藏族小型猪射血分数保留的心力衰竭手术模型
Summary
本方案描述了使用降主动脉收缩建立射血分数保留的心力衰竭迷你猪模型的分步程序。还介绍了评估该疾病模型的心脏形态学、组织学和功能的方法。
Abstract
全球超过一半的心力衰竭 （HF） 病例被归类为射血分数保留心力衰竭 （HFpEF）。大型动物模型在研究HFpEF的基本机制和确定潜在的治疗靶点方面受到限制。本工作详细介绍了藏族小型猪降主动脉收缩（DAC）的外科手术过程，以建立HFpEF的大型动物模型。该模型使用精确控制的降主动脉收缩来诱导左心室的慢性压力超负荷。超声心动图用于评估心脏的形态学和功能变化。DAC应激12周后，室间隔肥厚，但后壁厚度明显减少，伴有左心室扩张。然而，在 12 周期间，模型心脏的左心室射血分数保持在 >50%。此外，DAC模型显示心脏损伤，包括纤维化、炎症和心肌细胞肥大。DAC组的心力衰竭标志物水平显著升高。这种 DAC 诱导的小型猪 HFpEF 是研究这种疾病的分子机制和临床前测试的有力工具。
Introduction
射血分数保留的心力衰竭 （HFpEF） 占心力衰竭病例的一半以上，并已成为全球公共卫生问题1。临床观察表明 HFpEF 的几个关键特征：（1） 心室舒张功能障碍，伴有收缩刚度增加，（2） 静息时射血分数正常，运动表现受损，以及 （3） 心脏重塑2.提出的机制包括激素失调、全身性微血管炎症、代谢紊乱以及肌节和细胞外基质蛋白的异常3。然而，实验研究表明，射血分数降低的心力衰竭 （HFrEF） 会导致这些改变。临床研究探讨了血管紧张素受体抑制剂和药物治疗 HFpEF中 HFrEF 的治疗效果 4,5。然而，需要独特的HFpEF治疗方法。与了解临床症状相比，HFpEF在病理学、生物化学和分子生物学方面的改变仍然不明确。
已经开发了 HFpEF 的动物模型来探索其机制、诊断标志物和治疗方法。实验动物，包括猪、狗、大鼠和小鼠，可发生HFpEF，并选择高血压、糖尿病和衰老等多种危险因素作为诱导因素6,7。例如，单独使用醋酸脱氧皮质酮或与高脂肪/高糖饮食联合使用可诱导猪的 HFpEF 8,9。心室压力超负荷是另一种用于在大型和小型动物模型中开发 HFpEF的技术 10。此外，近年来，各大洲都采用了特定的EF临界值来定义HFpEF，如欧洲心脏病学会指南、美国心脏病学会基金会/美国心脏协会11、日本循环学会/日本心力衰竭学会12所示。因此，如果采用临床标准，许多先前建立的模型可能适用于 HFpEF 研究。例如，Youselfi等人声称转基因小鼠品系Col4a3-/-是一种有效的HFpEF模型。该菌株出现典型的 HFpEF 心脏症状，例如舒张功能障碍、线粒体功能障碍和心脏重塑13。之前的一项研究使用高能量饮食在老年猴子14 中诱导具有中等 EF 的心脏重塑，其特征是代谢紊乱、纤维化和心肌中肌动球蛋白 MgATP 酶减少。小鼠横主动脉收缩 （TAC） 是模拟高血压诱导的室性心肌病最广泛使用的模型之一。左心室从向心性肥大（EF升高）进展为扩张性重塑（EF降低15.16）。这两个典型阶段之间的过渡表型表明，主动脉收缩技术可用于研究 HFpEF。
猪 HFpEF 模型的病理特征、细胞信号转导和 mRNA 谱之前已发表17。在这里，提出了建立该模型的分步方案以及评估该模型表型的方法。该过程如 图 1 所示。简而言之，手术计划由主要研究者、外科医生、实验室技术人员和动物护理人员共同制定。这些迷你猪接受了健康检查，包括生化测试和超声心动图检查。手术后，进行了抗炎和镇痛手术。超声心动图、组织学检查和生物标志物用于评估表型。
Protocol
所有动物研究均获得广东省实验动物监测所机构动物护理和使用委员会的批准（批准号。IACUC2017009）。所有动物实验均按照《实验动物护理和使用指南》（第 8 版，2011 年，美国国家科学院）进行。这些动物被安置在广东省实验动物监测研究所（许可证号）AAALAC认可的设施中。SYXK （YUE） 2016-0122， 中国）。使用6只雄性藏族迷你猪（假猪组和DAC组各n=3头，体重25-30公斤）开发HFpEF模型。 <p class="jo…
Representative Results
超声心动图在第 0、2、4、6、8、10 和 12 周评估心脏结构和功能。胸骨旁短轴视图的 B 模式和 M 模式记录显示在图 4A 中。超声心动图测量包括室间隔厚度（VST）、后壁厚度（PWT）和左心室内部尺寸（LVID）。在观察期间，DAC心脏中舒张末期的VST增加，而舒张末期的PWT增加然后减少，这表明DAC迷你猪的左心室中存在肥厚重塑（图4B，C?…
Discussion
本研究利用DAC技术建立了藏小型猪的HFpEF模型。本文介绍了动物和器械的分步制备方案，包括镇静、气管插管、静脉插管、外科手术和术后护理。此外，还介绍了超声心动图B型和M型心脏图像的记录技术。DAC 后，心脏在第 4 周和第 6 周出现左心室肥厚，第 8 周后出现扩张。LVEF 在 12 周期间得以保存。在DAC心脏中观察到纤维化和炎症。
开胸手术和主动脉收缩的结合已被用于开?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了广东省科技计划（2008A08003、2016A020216019、2019A030317014）、广州市科技计划（201804010206）、国家自然科学基金（31672376、81941002）和广东省实验动物重点实验室（2017B030314171）的支持。
Materials
| Absorbable surgical suture | Putong Jinhua Medical Co. Ltd, China | 4-0 | |
| Aesthesia ventilator station | Shenzhen Mindray Bio-Medical Electronics Co., Ltd, China | WATO EX-35vet | |
| Aspirator | Shanghai Baojia Medical Apparatus Co., Ltd, China | YX930D | |
| Benzylpenicillin | Sichuan Pharmaceutical. INC, China | H5021738 | |
| Disposal endotracheal tube with cuff | Shenzhen Verybio Co., Ltd, China | 20 cm, ID 0.9 | |
| Disposal transducer | Guangdong Baihe Medical Technology Co., Ltd, China | ||
| Dissection blade | Shanghai Medical Instruments (Group) Co., Ltd, China | ||
| Electrocautery | Shanghai Hutong Medical Instruments (Group) Co., Ltd, China | GD350-B | |
| Enzyme-linked immunosorbent assay ELISA kit | Cusabio Biotech Co., Ltd, China | CSB-E08594r | |
| Eosin | Sigma-Aldrich Corp. | E4009 | |
| Flunixin meglumine | Shanghai Tongren Pharmaceutical Co., Ltd., China | Shouyaozi(2012)-090242103 | |
| Forceps | Shanghai Medical Instruments (Group) Co., Ltd.,China | ||
| Hematoxylin | Sigma-Aldrich Corp. | H3136 | |
| Isoflurane | RWD Life Science Co., Ltd, China | Veteasy for animals | |
| Laryngoscope | Taixing Simeite Medical Apparatus and Instruments Limited Co., Ltd, China | For adults | |
| LED surgical lights | Mingtai Medical Group, China | ZF700 | |
| Microplate reader | Thermo Fisher Scientific, USA | Multiskan FC | |
| Microscope | Leica, Germany | DM2500 | |
| Mobile restraint unit | Customized | N/A | A mobile restraint unit, made by metal frame and wheels, with a canvas cover |
| Oxygen | Local suppliers, Guangzhou, China | ||
| Paraformaldehyde | Sigma-Aldrich Corp. | V900894 | |
| Patient monitor | Shenzhen Mindray Bio-Medical Electronics Company, China | Beneview T5 | |
| Peripheral Intravenous (IV) Catheter | Shenzhen Yima Pet Industry Development Co., Ltd., China | 26G X 16 mm | |
| Propofol | Guangdong Jiabo Phamaceutical Co., Ltd. | H20051842 | |
| Rib retractor | Shanghai Medical Instruments (Group) Co., Ltd.,China | ||
| Ruler | Deli Manufacturing Company, China | ||
| Scalpel handles | Shanghai Medical Instruments (Group) Co., Ltd.,China | ||
| Scissors (g) | Shanghai Medical Instruments (Group) Co., Ltd.,China | ||
| Suture | Medtronic-Coviden Corp. | 3-0, 4-0 | |
| Ultrasonic gel | Tianjin Xiyuansi Production Institute, China | TM-100 | |
| Veterinary monitor | Shenzhen Mindray Bio-Medical Electronics Company, China | ePM12M Vet | |
| Veterinary ultrasound system | Esatoe, Italy | MyLab30 | Equiped with phased array transducer (3-8 Hz) |
| Xylazine hydrochloride injection | Shenda Animal Phamarceutical Co., Ltd., China | Shouyaozi(2016)-07003 | |
| Zoletil injection | Virbac, France | Zoletil 50 | Tiletamine and zolazepam for injection |
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Cite This Article
Li, X., Tan, W., Li, X., Zheng, S., Zhang, X., Chen, H., Pan, Z., Zhu, C., Yang, F. H. A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs. J. Vis. Exp. (180), e63526, doi:10.3791/63526 (2022).