脉收缩7。在这些模型中，血管紧张素II灌注伴有各种器官副作用，如肾脏7。诱导代谢紊乱通常需要相当长的时间。升主动脉收缩被认为与人类疾病相关性有限7.
TAC是一种可靠的模型，可增加后负荷并诱发心脏肥大和心力衰竭8。开胸TAC模型最早由Rockman等人描述，并在世界各地的众多实验室中使用9。然而，这种经典的TAC程序对小鼠造成相当大的创伤并改变其正常行为，这可能需要很长的恢复时间并干扰进一步的治疗10。其他改良的闭胸TAC手术确实减少了一些侵入性步骤，但需要显微外科手术技能或机械通气10，11。
本协议详细介绍了一种循序渐进的方法，该方法使用自制的牵开器通过胸骨上边缘的 3 毫米中线切口 对 主动脉弓进行微创方法。该模型不需要显微外科技术、机械通气或切开肋骨，从而提供了一种快速、手术创伤受限、简单、廉价的方式来进行 TAC 手术。
Protocol
目前的协议由中国武汉华中科技大学同济医学院同济医院伦理委员会批准。该程序在雄性成年C57 / BL6小鼠（>10周龄）上进行。所有手术器械在手术前均通过高压灭菌进行灭菌。 1. 手术器械的准备 准备一个 5 mL 注射器，用针架捏掉针尖以使其变钝。 准备一根 27 G 的针头，并用持针器将其钝化。用眼科剪刀剪针尖是使针头变钝的另一种方法。 …
Representative Results
TAC手术成功后，使用超声成像系统检测压力过载。手术四周后，小鼠心脏功能下降。在本研究中，通过4周后接受TAC手术的小鼠的射血分数（EF），分数缩短（FS），左心室质量（LV质量）和左心室内径（LVID） 验证 了TAC手术的有效性。与假小鼠相比，4周后TAC小鼠的EF显着降低（47%±10%对78%±4%， p <0.0001）（图4A）。TAC小鼠的左心室质量显着升高（158.1±50.5对91.8±21.7…
Discussion
持续压力超负荷的诱导可逐渐引起心脏肥大和心力衰竭。该模型已在世界各地的众多实验室中使用14，15，16。该方案提供了一种改进的TAC方法，不需要显微外科手术技能或机械通气。
该方案中最重要的一步是在主动脉弓下通过丝线。当圈套钩住主动脉弓时，所有动作都必须轻柔，以减少对动脉的不必要牵?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由中国国家自然科学基金（NSFC 81822002）资助。我们感谢参加这项工作的所有成员。
Materials
| 4-0 nonabsorbable suture | Jinhuan | HM403 | Used for suturing the skin |
| 5 mL syringe | Haifuda Technology Co., Ltd. | BD-309628 | Used for making snare containing retractor |
| 7-0 nonabsorbable suture | Jinhuan | HM701 | Used for aorta ligation |
| Animal temperature monitor | Kaerwen | FT3400 | Used for monitoring body temperature |
| Buprenorphine | Sigma | B-044 | Used for post-surgical pain treatment |
| Depilatory cream | Veet | N/A | Used for remove body hair from the surgical area |
| Heating Pad | Xiaochuangxin | N/A | Used for maintaining body temperature |
| Ibuprofen | MCE | HY-78131 | Used for post-surgical pain treatment |
| Iron wire (0.5 mm) | Qing Yuan | Iron wire #26 | Used for making snare containing retractor |
| Microscopic tweezers | RWD | F12006-10 | Used for penetrating and separating the tissue to open operation space |
| Needle holder | RWD | F12005-10 | Used for pinching off the tip of gauge needle and blunting it |
| Ophthalmic forceps | RWD | F14012-10 | Used for holding skin and other tissues |
| Ophthalmic scissors | RWD | S11001-08 | Used for making sking incision of mouse |
| Pentobarbital sodium | Sigma | P3761 | Used for mouse anesthesia |
| Sterile operating mat | Hale & hearty | 211002 | Used for placing animal during surgery |
| Ultra-sound imaging system | Fujifilm visualsonics | vevo1100 | Used for measure the blood flow velocity, left ventricular wall thickness and ejection fraction, https://www.visualsonics.com/product/imaging-systems/vevo-1100 |
References
- Heidenreich, P. A., et al. AHA/ACC/HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation. 145 (18), 895 (2022).
- McDonagh, T. A., et al. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. European Heart Journal. 42 (36), 3599 (2021).
- Lv, B., et al. Induction of myocardial infarction and myocardial ischemia-reperfusion injury in mice. Journal of Visualized Experiments. (179), e63257 (2022).
- Curaj, A., Simsekyilmaz, S., Staudt, M., Liehn, E. Minimal invasive surgical procedure of inducing myocardial infarction in mice. Journal of Visualized Experiments. (99), e52197 (2015).
- Nakamura, M., Sadoshima, J. Mechanisms of physiological and pathological cardiac hypertrophy. Nature Reviews Cardiology. 15 (7), 387-407 (2018).
- Wang, H., et al. Bibliometric analysis on the progress of chronic heart failure. Current Problems in Cardiology. 47 (9), 101213 (2022).
- Riehle, C., Bauersachs, J. Small animal models of heart failure. Cardiovascular Research. 115 (13), 1838-1849 (2019).
- Melleby, A. O., et al. A novel method for high precision aortic constriction that allows for generation of specific cardiac phenotypes in mice. Cardiovascular Research. 114 (12), 1680-1690 (2018).
- Rockman, H. A., Wachhorst, S. P., Mao, L., Ross, J. ANG II receptor blockade prevents ventricular hypertrophy and ANF gene expression with pressure overload in mice. The American Journal of Physiology. 266, 2468-2475 (1994).
- Eichhorn, L., et al. A closed-chest model to induce transverse aortic constriction in mice. Journal of Visualized Experiments. (134), e57397 (2018).
- Tavakoli, R., Nemska, S., Jamshidi, P., Gassmann, M., Frossard, N. Technique of minimally invasive transverse aortic constriction in mice for induction of left ventricular hypertrophy. Journal of Visualized Experiments. (127), e56231 (2017).
- Lang, R. M., et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. Journal of the American Society of Echocardiography. 28 (1), 1-39 (2015).
- Li, L., et al. Assessment of cardiac morphological and functional changes in mouse model of transverse aortic constriction by echocardiographic imaging. Journal of Visualized Experiments. (112), e54101 (2016).
- Wang, X., et al. ATF4 protects the heart from failure by antagonizing oxidative stress. Circulation Research. 131 (1), 91-105 (2022).
- Li, J., et al. GCN5-mediated regulation of pathological cardiac hypertrophy via activation of the TAK1-JNK/p38 signaling pathway. Cell Death & Disease. 13 (4), 421 (2022).
- Syed, A. M., et al. Up-regulation of Nrf2/HO-1 and inhibition of TGF-beta1/Smad2/3 signaling axis by daphnetin alleviates transverse aortic constriction-induced cardiac remodeling in mice. Free Radical Biology and Medicine. 186, 17-30 (2022).
- Zaw, A. M., Williams, C. M., Law, H. K., Chow, B. K. Minimally invasive transverse aortic constriction in mice. Journal of Visualized Experiments. (121), e55293 (2017).
- Lao, Y., et al. Operating transverse aortic constriction with absorbable suture to obtain transient myocardial hypertrophy. Journal of Visualized Experiments. (163), e61686 (2020).
- Veldhuizen, R. A., Slutsky, A. S., Joseph, M., McCaig, L. Effects of mechanical ventilation of isolated mouse lungs on surfactant and inflammatory cytokines. European Respiratory Journal. 17 (3), 488-494 (2001).
- Withaar, C., Lam, C. S. P., Schiattarella, G. G., de Boer, R. A., Meems, L. M. G. Heart failure with preserved ejection fraction in humans and mice: embracing clinical complexity in mouse models. European Heart Journal. 42 (43), 4420-4430 (2021).
Cite This Article
Abuduwufuer, K., Wang, J. J., Li, H., Chen, C. A Modified Technique for Transverse Aortic Constriction in Mice. J. Vis. Exp. (186), e64386, doi:10.3791/64386 (2022).