小鼠使用表面垫心电图心肌缺血再灌注损伤的确认
Summary
During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.
Abstract
Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.
Introduction
心脏疾病仍然死亡全世界1,2的主要原因。不仅是左心室（LV）的最肌肉室，负责从心脏泵血对整个主体3，它是一个常见的心脏损伤部位心肌梗死后4。左心室组织死亡往往会导致心脏收缩功能衰竭。心脏疾病的动物模型是当务之急生物医学心血管研究的进步。小鼠C57BL / 6株已用于动物模型的流行选择由于它们的快速繁殖的时间，成本低和易于在遗传改变。心脏疾病的研究大多数小鼠模型手术涉及到左冠状动脉的LAD支闭塞。在LAD有时称为左钝缘5,6。法援署提供血液到左室前壁和安特罗侧壁。结扎研究的目的是诱导前梗塞，有时延长INT澳劣质和侧壁区7。
经常使用心肌梗塞研究两个模型包括慢性阻塞心肌梗塞和心肌缺血/再灌注损伤。慢性阻塞是通过手术缝合周围，阻止永久通过LAD血流创建。缺血/再灌注损伤被创建以相同的方式多只用一个短暂的，通常是30-60分钟，局部缺血期。实现短暂缺血，周围LAD咬合缝合关系和一个小的PE-10管，其被平行放置在LAD心脏的心外膜表面上，随后再灌注期，其中管子和封闭缝合被去除，血液是使其再次通过动脉并进入心肌流动。缺血/再灌注手术已被认为是临床相关的因再灌注损伤人体平行梗塞治疗，其中包括舞会的性质PT冠状动脉成形术和动脉支架置入，或冠状动脉旁路。典型地，这些手术期间，在小鼠心脏左心室的缺血是由心肌壁的可见苍白证实。但是，通过简单地不断监测条件下的心电图（ECG）垫进行手术，可在ECG波形观察到明显的变化，从而证实小鼠心肌缺血再灌注损伤。
虽然小鼠心脏在许多方面，包括它的四腔结构类似于人类的心脏，心中也有差异。一个明显的区别是成年小鼠的平均静息心脏速率为600 -每分钟（BPM）700次，而成人人类是60-100〜8,9 BPM。此外，在小鼠复极化波，J和T，常合并与除极QRS波群作出明确的ST段难以辨别10。要复杂electrocardiographicall的过程ý确认心肌缺血，它是T波和用作缺血和心肌梗死损伤在人体中的诊断标志物的ST段的抬高，临床上称为STëlevation 米 yocardial 我 nfarction或STEMI。一个人和鼠的波形之间的主要区别是，S波之后紧接着是J-波直接传送到负T波。期间小鼠急性心肌缺血S波减小的幅度，并随后直接由异常的J-波和倒T波11。 T波似乎没有在小鼠中11来表示复极化的显著一部分。尽管命名和鼠标与人类不同，小鼠心肌缺血再灌注心电图确认是仍然可行和相对简单的。为了简化波形解释的缘故，SJT之间的段被称为ST-segmeNT于此。
发表于2013 STEMI指南推荐小于90分钟12患者门到气球时间。这意味着从患者的冠状动脉闭塞的识别的时间帧，直至动脉重新打开应小于90分钟。跳动的心脏不断工作，因此，具有较高的氧化代谢和氧耗3的较高水平。为了提供这一点，毛细血管的网络是可用于每个肌细胞3。只需要一个心脏跳动数耗尽它的氧气和营养供应。在90分钟的窗口，在一个人的缺血性心脏地区将被封锁接收5400名点附近摇摆心脏跳动的富含氧气的血液的价值。在同样的90分钟的窗口，鼠标将有54000 63000心脏跳动。对鼠缺血/再灌注损伤的实验时间点通常是30分钟和60分钟之间。
发展的重要性ING证实了小鼠模型心肌缺血再灌注的补充方法对心肌缺血/再灌注损伤的研究数据的一致性和可重复性产生深远的影响。目视观察心脏的组织颜色改变目前的做法是不够的作为一个独立的诊断。此外，除去管和缝合之后再灌注不能保证。虽然动脉不再打结，动脉可具有在手术期间持续伤害，并可能成为不可能reperfuse。这将是有益的的心电图变化的记录，以确认再灌注而不是依赖于心肌苍白和发红（红色）的意见。心不显示缺血/再灌注损伤的标记就可以迅速被标记，并就如何继续处理的决定可以由侦查进行。
最后，建立心电图的记录从基线整个日变化Ë缺血再灌注时间允许调查人员继续初次手术后监测心脏。目前调查的手术完成后尽快忘记的心脏。心电图是深入了解手术后在心肌小时发生至数天变化的简单方法。心电图记录的时间点，手术后可以揭示表明持续或恶化的组织死亡后发Q波。然而，为了有效地测厚仪新的或恶化的心电图标记，一个基线ECG必须可用于比较。
该协议将展示如何准备，获取和解读心电图，确认使用8小鼠心脏缺血再灌注 – 19周龄雄性C57BL / 6小鼠。
Protocol
Representative Results
Discussion
使用心电图改变为确认心肌缺血再灌注补充的方法，确保咬合结扎的准确位置。结扎的位置精度是减少动物之间的数据变化的关键。在小鼠心脏的LAD是一个困难的动脉可视化。因此，补充的视觉与苍白心电图改变将有助于确保结扎并导致组织损伤的正确位置。
自所述ECG垫提供心脏的非侵入性的观点，可以在研究过程中获得的多个心电图。这可以帮助提供更好的理解期间和手?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by Merit Review awards (BX002332 and BX000640) from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development, National Institutes of Health (R15HL129140), and funds from Institutional Research and Improvement account. The project is supported in part by the National Institutes of Health grant C06RR0306551.
Materials
| Vevo 1100 | Fujifilm Visual Sonics |
Echocardiography Machine | |
| Mouse Handling Plate | Fujifilm Visual Sonics |
Heated ECG plate | |
| Signa-Gel | Highly Conductive Multi- | ||
| Electrode Gel | Parker | 15-25 | Purpose Electrolyte |
| Transpore Medical Tape | 3M | 1527-0 | |
| PI-Spray II | Pharmaceutical Innovations | NDC 36-2013-25 | Cleaning agent for ECG plate |
| C57Bl6 Mice | The Jackson Laboratory | 000664 | Male, 8-12 wk |
| IsoThesia-Isoflurane | Henry Schein | NDC 1169-0500-1 | |
| Excel | Microsoft | ||
| Systane Nighttime Lubricant Eye Ointment | Alcon | 65050935 | |
| 7-0 Perma-Hand Silk Sutures | Ethicon | 640.O32 | |
| 5-0 Perma-Hand Silk Sutures | Ethicon | K809.O32 | |
| Surgical Scissors | ROBOZ | RS-5881 | |
| Forceps | Fine Science Tools | 11052-10 | |
| Gauze | Bio Nuclear Diagnostics Inc | DIS-022B | |
| Needle Holder | Fine Science Tools | 12565-14 | |
| Buprenex CIII | Patterson Veterinary | 0-891-9756 | Buprenorphine Hydrochloride Analgesic |
| Betadine | Purdue Products | 67618-150-08 |
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