Name: minimal invasive surgical procedure of inducing myocardial infarction in mice
Uploaded: 2015-05-04T11:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice at JoVE.com

This work was supported by Interdisciplinary Centre for Clinical Research IZKF Aachen (junior research group to E.A.L.) within the faculty of Medicine at RWTH Aachen University. We are grateful Dr. Rusu and Ashley Christina Vourakis for critical review of the manuscript and Mrs. Roya Soltan for the professional help with immunohistochemistry staining.

注:本文所提出的实验也相应进行德国低，欧洲动物护理准则。动物繁殖研究所在实验动物科学，Universiy医院亚琛，德国的动物设施的监督下教授R.·托尔巴博士和A.托伊布纳（动物福利主任）的。 1.动物护理<ol><li>保持老鼠在一个专门的监护病房，确保正确的获得食物和专业兽医的控制和治疗。如果动物被移动或从外部购买的，请接受手术前保证一周的住宿。 </li></ol> 2.插管<ol><li>麻醉8-10周龄雄性C57BL / 6野生型小鼠，25-27克用腹膜内注射100mg / kg的氯胺酮和10mg / kg的甲苯噻嗪。监测麻醉通过脚趾捏反射的水平。放置在眼睛兽医软膏，以防止在操作过程中干。 </li><l我&gt;开始手术，按照你的机构的动物护理准则之前管理的疼痛治疗丁丙诺啡与0.1毫克/千克体重皮下注射。 </li><li>保证无菌条件的维护，以避免在手术期间的感染通过使用无菌的材料和工具。 </li><li>将麻醉小鼠中在加热的手术台仰卧位置。使用小的剃刀除去从两个腹的颈部区域和胸部的左半头发和消毒之前用切口70％的酒精。 <ol><li>执行使用手术剪刀在颈部的中心0.5cm的小正中切口。在皮肤下，经过2脂肪体用无菌弯钳，并通过覆盖肌肉的透明可视化立体显微镜下的气管。 </li></ol></li><li>利用体视显微镜（图1A）口头介绍插管插管插入视图下气管。区分金属套管救援人员到场唉透明的组织。并检查，位置和定位的操作在任何时刻（ 图1B）中。 </li><li>套管连接到小动物呼吸机和通风根据设置来调整生产准则（100-150微升和100-150之间的呼吸速率每分钟之间的潮气量）。 </li></ol> 3.心肌梗死感应<ol><li>在xyphoid和左axila之间的线的中间执行一个皮肤切口小于0.5cm。使用镊子肌肉层从底层的肋骨分开。 </li><li>通过使用小剪刀，直到胸腔打开17执行的肋之间有一个小切口。从下面的2 个和第一个更简单的方法编号:慢性梗死，执行切口的第 5肋间空间（ 图1C）和/或用于局部缺血/再灌注模型中，在第 4 次肋间隙（ 图1D） 3 日 </suP&gt;肋间，分别地）。 </li><li>将拉钩插入切口打开胸腔，并以可视化的心脏。 </li><li>小心取出心包，以防止过度纤维化的进程。 </li><li>可视化的左冠状动脉前降支（LAD）作为深定位淡红色容器。如果法援署不能进行可视化，考虑一些参考点，以增加可重复性。 <ol><li>对于慢性梗死模型中，放置结扎在心脏（耳廓和顶点之间）的腹侧的中间，具有作为参考的静脉， 如图1C所示 。绑定使用0/7丝线获得透前部和后部梗死动脉的两个分支。灰度颜色指示结扎的位置，如果需要（ 图1C）可以重复。 </li><li>对缺血/再灌注模型中，放置结扎耳廓下，经LAD的主体（ 图1D）。该LIGATURE位于在硅管，以保护容器的完整性。灰色的颜色表明梗死面积，应该出现在整个心脏（ 图1D）。广场上的排骨时间缝线在缺血期和滋润使用压缩，以避免组织干燥。缺血后，除去硅管，并切断与小剪刀缝线以可视化的再灌注。 </li></ol></li><li>旁在程序开始时所使用的麻醉剂和止痛剂（步骤2.1和2.2），用外科手术过程中的0.5％的异氟烷​​，以确保动物的适当的舒适度，或者跟随的机构的动物护理准则。 </li></ol> 4.缝合和恢复<ol><li>通过填充用温等渗盐水消除胸部残留空气。 </li><li>关闭胸部用3缝线0/6（ 见图2A和2B）。定位内侧缝线以90°的角度，以确保如ealed肋的闭合，如图2（ 图2A，B）。 </li><li>关闭肌肉层用2缝线（ 图2C）中，用3-4缝合线0/6（ 图2D）的皮肤。分别执行这些缝合线以得到合适的窗口进行进一步的超声心动图测量。 </li><li>断开呼吸机插管插管，并允许自主呼吸。对于以后的识别，标记使用本地系统（请动物福利工作人员从您所在的机构）的鼠标。 </li><li>在红灯放下鼠标左侧，直到它唤醒。不要让动物无人值守，直到它已经恢复了神志充足。不允许已经历手术，可以在其他动物的公司，直到完全恢复的动物。 </li><li>管理疼痛治疗丁丙诺啡与0.1毫克/千克体重，皮下注射，未来3天，按照你的机构的动物护理准则。 </li&gt; </ol>心肌梗死5.分析<ol><li>定期监测通过的超声心动图（ 图3A）装置中的心脏功能:射血分数，缩短分数，心输出量和心脏的尺寸。 </li><li>麻醉用腹膜内注射100mg / kg的氯胺酮和10mg / kg的甲苯噻嗪的动物。由于缺乏反射的确认正确麻醉手术前。 </li><li>打开胸腔，并切除心脏，将其放置在无菌PBS液洗涤广泛的剩余的血液。 </li><li>如果需要，直接从心脏通过避免梗死区域的损伤采集血液，或摘取心脏后，从胸腔。 </li><li>清洗后，停在diastola饱和氯化钾溶液中的心脏（与不孕过滤3M氯化钾PBS）。组织学分析固定在心脏10％福尔马林并继续步骤5.7。 </li><li>如果必要的话，测量汽车的可行性DIAC细胞埃文斯蓝/三苯基氯化四氮唑（TTC）染色。重建结扎在初始位置后，用灌注主动脉插管用200微升1％埃文斯蓝溶液的心脏，并在-20℃冷冻的心脏在一个小塑料袋，无需清洗。 <ol><li> 2小时后，用锋利的解剖刀执行5横向滑动，并培育它们在TTC溶液10-15分钟，在37℃下，所描述的制造。固定的幻灯片在10％福尔马林10分钟，并把它们的显微镜载玻片进行进一步的分析之间。 </li><li>嵌入心脏组织中的石蜡，通过尖端定位的心脏，进行横向切片。执行的5微米连续切片。收集前20段和丢弃在下一个300微米。继续部分协议直到二尖瓣水平已经达到（ 图3A，B）。连续切片，400微米间隔在整个心脏被收集并可以被染色˚F或定性和定量分析。 </li></ol></li><li>使用莫里的一步法染色6-8测量梗死面积。 </li><li>使用通常的免疫组织染色中连续切片分析血管形成，胶原蛋白含量或炎症细胞募集。 </li></ol>

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The authors have nothing to disclose.

在实验过程中，也有要注意一些关键点:插管，所述打开胸腔，并在LAD结扎。第一个关键步骤是experiements前动物的插管。许多团体正在使用用于固定鼠标和光源直接插入套管插入气管竖直支撑。该方法具有涉及插管进入气管插入正确的不确定性，是最容易出现故障的新手。制造一个小切口，套管的位置可在整个动作过程中被控制，从而降低了默认速率。此外，气管切开被超过，从而减少并发症，减少操作的时间。 下一关键步骤是胸腔的开口。胸骨正中代表了高风险的机动延缓动物的恢复。横向切开左暗示的2-3肋15的切割</SUP&gt;，导致缺陷的恢复和死亡率上升。我们使用的肋骨提供了最小的负担之间的小模型，离散切口。这些动物在手术后很快恢复，不存在缺陷或干扰愈合。下部间肋软骨空间被取为参考点。考虑到这一点，适当的和差异化的进入慢性缺血/再灌注模型结扎的地方，不会引起严重的问题。 结扎本身代表了最关键的一步。左降支冠状动脉很难被可视化，并且常常需要不以约束。因此，一些解剖参考点指出，以帮助外科医生进行正确的连接。对于慢性梗死模型中，结扎被放置在心脏的腹侧的中间，耳廓和顶点之间，主要的前静脉（ 图2B）的结尾的上方。效率得以CONTRO通过可视化的灰色的在灾区的外观LLED。如果梗塞区域出现前，不包括后壁，一个新的缝合线可以放置到第一缝合的左侧。 LAD的主要根源总是可见耳廓18岁以下，因此不会检测这部分出现严重的问题。然而，耳廓呈现出血的主要危险，需要小心处理。 该程序是由存在适当的设备的限制。的通风机和适当的麻醉系统的小动物是昂贵的并且需要连接到该室的气体和通风系统。此外，动物的密切监督是必要的，在第一周后过程，以检测可能的临床。为了检验在试验过程中的心脏功能，高分辨率超声，复杂朗根多夫灌注的系统，或小室内导管测量是必需的，INVolving成本高，更多的专业知识。 考虑到心肌梗死，有可用来重现体外的事件的复杂性，没有可供选择的方法。根据感兴趣的点，分离的心脏中的Langendorff系统的离体灌注提供有关收缩，心脏功能和存活心肌响应于不同的刺激或药物的资料。然而，它不包括血液成分和免疫系统的所有的干扰，并且它不表示心肌梗死后长期研究重塑和愈合。 在进行心肌梗死过程之后，所有其它功能性分析，可以进行，例如脑室压力测量，超声（小动物超声系统）或分离的心脏的Langendorff灌注。此外，所有的生物和分子分析可以被执行，以确定细胞，蛋白质，的mRNA，微小RNA，锗未列名或其他生物标志物，其可以作为治疗目标开发用于心肌梗死新的治疗策略。

心肌梗死是死亡的主要原因工业化国家之一。尽管诊断和治疗方法的不可否认的进展，心血管疾病仍然是死亡的主要原因。鉴于预期提高生活和生命有关的风险，不断增加心血管疾病的发病率，预计在未来。因此，有强烈的需要建立和验证新的方法用于心血管疾病的治疗。人类研究的信息从它的局限性患，这些研究通常是不足以解释和理解的机制在分子水平上，无法提供解决这些严重的健康问题。 此外，基础研究已因限于复杂性和难度重现心血管疾病的机制，在实验室。因此，为了增加约心肺的病理生理我们的知识血管疾病，它是必不可少的，以验证动物模型1,2。然而，为了鉴定参与愈合心肌梗塞，分析在不同时间点后是必要的分子事件的所有级联，导致了大量的动物实验。 心肌梗塞实验通常通过使用动物模型进行的。诱发心肌梗死的小动物是3-11来探讨比大动物模型细胞和分子事件的最合适和最有效的模式。而且，没有其他的物种呈现的转基因或基因敲除品系小鼠12的可用性。这些小鼠模型在其它疾病，包括心血管病症非常有用的（如动脉粥样硬化，在支架内再狭窄）13,14。此外，低怀孕期间和高数后代资格小鼠模型作为最有吸引力的系统来研究心肌INFA的分子机制rction 12。 尽管如此，心脏小鼠的规模预计操纵显微过程中精度高。这样的教学资质和熟练的手术人员是一个耗时和工作的过程。因此，我们在此提出一个详细的显微程序，包括技巧和窍门来引导甚至平均资历的合作者，如学生或技术人员进行小鼠复杂的心肌梗死模型。 最初，插管是由短插管的装置，而无需使用气管切开进行。胸切口位于肋间区域，避免肋或/和周围组织的损伤。该子步骤是高度相关的，以确保快速恢复和愈合15。结扎由差动对慢性缺血和缺血/再灌注模型中，对于高存活率，同时仍保持一个显著梗塞大小。我们的经验表明，塔用丝线缝合吨相比，低温伤害16保证了较高的可重复性。 总之，这里所描述的方法适用于既慢性缺血和缺血/再灌注模型中的小动物。在此过程中提出的技巧和窍门，旨在实现与即使是低或平均合格人员将其应用在小动物模型。

<table><tbody><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZ/X9</td><td></td></tr><tr><td>Mouse ventilator</td><td>Harvard Apparatus</td><td>730043</td><td>Model Minient 845</td></tr><tr><td>Dual Anesthesia System (Tabletop Version)</td><td>Harvard Apparatus</td><td></td><td>Selfcontained isofluranebased anesthesia unit for use on lab tables, with a compact 8&quot; x 11&quot; footprint.&amp;nbsp;</td></tr><tr><td>Intubation cannula</td><td>Harvard Apparatus</td><td>732737</td><td></td></tr><tr><td>Forceps</td><td>FST, Germany</td><td>9119700</td><td>standard tip curved 0.17 mm x 0.1 mm</td></tr><tr><td>Scissors</td><td>FST, Germany</td><td>9146011</td><td>straight</td></tr><tr><td>Vannas scissor</td><td>Aesculap, Germany</td><td>OC 498 R</td><td></td></tr><tr><td>Retractors</td><td>FST, Germany</td><td>1820010</td><td>2.5mm wide</td></tr><tr><td>Retractors</td><td>FST, Germany</td><td>1820011</td><td>5 mm wide</td></tr><tr><td>Wire handles</td><td>FST, Germany</td><td>1820005</td><td>&amp;nbsp;10 cm</td></tr><tr><td>Wire handles</td><td>FST, Germany</td><td>1820006</td><td>&amp;nbsp;14 cm</td></tr><tr><td>Ketamine 10%</td><td>CEVA, Germany</td><td></td><td></td></tr><tr><td>Xylazine 2%</td><td>Medistar, Germany</td><td></td><td></td></tr><tr><td>Bepanthene eye and nose cream</td><td>Bayer, Germany</td><td></td><td></td></tr><tr><td>Silicon tube</td><td>IFK Isofluor, Germany</td><td>custommade&amp;nbsp; product</td><td>diameter 500 &amp;micro;m</td></tr><tr><td>section thickness 100 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>polytetrafluorethylene catheter</td><td></td><td></td><td></td></tr><tr><td>PROLENE Suture 6/0&amp;nbsp;</td><td>ETHICON</td><td>8707H</td><td>polypropylene monofilament suture, unresorbable, needle CC1, 13 mm, 3/8 Circle</td></tr><tr><td>7/0 Silk</td><td>Seraflex</td><td>IC 1005171Z</td><td></td></tr><tr><td>Ultrasound&amp;nbsp;</td><td>Vevo, Canada</td><td>770 Vevo</td><td></td></tr></tbody></table>

minimal invasive surgical procedure of inducing myocardial infarction in mice

心肌梗死仍然死在西方国家的主要原因，尽管支架的发展区域，在过去的几十年长足的进步。对于澄清潜在机制和新的治疗策略的发展，有效的动物模型的有效性是强制性的。由于我们在体内条件需要新的见解心血管疾病的病理机制，以打击心肌梗死，动物模型的有效性是一个重要方面。然而，保护动物是在这样的背景下高度相关。因此，我们建立了心肌梗死的小鼠微创和简单的模型，它保证了高重复性和动物的存活率。因此，这种模式满足的3R原则动物实验的要求（更换，细化和还原），并保证所需的cardiov治疗策略进一步发展的科学信息血管性疾病。

心肌梗死过程发生在25-30分钟，并示出了死亡率为10％。手术后，小鼠从麻醉中恢复的下一个15分钟之内。任何物理损伤，观察到鼠标操作。然而，有心脏破裂一个星期后慢性心肌梗死后的风险较高，如果修复过程期间炎症阶段被扰乱。因为心脏是能够泵送期间显著改变其尺寸，重要的是对所有所收集的心要被停止在相同的位置，例如在diastola。这可以通过用饱和氯化钾溶液灌注心脏来实现。增加外K +浓度的块中的离子泵，降低心肌细胞的膜静息电位，造成心脏活动的舒张期停滞。 梗塞面积可以看出，在超声分析（ 图3A，下图）。在比较正常心肌，缺血区出现薄和运动减弱（ 图3A，上图）。根据所使用的模型，该梗塞大小会有所不同。慢性梗死模型诱导的顶点（ 图3B）的圆形，透梗死，而缺血/再灌注诱导的薄的中间壁和在所有心脏（ 图3C）。有许多方法来确定梗死面积。如果目的是要分析对心脏存活的直接作用，伊文思蓝/ TTC染色18被指示要进行再灌注至少2小时后，才能够看到在心肌的任何变化。部分可立即染色后进行分析（ 图3B，中图）或可以保持载玻片之间在福尔马林2-3天（ 图3C，中图）。蓝色区域代表健康心肌，不受缺血。红色区域代表第里面存活心肌ë缺血区（风险心肌），而白色区域表示的死组织。通常，梗塞大小被表示为百分比从风险区。 成熟瘢痕所得重构处理之后可以很容易地通过immunohistolgy使用Gomori法的一步法染色测定。被蓝染梗死和染红健康心室区（ 图3B和C，右图）从每个级别的第一部分，直到二尖瓣确定。为了避免由于LAD的不同层次的结合的变化，从所有部分中的梗塞被认为并表示为总左心室体积的百分比。可以达到15-20％的慢性梗塞模型和10-15％后缺血/再灌注模型中的梗死体积。此外，慢性梗死模型将诱发加重扩张，在缺血/再灌注模型没有观察到（ 图3B和C右击牛逼面板）。 常规染色程序都可以使用，例如:用于揭示血管生成CD31染色（红色， 图4A）或平滑肌肌动蛋白染色来确定肌纤维母细胞（绿色， 图4B）。双荧光染色也可应用，以确定不同的目标分子在梗死区域中，由于不存在心肌没有给出自动免疫荧光（ 图4C）。 <img alt="图1" src="/files/ftp_upload/52197/52197fig1.jpg" /> 图1: 内侧切口和插入气管插管的套管（一）。金属套管穿过组织（B）的透明的stereomicroscopic可视化。的气管环（蓝色箭头）和套管（黑色箭头）被指出。肋间切口慢性心肌梗塞莫德尔和LAD（三）结扎。结扎位于心脏（耳廓和先端，黑色在下部面板之间）的中间，以作为参考的静脉（示意蓝色，下图）的末端。动脉的两个分支应绑定（红色中下图）。灰色的颜色表明梗死面积，它出现在心脏（右下图）的下半部分。结扎的缺血/再灌注模型耳廓下进行，装订LAD的主体（红色中下图）在硅管（右侧）（D）中 。灰色的颜色表示梗死面积，这是目前在整个心脏（右下图）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52197/52197fig1large.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/52197/52197fig2.jpg" /> 图2: </str翁&gt;肋缝线密封件，如果内侧缝线定位在90°在两个慢性（A）一种角胸切口和缺血/再灌注模型（B，左图）。 在体内成像的肋缝合（C，左面板），肌肉缝合（C，中图）和皮肤缝合（C，右图）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52197/52197fig2large.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/52197/52197fig3.jpg" /> 图3:超声心动图图像 。普通（A，上图）和梗死区（A，下图）的照片，在长轴采集（纵向，左图）或短轴（横向，右图）..脑梗塞引起的慢性结扎（B）和由1小时缺血再灌注（℃）。伊文思蓝/ TTC染色可以识别灌注（蓝色）/非灌注区域以及可行的（红色）/死（白色）心肌（B，C，中图）。莫里的一步法染色允许梗死区（蓝色）的识别，并从正常区域（红色）（B，C，右图）区分它们。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52197/52197fig3large.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/52197/52197fig4.jpg" /> 图4:不同的染色可以在梗死区中进行，如CD31，以描述新血管生成（A，红色，简单箭头），或平滑肌肌动蛋白为肌成纤维细胞（B，克颖，简单的箭头），以及双重染色（C，CD31-红/平滑肌肌动蛋白绿色），counterstained用DAPI的细胞核（蓝色）。肌纤维母细胞可以很容易地从大或小动脉平滑肌细胞，它们总是伴随着内皮细胞层（C，箭头）进行区分。双箭头指向红细胞自体荧光。规模杆50微米。

Watch this Scientific Journal Video about Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice at JoVE.com

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

在诱导小鼠心肌梗死的微创手术过程

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

minimal-invasive-surgical-procedure-inducing-myocardial-infarction

Universidad San Sebastian

Research

JoVE Journal

Medicine

27.5K 次观看.  RWTH Aachen University, Germany. A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

视频: Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

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 ...

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 ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

生物工程

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

一种改良的小鼠心肌梗死诱导简易方法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

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 ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

超声引导诱导多能干细胞来源心肌细胞植入心肌梗死小鼠

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

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 ...