标准化大鼠冠状动脉环制备及实时记录血管直径动态张力变化
Summary
本方案描述了用于测量大鼠冠状动脉血管反应性的线肌图技术。
Abstract
冠状动脉疾病（CAD）作为心血管系统疾病的重要事件，被广泛认为是动脉粥样硬化、心肌梗塞、心绞痛等严重威胁全世界人民生命和健康的罪魁祸首。然而，如何记录孤立血管的动态生物力学特征，长期以来一直困扰着人们。同时，精确定位和分离冠状动脉以测量 体外 动态血管张力变化已成为CAD药物开发的趋势。本方案描述了大鼠冠状动脉的宏观鉴定和微观分离。使用已建立的多肌图系统监测冠状动脉环沿血管直径的收缩和扩张功能。冠状动脉环张力测量的标准化和编程方案，从采样到数据采集，极大地提高了实验数据的可重复性，保证了生理、病理和药物干预后血管张力记录的真实性。
Introduction
冠状动脉疾病（CAD）已被广泛认为和关注为一种典型和具有代表性的心血管疾病，是发达国家和发展中国家的主要死因1，2。作为正常心脏生理功能的血液和氧气供应途径，循环血液通过心肌表面的两个主要冠状动脉和血血管网络3，4进入并滋养心脏。冠状动脉中的胆固醇和脂肪沉积物切断了心脏的血液供应和血管系统的剧烈炎症反应，导致动脉粥样硬化，稳定型心绞痛，不稳定型心绞痛，心肌梗塞或心源性猝死5，6。响应冠状动脉的病理性狭窄，代偿性加速生理心跳通过增加左心室7的输出来满足心脏本身或身体重要器官的血液供应。如果长时间的冠状动脉狭窄不能及时缓解，心脏的某些区域可能会出现广泛的新血管8。目前，CAD的临床治疗往往采用药物溶栓或手术机械溶栓和外源性仿生血管旁路，用药频繁，手术残疾很大9.因此，冠状动脉功能研究生理活动仍是心血管疾病10项的急需突破口。
除了无线遥测系统，没有可用的技术手段来检测冠状动脉生理活动，它可以动态记录 体内 冠状动脉压力，血管张力，血氧饱和度和pH值11。因此，考虑到冠状动脉的质地隐秘性和复杂性，冠状动脉的准确识别和分离无疑是探索CAD 体外4多种机制的最佳选择。
一系列多肌图系统，特别是线材显微照片微血管张力检测仪（见 材料表），是一种非常成熟的市场化设备，用于记录小血管、淋巴管和支气管的 体外 组织张力变化，具有高精度和连续动态记录12的特点。该系统已被广泛用于记录直径为60μm至10mm的腔体结构的 体外 组织张力特性。线材显微照片平台的连续加热特性在很大程度上抵消了对不利外部环境的刺激。同时，气体混合物的恒定输入和pH值使我们能够在相似的生理状态下获得更准确的血管张力数据13。然而，考虑到大鼠冠状动脉解剖定位的复杂性（图1），其分离一直令人困惑，并限制了其对多样化心血管疾病和药物开发的机制的探索。因此，本方案详细介绍了大鼠冠状动脉的解剖位置和分离过程，随后在钢丝显微照片14的平台上进行张力测量。
Protocol
该动物方案由成都中医药大学管理委员会审查批准（备案号：2021-11）。雄性Sprague Dawley（SD）大鼠（260-300g，8-10周龄）用于本研究。这些老鼠被关在动物室里，在实验期间可以自由饮水和进食。 1. 溶液制备 通过溶解118 mM的NaCl，4.7 mM的K +，2.5 mM的CaCl2，1.2 mM的KH2PO4，1.2 mM的MgCl2∙6H2O，25 mM的NaHCO3，11 m…
Representative Results
解剖学上定位，大鼠冠状动脉分布并隐藏在心肌组织深处不容易识别。通过比较人（图1A）和大鼠的冠状动脉（图1B），根据 图2中的采样过程进行大鼠冠状动脉的快速准确分离。在光学显微镜下从前部精确定位右耳廓，肺动脉和顶点后，沿着 图2A所示的实心黑线解剖心肌。冠状动脉脑室间分支的约5毫米明显…
Discussion
冠状动脉微循环紊乱涉及广泛的CAD患者，已逐渐得到认可，并关注充分心肌灌注的基础。考虑到突发性冠心病和心血管疾病的严重并发症，及时药物预防和治疗对于CAD17的临床个体非常重要。不可避免地，冠状动脉解剖结构的保密性及其生理结构的复杂性严重制约了对CAD18、19、20、21、22<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了四川省科技计划重点研发项目（2022YFS0438）、国家自然科学基金（82104533）、中国博士后科学基金（2020M683273）和四川省科技厅（2021YJ0175）的支持。
Materials
| Apigenin | Sangon Biotech Co., Ltd., Shanghai, China | 150731 | |
| CaCl2 | Sangon Biotech Co., Ltd., Shanghai, China | A501330 | |
| D-glucose | Sangon Biotech Co., Ltd., Shanghai, China | A610219 | |
| HEPES | Xiya Reagent Co., Ltd., Shandong, China | S3872 | |
| KCl | Sangon Biotech Co., Ltd., Shanghai, China | A100395 | |
| KH2PO4 | Sangon Biotech Co., Ltd., Shanghai, China | A100781 | |
| LabChart Professional version 8.3 | ADInstruments, Australia | — | |
| MgCl2·6H2O | Sangon Biotech Co., Ltd., Shanghai, China | A100288 | |
| Multi myograph system | Danish Myo Technology, Aarhus, Denmark | 620M | |
| NaCl | Sangon Biotech Co., Ltd., Shanghai, China | A100241 | |
| NaHCO3 | Sangon Biotech Co., Ltd., Shanghai, China | A100865 | |
| Steel wires | Danish Myo Technology, Aarhus, Denmark | 400447 | |
| U46619 | Sigma, USA | D8174 |
References
- Malakar, A. K., et al. A review on coronary artery disease, its risk factors, and therapeutics. Journal of Cellular Physiology. 234 (10), 16812-16823 (2019).
- Murray, C., et al. national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: A systematic analysis for the global burden of disease Study 2013. The Lancet. 385 (9963), 117-171 (2015).
- Zhang, Y., et al. Adenosine and adenosine receptor-mediated action in coronary microcirculation. Basic Research in Cardiology. 116 (1), 22 (2021).
- Allaqaband, H., Gutterman, D. D., Kadlec, A. O. Physiological consequences of coronary arteriolar dysfunction and its influence on cardiovascular disease. Physiology. 33 (5), 338-347 (2018).
- Minelli, S., Minelli, P., Montinari, M. R. Reflections on atherosclerosis: Lesson from the past and future research directions. Journal of Multidisciplinary Healthcare. 13, 621-633 (2020).
- Alvarez-Alvarez, M. M., Zanetti, D., Carreras-Torres, R., Moral, P., Athanasiadis, G. A survey of sub-saharan gene flow into the mediterranean at risk loci for coronary artery disease. European Journal of Human Genetics. 25 (4), 472-476 (2017).
- LaCombe, P., Tariq, M. A., Lappin, S. L. Physiology, Afterload Reduction. StatPearls [Internet]. , (2022).
- Gutterman, D. D., et al. The human microcirculation: regulation of flow and beyond. Circulation Research. 118 (1), 157-172 (2016).
- Wang, G., Li, F., Hou, X. Complementary and alternative therapies for stable angina pectoris of coronary heart disease: A protocol for systematic review and network meta-analysis. Médecine. 101 (7), 28850 (2022).
- Markousis-Mavrogenis, G., et al. Coronary microvascular disease: the "meeting point" of cardiology. European Journal of Clinical Investigation. 52 (5), 13737 (2021).
- Allison, B. J., et al. Fetal in vivo continuous cardiovascular function during chronic hypoxia. The Journal of Physiology. 594 (5), 1247-1264 (2016).
- Wenceslau, C. F., et al. Guidelines for the measurement of vascular function and structure in isolated arteries and veins. American Journal of Physiology-Heart and Circulatory Physiology. 321 (1), 77-111 (2021).
- Liu, L., et al. Comparison of Ca2+ handling for the regulation of vasoconstriction between rat coronary and renal arteries. Journal of Vascular Research. 56 (4), 191-203 (2019).
- Sun, J., et al. Isometric contractility measurement of the mouse mesenteric artery using wire myography. Journal of Visualized Experiments. (138), e58064 (2018).
- Guo, P., et al. Coronary hypercontractility to acidosis owes to the greater activity of TMEM16A/ANO1 in the arterial smooth muscle cells. Biomedicine & Pharmacotherapy. 139, 111615 (2021).
- Ping, N. N., Cao, L., Xiao, X., Li, S., Cao, Y. X. The determination of optimal initial tension in rat coronary artery using wire myography. Physiological Research. 63 (1), 143-146 (2014).
- Niccoli, G., Scalone, G., Lerman, A., Crea, F. Coronary microvascular obstruction in acute myocardial infarction. European Heart Journal. 37 (13), 1024-1033 (2016).
- Mumma, B., Flacke, N. Current diagnostic and therapeutic strategies in microvascular angina. Current Emergency and Hospital Medicine Reports. 3 (1), 30-37 (2015).
- Lanza, G. A., Parrinello, R., Figliozzi, S. Management of microvascular angina pectoris. American Journal of Cardiovascular Drugs. 14 (1), 31-40 (2014).
- Zhu, T. Q., et al. Beneficial effects of intracoronary tirofiban bolus administration following upstream intravenous treatment in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention: The ICT-AMI study. International Journal of Cardiology. 165 (3), 437-443 (2013).
- Huang, D., et al. Restoration of coronary flow in patients with no-reflow after primary coronary intervention of acute myocardial infarction (RECOVER). American Heart Journal. 164 (3), 394-401 (2012).
- Fu, W. J., et al. Anti-atherosclerosis and cardio-protective effects of the Angong Niuhuang Pill on a high fat and vitamin D3 induced rodent model of atherosclerosis. Journal of Ethnopharmacology. 195, 118-126 (2017).
- Li, J., et al. Chinese medicine She-Xiang-Xin-Tong-Ning, containing moschus, corydalis and ginseng, protects from myocardial ischemia injury via angiogenesis. The American Journal of Chinese Medicine. 48 (1), 107-126 (2020).
- Wu, W., et al. Three dimensional reconstruction of coronary artery stents from optical coherence tomography: Experimental validation and clinical feasibility. Scientific Reports. 11 (1), 1-15 (2021).
- Liu, M., et al. Janus-like role of fibroblast growth factor 2 in arteriosclerotic coronary artery disease: Atherogenesis and angiogenesis. Atherosclerosis. 229 (1), 10-17 (2013).
- Hu, G., Li, X., Zhang, S., Wang, X. Association of rat thoracic aorta dilatation by astragaloside IV with the generation of endothelium-derived hyperpolarizing factors and nitric oxide, and the blockade of Ca2+ channels. Biomedical reports. 5 (1), 27-34 (2016).
- Guo, Y., et al. Anticonstriction effect of MCA in rats by danggui buxue decoction. Frontiers in Pharmacology. 12, 749915 (2021).
- Jing, Y., et al. Apigenin relaxes rat intrarenal arteries, depresses Ca2+-activated Cl− currents and augments voltage-dependent K+ currents of the arterial smooth muscle cells. Biomedicine & Pharmacotherapy. 115, 108926 (2019).
Citer Cet Article
Guo, P., An, W., Guo, Y., Sun, Z., Wang, X., Zhang, S. Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter. J. Vis. Exp. (184), e64121, doi:10.3791/64121 (2022).