Name: murine left anterior descending (lad) coronary artery ligation: an improved and simplified model for myocardial infarction
Uploaded: 2017-04-02T15:00:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction at JoVE.com

This model was developed with the support of the National Institute of General Medical Sciences (NIGMS)/the National Institute of Health (NIH) grant 1P20GM103652 (Project# 3) (to MRA) and the American Heart Association (AHA) Grant-in-Aid 14GRNT20460291 (to MRA); the Brazilian government grant CAPES (to KR and FR); and a Brown University LINK award (to IM). We also acknowledge the outstanding technical support from our veterinarians and animal facility staff.

这种动物的协议已经通过审查，机构动物护理和使用委员会（IACUC）在罗德岛医院批准。 1.麻醉插管<ol><li>称重鼠标来计算手术后止痛药的用量。 </li><li>鼠标放置在感应腔室，并提供4％异氟烷9 - 10分钟，监测整个动物。打开热珠灭菌，使得该装置可以预热到约250℃。预热需要15 - 20分钟。 </li><li>一旦小鼠达到麻醉的深平面，具有大约32次呼吸/分钟的呼吸速度，放置在泡沫聚苯乙烯板鼠标仰卧和使用顶部门齿下固定的弹性带保持嘴巴张开。通过执行脚趾掐确认镇静。使得口咽可以显现高强度照明鼠标在上面进行定位。 </li><li>使用弯钳打开颚和另一对钳解除舌头的方式。一定要插管，而位于或略低于眼水平的小鼠的身体。推荐使用手术放大镜的。 </li><li>可视化声带的开启和关闭。当打开时，插入20号，1-在静脉内（IV）导管用钝尖针导引器。使用针，引导导管至气管开口，但要避免将针插入气管。正确位置的验证可以使用塑料移液管来完成。 </li><li>所述插管小鼠转移到配备有加热装置的操作表面。将鼠标连接到一个小动物呼吸机设置为150μL/冲程的冲程容积为130个冲程/分钟的行程速度。 </li><li>交付2.5％的异氟醚。通过检查双侧挺胸抬头验证插管。通过执行脚趾掐确认的麻醉。鼠标可能需要5 - 呼吸机10分钟才能完全麻醉。 </li></ol><ol><li>胶带向下在呼吸机和IV导管之间的连接部位的插管。大盘回落的四肢。广场上的眼睛无菌润滑油滴。 </li><li>修剪胸廓的腹侧左侧用电动剃刀。将灰尘从剃毛皮用干擦拭物，并使用无菌棉签申请的脱毛膏一小层。霜剂应保持与毛囊约30联系人 - 45秒。 </li><li>虽然霜过程，放置3个无菌棉签在三个1.5毫升的管中填充有优碘浸泡。用蘸有蒸馏水纸巾，轻轻擦去奶油和皮毛。 </li><li>清洁外科领域三次，交替聚维酮碘和无菌70％的异丙醇制备垫，清洗在从中心移动到外周的圆周运动。将无菌悬垂超过鼠标的外科手术领域的四分之一大小的洞。 </li><li>清理周围的谅解备忘录的区域e为70％的乙醇。验证与脚趾捏一次麻醉。 </li></ol> 3. LAD结扎<ol><li>置于热珠灭菌蒸压手术器械预热至250℃下进行约20秒。放在无菌高压加热的手术铺巾的无菌器械。唐手术手套。 </li><li>用细尖镊子轻轻地在一个点大约5 mm提升肌肤突出剑突软骨的左侧。使用手术刀用10号刀片从这个点上创建的皮肤切口垂直向上，以胸骨柄的水平。 </li><li>使用弯钳皮肤和肌肉层轻轻分开。打开肌肉层，下面的皮肤切口。穿过肌肉层，一个在切口两侧插入两个5-0聚丙烯缝线，并用夹子固定保持肌肉层开放暂时固定缝合。 </li><li>识别，并在第三肋间切口，继胸腔的自然角。从鼠标的左侧末端取下胶带和左后脚固定到其右后脚用胶带。切割较长的胶带片和在稍微升高的位置其左侧前脚固定到操作表面。清洁用70％乙醇的手套。 </li><li>使用牵开器轻轻拨开3 和 第 4肋骨。切无菌纱布的一小部分，以x½在大约1，并在无菌0.9％盐水蘸。挤出多余的盐水并使用镊子轻轻插入纱布针对左肺，以防止在手术过程中偶然的肺损伤。 </li><li>轻轻取出薄心包用钳子。 </li><li>撕裂棉少量断无菌棉签把它卷成小球。这浸的棉花球到0.9％的无菌生理盐水轻轻擦拭心脏的表面上欣赏到动脉。轻轻向上推起左耳廓和定位下的冠状动脉尼思。 </li><li>识别LAD并通过LAD下8-0尼龙缝线;完成两个单结，确保结扎。如果连接成功，从结扎意志灼左心室远端。 </li><li>使用镊子，删除之前插入的纱布，然后轻轻取出牵引器。插入连接于25号针插入通过开胸开口胸腔6中，25号柔性管。推进约1 - 2在管道的进左肺上方的空间。返回鼠标仰卧位置和清洁用70％乙醇的手套。 </li><li>使用一个简单的中断模式5-0聚丙烯缝合关闭胸腔，保持胸腔引流管到位。取下两条缝合线保持肌肉层开放。使用在一个简单的连续图案5-0聚丙烯缝合关闭肌肉层，再次保持胸管就位。 </li><li>附加的1毫升注射器，以在胸部管25号针头。轻轻向上拉急跌ř同时逐渐提取胸腔用镊子胸管。提取管缓慢，因为这步骤除去过量的空气和血液，否则将被捕获到胸腔和结果在气胸。 </li><li>一旦注射器是满的，从针取下注射器，并在浪费烧杯或水槽的垃圾处置。继续这个过程，直到胸管被完全萃取。确保胸部紧密地密封。 </li><li>降低异氟醚1.5％。关闭与4-0聚丙烯缝合皮肤在简单的中断模式。关闭异氟醚蒸发掉。 </li><li>通过腹膜内（IP）注射施用0.9％盐水中0.1毫克/毫升的丁丙诺啡。在0.9％的盐水局部施用2毫克/毫升利多卡因用2毫克/毫升布比卡因的切口。通过皮下注射μL0.9％盐水500，缩放盐水量到鼠标的重量 - 管理200之间。 </li><li>等待ADMINI后5分钟stering的止痛药，以除去从插管鼠标。这有助于关闭呼吸机的过渡。 <ol><li>如果鼠标没有一个双边挺胸抬头，一旦关闭呼吸机，进行穿刺减压。要做到这一点，引入一个25号无菌针和3 和 第 4肋骨间的1毫升注射器，直到它进入胸腔，通过在电阻突然降低表示。轻轻向上拉柱塞，以去除多余的空气。 </li></ol></li><li>当鼠标演示了足够的双边呼吸频率和深度，响应一个脚趾捏，放置在干净的恢复笼鼠标加热灯下。提供与潮湿的食物和水瓶鼠标，在层流通风橱监测15 - 20分钟。监测夸张的呼吸努力，出血过多，或其他可能危及生命的并发症。 </li><li>在接下来的三天，通过在IP管理为0.1mg / mL丁丙诺啡止痛药jection每日两次。日常监控鼠标。 </li></ol>

<ol>
	<li>Hausenloy, D. J., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+directions+for+protecting+the+heart+against+ischaemia-reperfusion+injury:+targeting+the+Reperfusion+Injury+Salvage+Kinase+(RISK)-pathway.">New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway.</a> Cardiovasc Res. 61 (3), 448-460 (2004).</li><li>Roffi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+ESC+Guidelines+for+the+management+of+acute+coronary+syndromes+in+patients+presenting+without+persistent+ST-segment+elevation.">2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation.</a> Eur Heart J. 37 (3), 267-315 (2015).</li><li>Kumar, A., Cannon, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Coronary+Syndromes:+Diagnosis+and+Management,+Part+I.">Acute Coronary Syndromes: Diagnosis and Management, Part I.</a> Mayo Clin Proc. 84 (10), 917-938 (2009).</li><li>Eitan, A., Nikolsky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+therapy+in+patients+with+acute+coronary+syndromes:+how+to+make+the+right+choice.">Antithrombotic therapy in patients with acute coronary syndromes: how to make the right choice.</a> Minerva Med. 104 (4), 357-381 (2013).</li><li>Abbate, A., Bussani, R., Amin, M. S., Vetrovec, G. W., Baldi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+and+heart+failure:+role+of+apoptosis.">Acute myocardial infarction and heart failure: role of apoptosis.</a> Int J Biochem Cell Biol. 38 (11), 1834-1840 (2006).</li><li>Zheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nebivolol+protects+against+myocardial+infarction+injury+via+stimulation+of+beta+3-adrenergic+receptors+and+nitric+oxide+signaling.">Nebivolol protects against myocardial infarction injury via stimulation of beta 3-adrenergic receptors and nitric oxide signaling.</a> PLOS ONE. 9 (5), 98179 (2014).</li><li>Tarnavski, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16 (3), 349-360 (2004).</li><li>Buitrago, S., Martin, T. E., Tetens-Woodring, J., Belicha-Villanueva, A., Wilding, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Efficacy+of+Various+Combinations+of+Injectable+Anesthetics+in+BALB/c+Mice.">Safety and Efficacy of Various Combinations of Injectable Anesthetics in BALB/c Mice.</a> J Am Assoc Lab Anim Sci. 47 (1), 11-17 (2008).</li><li>Kolk, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAD-Ligation:+A+Murine+Model+of+Myocardial+Infarction.">LAD-Ligation: A Murine Model of Myocardial Infarction.</a> J Vis Exp. (32), e1438 (2009).</li><li>Wu, Y., Yin, X., Wijaya, C., Huang, M. H., McConnell, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+in+rats.">Acute myocardial infarction in rats.</a> Journal of visualized experiments : J Vis Exp. (48), (2011).</li><li>Ferrera, R., Benhabbouche, S., Bopassa, J. C., Li, B., Ovize, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hour+reperfusion+is+enough+to+assess+function+and+infarct+size+with+TTC+staining+in+Langendorff+rat+model.">One hour reperfusion is enough to assess function and infarct size with TTC staining in Langendorff rat model.</a> Cardiovasc Drugs Ther. 23 (4), 327-331 (2009).</li><li>Zeng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+5-ethynyl-2'-deoxyuridine+staining+as+a+sensitive+and+reliable+method+for+studying+cell+proliferation+in+the+adult+nervous+system.">Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.</a> Brain Res. 1319, 21-32 (2010).</li></ol>

The authors declare that they have no competing financial interests.

随着越来越多地使用在实验室MI模型，所描述的过程旨在提高小鼠的效率和生存率，同时尽量减少他们的手术后疼痛和不适。该协议努力通过使许多改进到LAD结扎过程的各个方面，以尽量减少死亡率。有一些区别。利用氯胺酮和赛拉嗪与异氟醚诱导以来，由于麻醉的持续时间较长的利益有些小鼠气管插管的研究，表明死亡率增加8。我们的方法只使用异氟烷诱导，大大减少了与毒品有关的并发症的可能性。另一个类似的LAD结扎协议包括气管切开，产生延长的恢复时间和用于精确训练9的需要增加。我们在这里描述的过程利用气管插管的非侵入性方法，产生LOWER死亡率和高重复性的结果。此外，而不是仅使用电动剃刀，我们的程序也使用脱毛膏毛皮移除，在不到1分钟，提供无菌区域的完全清晰的可视化。 另一个主要区别是鼠标，这之后发生的，而不是之前，初始切口的重新定位。使切口而鼠标是仰卧允许的地标更直接和准确的可视化，例如剑突软骨，从而导致更高的结果的可重复性。我们的方法也使用无菌棉签，而不是出血控制，减少医源性烧伤和感染的风险考特。除了这些差异之外，胸管插入了胸腔穿刺是特别要注意，因为我们的方法不涉及创建管新的切口。更确切地说，它涉及将所述管插入之前的切口，再次降低MORtality。我们也描述了该程序包括:（1）使用的牵开器，可实现冠状动脉的更精确和稳定的可视化; （2）无菌纱布插入到胸腔手术期间，从而减少医源性肺损伤的风险;和（3）以下的程序，其中已显示出两个盐水给药缩短恢复时间并防止体温过低。 虽然我们描述了一个永久的结扎后的模型，这个过程也可以被修改的急性心肌梗死模型。急性MI模型，也被描述为缺血和再灌注，是指30 -缺血60分钟，然后在心脏组织7再灌注。一种替代的方法来评估梗死大小或以危险区后缺血再灌注是2％三苯基氯化四唑（TTC）10的染色。 TTC染色是基于缺血损伤后染色的活组织的能力由于存在于心脏组织脱氢酶。这些酶转换可溶成分成不溶性红色成分，从而划定梗塞区域11。急性心肌梗死模型可以模拟发生在人类的心脏疾病，因此可能阐明心肌缺血10事件的有用工具的机制。法援署结扎可以通过观察组织颜色立即改变，诱发心肌梗死的这种方法提供了一个优势进行验证。检查结扎成功的另一种方法是使用心电图，虽然这涉及到使用昂贵的设备，并且可能不适合所有实验室是可行的。 如上所述，有几种简单的和负担得起的分子生物学技术，以确认收获后心脏结扎。上面显示的两种技术是伊文思蓝染色和三色染色。埃文的蓝色染料直接注入AO拱RTA，表明其中存在血流量不足。这是收获的心脏检验模型是否成功，并测量冠状动脉阻塞的程度后，立即采用一种快速而有效的方法。对于三色染色，心脏必须进行切片，然后进行免疫组化。三色染色可以表明缺血后纤维化领域或受其影响的慢性缺血心脏地区。的手术后的小鼠2所述的注射 - 之前24小时用5-乙炔基-2&#39;-脱氧尿苷（EDU），用于胸苷类似物，牺牲是用于表示以下缺血DNA复制和细胞增殖的区域的有效方法，特别是在研究涉及到血管再生12。 在一般情况下，LAD结扎研究的局限性包括手术后死亡的发生，造成主要由心律失常，出血，气胸的存在。一个有效的胸腔穿刺，机智豪特附加胸部切口（在当前的方法中所描述），和适当的术后护理是必要的，以避免在动物的发病率和死亡率。手术后低体温的非常仔细的监测也很关键。在颈部和胸部切口（气管切开胸腔穿刺术）的数量的减少在当前的方法描述有利于提高生存率。本文中所描述的可注射的术前麻醉剂的回避也将改善动物的术后恢复。 为了获得高重复性，法援署结扎模型需要严格的训练和经验。运营商需要进行手术的几个星期，以获得在对心脏所需部位可重复使梗死的能力。培训和经验是成功LAD结扎手术存活的两个关键因素。

冠状动脉疾病，或ACS，是最常见的心血管事件，将被认为是发病率和死亡率世界各地的主要原因在2020年1。 ACS的原因是心肌血栓的存在由于冠状动脉粥样硬化斑块，阻止或减少血液流向心脏组织2的破裂。因此，有临床症状的急性心肌缺血的存在一致，如心肌梗死（MI）3,4。 MI导致心肌细胞的质量损失和进展为病理性心室重构，从而导致心功能不全和心脏衰竭5,6。 其中一个最有效的方法来研究IHD一直模仿人类心肌梗死的动物模型。这是由闭塞的LAD实现老鼠。使用这个模型，我们研究如何心脏可以从IHD造成的损害进行保护。 在过去的十年中，研究人员使用较大的动物模型，以较小的动物，包括大鼠小鼠移位移动。较小的小鼠模型开始被优选的原因很多，其中包括它们的小尺寸，大产仔数，维护成本低，和短的怀孕期，以及用于转基因和基因敲除模型7的膨胀可用性。虽然小鼠体积小，专为他们设计的新的手术器械已经在这方面的发展援助。我们的方法利用这些新的手术器械。 虽然一些方法来实现的侵入气管切开术，我们使用气管插管的微创方法。使用口咽开销照明，我们插管不会造成任何切口，提供T A更安全，创伤小经验他的动物。然后将鼠标放置在呼吸机和在整个过程中保持在异氟醚。由于药物产生的麻醉时间短，只需要几分钟，让动物从麻醉一旦停产恢复。我们的手术模型还包括微创胸腔穿刺。从胸腔胸腔穿刺术使用仔细取出血液和过量空气通过原开胸切口已经解决了LAD结扎的共同术后并发症:张力气胸。该方法中，这消除了在其它方法中，一个用于气管切开术，另一个用于胸腔-已经产生了更少的外科手术后的并发症中使用的两个附加的切口的需要，并大幅度降低了死亡率。

<table><tbody><tr><td>High-Intensity Light Source</td><td>Harvard Apparatus</td><td>72-0215</td><td></td></tr><tr><td>SurgiSuite Operating Platform</td><td>Kent Scientific Corporation</td><td>SurgiSuite</td><td>Uses a rechargeable, battery-operated far infrared warming pad. Charge overnight before surgery.&amp;nbsp;</td></tr><tr><td>SurgiSuite LED Lighting Kit</td><td>Kent Scientific Corporation</td><td>SURGI-5003</td><td></td></tr><tr><td>Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Preheating takes 15 - 20 min. Instruments take 20 s to sterilize.</td></tr><tr><td>Small Rodent Anesthesia System</td><td>VetEquip Inc.</td><td>901810</td><td></td></tr><tr><td>Isofluorane</td><td>Piramal Enterprises</td><td>66794-017-10</td><td></td></tr><tr><td>Buprenorphine</td><td>Rhode Island Hospital Pharmacy</td><td>NDC 12496-0757-1, 12496-0757-5</td><td></td></tr><tr><td>Surgical Loupes</td><td>Roboz</td><td>RS-6687</td><td></td></tr><tr><td>Small Rodent Ventilator</td><td>Harvard Apparatus</td><td>73-0043</td><td></td></tr><tr><td>Lubricating Drops</td><td>Thermo Fisher Scientific</td><td>19-898-350</td><td></td></tr><tr><td>Electric Razor</td><td>Kent Scientific Corporation</td><td>CL 9990-1201</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Medical Tape</td><td>Thermo Fisher Scientific</td><td>18-999-380</td><td></td></tr><tr><td>Betadine</td><td>Thermo Fisher Scientific</td><td>19-027136</td><td></td></tr><tr><td>70% Isopropanol Wipes</td><td>Thermo Fisher Scientific</td><td>22-363-750</td><td></td></tr><tr><td>Surgical Drapes</td><td>Braintree</td><td>SP-TS</td><td></td></tr><tr><td>Surgical Gloves</td><td>Thermo Fisher Scientific</td><td>18999102D</td><td></td></tr><tr><td>5-0 Polypropylene Sutures&amp;nbsp;</td><td>Ethicon</td><td>8630G</td><td></td></tr><tr><td>8-0 Nylon Sutures</td><td>Fine Science Tools</td><td>12051-08</td><td></td></tr><tr><td>Platinum-Cured Tubing</td><td>Harvard Apparatus</td><td>72-1042&amp;nbsp;</td><td>0.3 mm inside diameter x 0.6 mm outside diameter</td></tr><tr><td>0.9% Saline</td><td>Thermo Fisher Scientific</td><td>19-310-207</td><td></td></tr><tr><td>4-0 Polypropylene Sutures</td><td>Ethicon</td><td>8631G</td><td></td></tr><tr><td>1 CC Syringe with 25-Gauge Needle&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>14-826-100</td><td></td></tr><tr><td>Scissors</td><td>Kent Scientific Corporation</td><td>INSS600225</td><td></td></tr><tr><td>Forceps</td><td>Kent Scientific Corporation</td><td>INS700100</td><td></td></tr><tr><td>Cotton Swabs</td><td>Thermo Fisher Scientific</td><td>23-400-118</td><td></td></tr><tr><td>IV Catheter, 20-Gauge</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;NC9892181</td><td></td></tr><tr><td>Retractor</td><td>Kent Scientific Corporation</td><td>INS 750369</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11003-12</td><td></td></tr><tr><td>Dissecting Forceps, Straight</td><td>Kent Scientific Corporation</td><td>INS 700101</td><td></td></tr><tr><td>Dissecting Forceps, Curved</td><td>Kent Scientific Corporation</td><td>INS 700103</td><td></td></tr><tr><td>Hemostatic Forceps, Straight</td><td>Kent Scientific Corporation</td><td>INS 750451</td><td></td></tr><tr><td>Hemostatic Forceps, Curved</td><td>Kent Scientific Corporation</td><td>INS 750452</td><td></td></tr><tr><td>Tissue Forceps</td><td>Kent Scientific Corporation</td><td>INS 700131</td><td></td></tr><tr><td>Needle Holder</td><td>Kent Scientific Corporation</td><td>INS 600109</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Kent Scientific Corporation</td><td>INS 600225</td><td></td></tr></tbody></table>

murine left anterior descending (lad) coronary artery ligation: an improved and simplified model for myocardial infarction

缺血性心脏疾病（IHD），或急性冠脉综合征（ACS），是死亡的在美国的主要原因之一。 IHD的特征在于血液供应减少到心脏，导致氧气的损失和心脏肌肉的随后的坏死。该MI模型已经得到普及其作为短期缺血再灌注模型和一个长期的永久的结扎模型使用。下面，我们描述了LAD的结扎的可靠方法。随着鼠标基因工程技术变得越来越先进，并与质量鼠手术器械的日益普及，鼠标已经成为MI手术的热门机型。我们的手术模型包括使用对鼠标的快速恢复的易可逆麻醉剂;微创气管内插管，而不涉及气管切开术;并通过原开胸部位的胸腔而不在胸部额外的切口，如在一些其他方法来完成，从胸腔至有效地去除过量的血液和空气。此方法相对比其它方法，这极大地减少了外科手术和外科手术后的并发症和死亡率及改善再现性侵入性更小。

小鼠在手术后28天安乐死，心中的收获和检查。将小鼠麻醉，用50 - 75毫克/千克氯胺酮和5 - 10mg / kg的甲苯噻嗪。当动物是足够的麻醉下，胸腔打开，并使用23号针头，冷氯化钾（KCL，30mM的）被注入到心脏的后底下区域。在心脏处于舒张期被捕。对于结扎的进一步验证，心脏从动物取出，被注射4％多聚甲醛，然后1％伊文思蓝染料。 图1显示了在缺血性左心室缺乏Evan的蓝。 图2展示了一个适当的气管内插管设置。 图3显示了胸管在初始切口部位的胸腔穿刺的位置，与缝合肌肉层封闭管周围从c空气提取前HEST腔。三色染色显示了在梗塞区域的增加胶原（ 图4）。 <img alt="图1" src="/files/ftp_upload/55353/55353fig1.jpg" /> 图1:文思蓝注射。埃文的蓝色注射揭示了缺血组织缺乏染料，定位于左心室梗死区。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55353/55353fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55353/55353fig2.jpg" /> 图2:气管插管。正确的气管插管设置的演示。操作员，穿着手术放大镜，在眼平用鼠标就位。一种高强度照明器被向下聚焦到气管区域，透照邻ropharynx。弹性带被钩上门牙的后面，从而允许操作员与弯钳打开嘴巴。所述弯钳用于保持舌头一侧为清晰的可视化。用钝尖端针导引器的静脉导管插管套管在一个稍微向上的角度，而声带的打开和关闭被可视化推进。声带开，气管插管收盘前的可视化是一个成功的插管的关键点之一。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55353/55353fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55353/55353fig3.jpg" /> 图3:胸腔。用于胸腔穿刺胸管，插入到原切口部位的放置。肌肉层被缝合关闭围绕胸腔内的空气之前，管与注射器萃取，然后胸管被去除。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55353/55353fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55353/55353fig4.jpg" /> 图4:三色染色。左图:控制，非LAD结扎心脏。右图:LAD结扎，梗塞心脏。采用布里希猩红酸性品红溶液，磷钨/磷钼酸溶液，苯胺蓝三色染色（马森）揭示了增加的胶原（蓝色）作为在横截面的左心室梗塞区域纤维化的标志物。条=500μm左右。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55353/55353fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction at JoVE.com

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

鼠左前降支（LAD）冠状动脉结扎:改进和简化模型的心肌梗死

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

murine-left-anterior-descending-lad-coronary-artery-ligation-an

Universidad San Sebastian

Research

JoVE Journal

Medicine

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

24.4K 次观看.  Rhode Island Hospital, Brown University Warren Alpert Medical School. We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

视频: Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

科研

医学

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

改性技术在小鼠冠状动脉结扎

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

生物学

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

左冠状动脉前降支的小鼠结扎:对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

左冠状动脉结扎术:心肌梗死的外科小鼠模型

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation myocardial infarction (STEMI), resulting in cardiogenic shock and fatal arrhythmia, which are associated with high mortality. Primary coronary intervention (PCI) for recanalizing the coronary artery significantly improves the outcomes of STEMI, but advancements made in shortening the door-to-balloon time have failed to reduce in-hospital mortality, suggesting that additional therapeutic strategies are required. Left anterior descending coronary artery (LAD) ligation in rats is an animal model for acute myocardial IR research that is comparable to the clinical scenario in which rapid coronary recanalization through PCI is used for STEMI; however, PCI-induced STEMI is a technically challenging and complicated operation associated with high mortality and great variation in infarction size. We identified the ideal position for LAD ligation, created a gadget to control a snare loop, and supported a modified surgical maneuver, thereby reducing tissue damage, to establish a reliable and reproducible acute myocardial ischemia-reperfusion (IR) research protocol for rats. This is a non-survival surgery. We also propose a method for validating the quality of study results, which is a critical step for determining the accuracy of subsequent biochemical analyses.

Herein, we present a protocol focusing on the quality control of the left anterior descending coronary artery ligation by technically modifying the traditional procedure in rats for acute myocardial ischemia-reperfusion research.

用于缺血再灌注研究的左前降冠状动脉结扎术:通过技术修改和质量控制改进模型

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...