在大鼠中建立深低温循环停滞
Summary
该协议提出了大鼠深低温循环停搏的建立，可用于研究全身炎症反应综合征，缺血/再灌注损伤，氧化应激，神经炎症等。
Abstract
深度低温循环骤停（DHCA）常规应用于复杂先天性心脏病和主动脉弓疾病的手术。本研究旨在提供一种在大鼠中建立DHCA的方法。为了评估DHCA过程对生命体征的影响，使用没有循环停止的常温体外循环（CPB）大鼠模型作为对照。正如预期的那样，DHCA导致体温和平均动脉血压显着降低。血气分析表明，DHCA增加了乳酸水平，但不影响血液pH值和血红蛋白、血细胞比容、Na+、Cl−、K+和葡萄糖的浓度。此外，与常温CPB大鼠相比，透射电子显微镜结果显示DHCA大鼠海马自噬体轻度增加。
Introduction
自1953年以来，深低温循环骤停（DHCA）已用于心脏手术1。DHCA涉及将患者的核心温度降低到严重低温水平（15-22°C），然后全局中断流向身体的血液2。循环停滞可以提供相对不流血的操作场。深度低温会降低新陈代谢，特别是在大脑和心肌中，这是防止缺血的有效方法3。DHCA通常用于复杂先天性心脏病，主动脉弓疾病，甚至带有腔静脉血栓的肾脏或肾上腺肿瘤4，5的手术中。因此，建立DHCA动物模型为临床环境中程序的细化和并发症的预防提供了重要参考。
虽然可以用犬6、兔7和其他动物建立模型，但由于它们的可操作性和低成本，最好使用大鼠。DHCA大鼠模型于2006年由Jungwirth等人首次描述8。结果发现，循环停止的持续时间对神经系统结局有影响。从那时起，DHCA大鼠模型得到了广泛的研究。已经澄清，DHCA可能引发全身炎症反应综合征（SIRS）9。在随后的研究中，药理学家发现，由SIRS诱导的DHCA相关神经炎症可以通过白藜芦醇10和雷公藤内酯11减弱。我们的团队还发现，DHCA相关的神经炎症可以通过抑制冷诱导的RNA结合蛋白12来减弱。在心血管系统中，超氧化物歧化酶对DHCA13期间的缺血/再灌注（I / R）损伤具有心脏保护作用。这些结果扩大了对DHCA相关病理生理过程的理解，并为改善DHCA的结局提供了新的方向。然而，关于DHCA后内毒素血症，氧化应激和自噬的结果尚无定论。DHCA使用与体外循环（CPB）相同的操作技术14，但其管理策略不同，并且不同团队产生DHCA的步骤不同8，9，10，11。本研究旨在提供一种在大鼠中建立DHCA程序的方法。
Protocol
该方案经过机构审查，并获得了中国医学科学院阜外医院机构动物护理和使用委员会的批准（FW-2021-0005）。所有实验程序均按照美国国立卫生研究院出版的实验动物护理和使用指南进行。 注意：雄性Sprague-Dawley大鼠（重量：500-600克，年龄：12-14周）保持在标准实验室条件下，自由获取食物和水。将大鼠随机分为两组（n = 6，每组）：DHCA组和常温CPB组（NtCPB组）。 <p class="jove…
Representative Results
作为对照组，未发生循环骤停的常温CPB（NtCPB）大鼠在整个手术过程中平均动脉血压（MAP）和体温稳定，而DHCA大鼠心脏骤停期间的MAP下降（p <0.01， 图3A）。DHCA大鼠的温度在冷却阶段迅速下降，并在复温阶段逐渐恢复。当将大鼠从DHCA回路断奶时，DHCA大鼠的温度恢复正常（图3B）。 通过血气分析研究了DHCA过程对大鼠的影响。?…
Discussion
插管是在大鼠中建立DHCA的最基本程序。插管前，用 0.5 mL 的 2% 利多卡因浸泡动脉将使其更容易插管。插管后，需要通过颈外静脉 用 500IU / kg肝素进行肝素化，以避免微血栓形成17。我们反复发现，这种剂量的肝素可以达到激活凝血时间（ACT）>480秒的目标。复温期是最困难的部分。在我们的实验中，温度从18°C上升到34°C需要60多分钟，而在其他一些实验中，复温期可以在30?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢张亮在实验过程中帮助收集视频数据。本研究得到了国家自然科学基金（批准号：82070479）和中央高校基本科研业务费（批准号：3332022128）的支持。
Materials
| Heat Exchanger | Xi’an Xijing Medical Appliance Co., Ltd | Animal-M | |
| Membrane Oxygenator | Dongguan Kewei Medical Instrument Co., Ltd. | Micro-M | |
| Monitor | Chengdu Techman Co., Ltd | BL-420s | |
| Roller Pump | Changzhou Prefluid Technology Co.,Ltd | BL100 | |
| SD Rat | HFK Bioscience Co.,Ltd. | / | |
| Sevoflurane | Maruishi Pharmaceutical Co. Ltd | H20150020 | |
| Shaver | Hangzhou Huayuan Pet Products Co.,Ltd. | / | |
| Vaporizer | SPACECABS | / | |
| Ventilator | Shanghai Alcott Biotech Co., Ltd | ALC-V8S | |
| Water Tank | Maquet Critical Care AB | Jostra HCU20-600 |
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Citazione di questo articolo
Yan, W., Ji, B. Establishment of Deep Hypothermic Circulatory Arrest in Rats. J. Vis. Exp. (190), e63571, doi:10.3791/63571 (2022).