孤立大鼠心脏长期心室颤动模型
Summary
该协议提出了由低压交流电连续刺激引起的大鼠心脏长期心室颤动的模型。该模型成功率高，稳定可靠，可重复，对心脏功能影响小，仅引起轻度心肌损伤。
Abstract
心室颤动（VF）是一种致命的心律失常，在心脏病患者中发病率很高，但灌注下的VF骤停是心脏外科领域被忽视的术中停搏方法。随着心脏手术的最新进展，对灌注下长期VF研究的需求有所增加。然而，该领域缺乏简单、可靠和可重复的慢性心室颤动动物模型。该方案通过交流电（AC）电刺激心外膜诱导长期VF。采用不同条件诱导VF，包括用低电压或高电压连续刺激以诱导长期VF和用低电压或高压刺激5 min以诱导自发的长期VF。比较不同情况的成功率，以及心肌损伤和心脏功能恢复的发生率。结果表明，连续低压刺激诱导长期VF，5 min低压刺激诱导自发性长期VF，伴轻度心肌损伤，心功能恢复率高。然而，低电压、连续激励的长期VF模型具有较高的成功率。高压刺激提供了更高的VF诱导率，但除颤成功率低，心脏功能恢复不良，心肌损伤严重。基于这些结果，推荐连续低压心外膜交流电刺激，其成功率高，稳定性高，可靠性高，重现性强，对心脏功能影响小，心肌损伤轻。
Introduction
心脏手术通常通过开胸术进行，阻塞主动脉并灌注心脏停滞溶液以阻止心脏。重复心脏手术可能比初始手术更具挑战性，并发症和死亡率更高1，2，3。此外，传统的正中胸骨切开术可能会对胸骨后面的桥血管、升主动脉、右心室和其他重要结构造成损害。结缔组织分离引起的大量出血、胸骨伤口感染和胸骨切开术引起的胸骨骨髓炎都是可能的并发症。大量解剖会增加重要心脏结构病变和出血的风险。
随着微创心脏手术的发展，切口越来越小，有时难以实现心脏骤停。在心室颤动（VF）下重复心脏手术4，5是安全、可行的，并且可以提供更好的心肌保护。因此，该协议介绍了微创体外循环手术中VF心脏骤停的方法。心脏在VF期间失去有效收缩，因此在手术过程中无需缝合和阻塞升主动脉，这简化了手术。然而，即使心脏连续灌注，长期VF仍然可能对心脏有害。
随着这种方法的使用越来越广泛，如何在VF期间保护心脏的问题变得越来越重要。这将需要使用长期VF的动物模型进行广泛而深入的研究。过去，该领域的研究主要使用大型动物6，7，并且需要外科医生，麻醉师，灌注师和其他研究人员之间的合作。这些研究耗时太长，样本量通常很小，研究通常侧重于心脏功能，而不是机制和分子评估。迄今为止，还没有研究报告建立长期VF模型的详细方案。
因此，该协议提供了使用Langendorff装置开发长期VF大鼠模型所需的详细信息。该方案简单、经济、可重复且稳定。
Protocol
本次调查中使用的所有实验程序和方案均由解放军总医院动物护理和使用委员会审查批准。 1. 准备朗根道夫仪器 准备克雷布斯-亨塞莱特（K-H）缓冲液。要制备 K-H 缓冲液，请在蒸馏水中加入以下内容：118.0 mM NaCl、4.7 mM KCl、1.2 mM MgSO 4、1.2 mM NaH 2 PO4、1.8 mM CaCl2、25.0 mM NaHCO3、11.1 mM 葡萄糖和 0.5 mM EDTA。 准备?…
Representative Results
实验共使用57只大鼠，其中30只符合纳入标准。将纳入的动物分为5组，每组6只：对照组（C组）、低压持续刺激长期VF组（LC组）、高压持续刺激长期VF组（HC组）、低压诱导自发长期VF组（LI组）和高压诱导自发长期VF组（HI组）。每组的实验过程如图 2所示。 VF模型的成功率VF的比率，除颤的成功率和VF模型的成功率如 表1所示。…
Discussion
该协议在孤立的大鼠心脏中建立了长期VF的动物模型，该模型以前未报道过。此外，本研究还比较了不同的电刺激条件。本研究为心脏手术期间心室颤动骤停相关的研究提供了一个模型。
模型的成功率是非常重要的指标，与人员、时间、经济成本有关。在VF模型中，成功率包括VF是否可以在心脏中诱导以及除颤后心脏是否可以恢复正常跳动。此外，应考虑心功能恢复率和心肌?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国人民解放军总医院第一医学中心心血管外科和中国人民解放军总医院实验动物中心的支持。
Materials
| 0 Non-absorbable suture | Ethicon, Inc. | Preparation of the isolated heart | |
| 95% O2 + 5% CO2 | Beijing BeiYang United Gas Co., Ltd. | K-H buffer | |
| AcqKnowledge software | BIOPAC Systems Inc. | Version 4.2.1 | Software |
| Automatic biochemistry analyzer | Rayto Life and Analytical Sciences Co., Ltd. | Chemray 800 | CK-MB assay |
| BIOPAC research systems | BIOPAC Systems Inc. | MP150 | Hardware |
| Blunt needle (20 G, TWLB) | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | Modified Langendorff perfusion system |
| Calcium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10005861 | K-H buffer |
| CK-MB assay kits | Changchun Huili Biotech Co., Ltd. | C060 | CK-MB assay |
| Curved forcep | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| EDTA | Sinopharm Chemical Reagent Co.,Ltd | 10009717 | K-H buffer |
| Electrical stimulator | BIOPAC Systems Inc. | STEMISOC | Hardware |
| Filter | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | |
| Glucose | Sinopharm Chemical Reagent Co.,Ltd | 63005518 | K-H buffer |
| Heparin sodium | Tianjin Biochem Pharmaceutical Co., Ltd. | H120200505 | Preparation of the isolated heart |
| Isoflurane | RWD Life Science Co.,LTD | 21082201 | Preparation of the isolated heart |
| Magnesium sulfate | Sinopharm Chemical Reagent Co.,Ltd | 20025118 | K-H buffer |
| Needle electrodes | BIOPAC Systems Inc. | EL452 | Hardware |
| Ophthalmic clamp | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ophthalmic forceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ophthalmic scissors | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Perfusion tube | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | Modified Langendorff perfusion system |
| Potassium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10016318 | K-H buffer |
| Sodium bicarbonate | Sinopharm Chemical Reagent Co.,Ltd | 10018960 | K-H buffer |
| Sodium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10019318 | K-H buffer |
| Sodium dihydrogen phosphate dihydrate | Sinopharm Chemical Reagent Co.,Ltd | 20040718 | K-H buffer |
| Sprague-Dawley (SD) rats | SPF (Beijing) biotechnology Co., Ltd. | Male, 300-350g | Preparation of the isolated heart |
| Thermometer | Jiangsu Jingchuang Electronics Co., Ltd. | GSP-6 | Modified Langendorff perfusion system |
| Tissueforceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Tissue scissors | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Toothed forceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ventilator | Chengdu Instrument Factory | DKX-150 | Preparation of the isolated heart |
| Water bath1 | Ningbo Scientz Biotechnology Co.,Ltd. | SC-15 | Modified Langendorff perfusion system |
| Water bath2 | Shanghai Yiheng Technology Instrument Co., Ltd. | DK-8D | Modified Langendorff perfusion system |
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Citer Cet Article
He, X., Li, L., Xu, W., Jiang, S. A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts. J. Vis. Exp. (192), e65101, doi:10.3791/65101 (2023).