Name: myocardial infarction in neonatal mice, a model of cardiac regeneration
Uploaded: 2016-05-24T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration at JoVE.com

This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to Q.F. (grant #MOP-119600).

的C57BL / 6和CD-1 IG-S小鼠繁殖对从Charles River购得。在这项研究中使用的动物，根据加拿大议会关于动物保护的准则，处理，研究方案被动物利用小组委员会在西部大学，伦敦，加拿大的批准。 1.动物护理<ol><li>分娩是完整的，幼仔已初步母乳喂养由他们的母亲了几个小时后，将它们放置在不同的笼子里带有CD-1养母。 CD-1母亲显示具有较强的本能培养一个平静的表型，并有较低的倾向蚕食受伤的幼崽15。 </li></ol> 2.手术<ol><li>通过将小狗在一个密封的腔异氟醚麻醉诱导（约500微升100％V / V异氟烷允许耗散超过1300 立方厘米室）。保持在腔室中的小鼠直到运动（约30秒）停止。 </li><li>莫删除从异氟醚室使用，并通过将鼠标放在湿冰上诱发低温麻醉。为了避免冻伤，把冰无菌手术手套里面，手套包裹内的鼠标和覆盖冰手套或包裹鼠标在无菌湿纱布，将它放在冰上。 注:小鼠更快地冷却时用冰冷的接触是在一个较大的表面面积，并且因此熔融的冰可以缩短低温诱导时间。 </li><li>缺乏运动反应到脚趾和尾巴捏麻醉确认。 注:挑起动作的停止发生在15℃和8℃之间的体温。 （冷却时间到该温度约1分钟）。完整的呼吸暂停和心脏停搏不需要 LAD结扎。 </li><li>移动至冰床装有手术显微镜手术区。确保鼠标小狗在整个手术过程中保持在冰上。 注:手术区，纱布和手术器械应灭菌。 Mainta在整个手术过程中无菌区，和穿一次性使用无菌手术手套。小鼠出生与他们的眼睛闭17不需要眼膏。 </li><li>放置在右侧卧位鼠标小狗。用聚维酮碘溶液，然后用乙醇棉签轻轻擦拭消毒的胸部。 </li><li>通过切割皮肤xyphoid和左腋下之间的左前足下面几毫米的左胸部进行皮肤切口沿腋中线。还用剪刀通过底层胸肌层进行切割。 </li><li>用钳分离肋骨，肋间肌请在第 4肋间左侧开胸。 注:新生儿肋是很脆弱的，并且可以容易破裂。要沿肋间肌开钳（而不是抓筋）避免这种情况，轻轻分开肋骨。 </li><li>可视化的左冠状动脉前降支（LAD）从左边出现AURICLE肺静脉后面和降超出心大静脉。 注意:在这个早期新生儿窗口，胸腺常常可视化并可以覆盖心脏基部的一部分。在LAD可以看出出现的胸腺的位置的旁边，这取决于胸椎切口的角度。 </li><li>通过使动脉下方11-0尼龙缝线（0.007毫米直径针）穿过中间心室左外耳（ 图1C）下面结扎LAD。缺血被确认与缝合部位（ 图1G）下方的心肌的热烫。 </li><li>关闭使用8-0尼龙缝合线（0.15毫米直径的针）胸切口​​。用两个缝合关闭肋骨。结扎，以确保针头穿过体腔时，不会刺破肺之前放置两个肋缝线。 </li><li>关闭肋骨后，从冰床鼠标。保持手术区域内的鼠标和直接将其放置在无菌手术以逢过一个温暖的加热垫在37℃开始升温。 </li><li>一旦在无菌受热面积，关闭使用8-0尼龙缝合线（0.15毫米直径的针），肌肉和皮肤层。使用一个缝合肌肉层和使用两个缝合的皮肤切口。 注:肌肉和皮肤层可以在加热垫以减少低温曝光时间关闭。鼠标温度开始上升，一旦放置在加热垫，但肌肉和皮肤缝合过程中保持足够维持麻醉足够低。 </li></ol> 3.手术恢复<ol><li>继续在一个温暖的加热垫迅速升温，直到自发运动的回报。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。用乙醇棉签轻轻擦拭损伤部位取下聚维酮碘和血。 </li><li>从麻醉中足够的恢复后，从无菌手术区域中删除，从培育被褥把它涂抹妈妈的笼子里，和返回的小狗养母。这有助于防止产妇拒绝或拆。 注意:不要幼仔返回与其他动物的笼子，直到完全康复。如果整个垃圾没有被使用，并同窝留在养母，将恢复幼仔垃圾的中间。如果执行一个以上的手术，完整的老鼠返回自己的养母之前单个垃圾所需的所有手术。 </li><li>观察养母的行为向小狗每10 - 15分钟2 - 3小时，以确保接受小狗。如果母亲显示侵略朝受伤小狗，取出小狗和异氟烷过量安乐死（&gt; 5％;直到呼吸停止），随后断头。不需要围手术期镇痛药物的集中疼痛反射没有完全在这个15岁早期开发:注意。 </li></ol> 4.心肌的测量梗死面积4 -6小时后心肌梗死<ol><li>让幼崽在护理CD-1养母的恢复期限4 - 6小时。从笼中取出母亲的小狗和异氟醚过量，然后用剪子斩首安乐死吧。 </li><li>切除心脏解剖显微镜下，注意不要撕心肌。 <ol><li>切断从xyphoid过程皮肤对胸部的顶部。打开腹壁胸腔下方。抓住较低的肋骨，并通过肋骨和肌肉切断沿纵向从隔膜到腋下左侧中线，腋。 </li><li>拿在横向平面剪刀，并通过隔膜精心切割从左右侧。请务必将低于心脏水平剪刀，以免到顶点的任何损害。 </li><li>把握胸廓和切割肋右侧和肌肉沿右腋中线。用剪刀删除对心脏血管的所有连接。抓住卸下胸腔的心脏的基础。 </li></ol></li><li>部分使用手术​​碳钢刀片的心脏成三块。使沿心脏的短轴首次下调的缝合和心尖之间的中点。使在缝合（ 图3A）的电平的第二切口。 注:此留下一个顶点部分约为0.75毫米厚的重0.0018克;中间基座部分约为1毫米厚，重0.0065克;和心脏的基地。 </li><li>放置在1％2,3,5-氯化三苯基四唑（TTC）的心脏切片在室温下进行10 - 15分钟。仔细观察标本，以避免过度染色。 </li><li>为了增加对比度，用4％多聚甲醛的染色心脏切片在4℃过夜。要计算％梗死面积，心脏照片节和测量成像软件梗死面积。而梗死区域划定为白色的18存活心肌污渍红色。 </li></ol> 5.测量心功能的超声心动图后MI <ol><li>让幼崽在护理CD-1养母的恢复，为期24 - 48小时。通过将小狗在密封腔异氟醚（5％异氟醚）诱导麻醉。保持在腔室中的小鼠直到运动（约30秒）停止。 </li><li>固定小狗中在加热的基座（温度37℃），其在一锥形鼻部仰卧位以提供0.5 - 1％异氟烷（麻醉维护）。放置预温热回波凝胶左侧胸部区域。 </li><li>获取左心室（LV）的胸骨旁长轴观。确保图像被获得下面在LV缝合线的水平。获取位置后，转动超声探头（40 MHz）的90°，以获得胸骨旁短轴观，并记录M型超声心动图图像。 </li><li>测量端舒张压和结束从短轴线M模式图像收缩期左室内部直径。计算射血分数和fractio最终缩短。 </li></ol> 6.心肌的测量梗死面积24小时后MI <ol><li>让幼崽在护理CD-1养母的恢复，为期24小时。从笼中取出母亲的小狗和异氟醚过量，然后用剪子斩首安乐死吧。 </li><li>打开胸腔，如步骤4.2。切除心脏前，请认真把握只是用细镊子膜片上方的胸主动脉。持在冠状面细剪刀，平贴胸壁，并切断胸主动脉关闭后胸壁在颅方向移动剪刀。 </li><li>切断从胸壁和任何其他连接游离的主动脉，直到它到达心脏。通过抓住上腔静脉和切割到身体的所有其它血管连接切除心脏。 </li><li>冲洗心脏（附主动脉）在盐水中。仔细导管插入用30号针头胸主动脉和扎奥尔塔用8-0尼龙缝合线套管。 </li><li>以1毫升/分钟的速率灌注通过经由30号针头的主动脉内皮素-1 5ml盐水的心脏。以1毫升/分钟的速率灌注用150微升的2％伊文思蓝溶液的心脏。从套管中取出心脏，部分为三个部分和第4节与TTC留下污点。 </li><li>要计算梗死面积，心脏照片节和测量成像软件梗死面积。存活心肌渍蓝，在危险中的污渍面积红色，梗死面积划定为白色。 </li></ol>

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The authors have nothing to disclose.

这里展示了外科LAD结扎是产生的MI在新生小鼠的可靠方法。这个模型提供了一个可重复的模型来研究哺乳动物心脏再生的研究人员。冠状血管的可视化是此方法的一个关键组成部分，以确保正确的缝线放置，从而保证重复性。而成年小鼠不具有变温功能，体温和新生小鼠的代谢率是密切随环境温度相关联。此外，新生小鼠的小尺寸使其非常适用于低温诱导表面冷却。手术和鼠标体温的定时是在复?...

心肌梗死（MI）是死亡的主要原因全世界，并且仍然负责大约三分之一的心脏衰竭病例1。而经皮介入和使用血栓溶解的不断优化的出现增加了再灌注MI后，心肌细胞死亡和收缩心肌的损失仍然发生。此外，还留了大量的"无选项"病人谁不是候选人或没有看到这些措施的好处。这些患者继续经历禁用缺血导致瘢痕形成和有害的心室重构的脑梗死愈合的机制。该方法最终导致心脏衰竭，为此，预后仍然尽管与血管紧张素转换酶（ACE）抑制剂和β-阻断剂的最佳药物管理不善。遗憾的是，一年期死亡率为患者严重受损左心室功能仍然为高达26％2。心脏移植是治疗心脏衰竭的最终治疗方案。然而，对于心脏移植的有限施主池不使这对大多数患者一个可行的选择。因此，新的治疗剂的发现，以恢复受损心肌仍然是最重要解决心脏疾病的问题。因此，心脏损伤可靠的动物模型需要作为这一进程的一个重要组成部分。 传统的教条已经决定了成人的心肌细胞的有丝分裂后，终末分化细胞，不能分割或去区分，以取代受损心肌3。因此，成年哺乳动物心脏永远无法完全从伤病中恢复，失去了心肌细胞将与纤维组织所代替。因此，研究主要集中在治疗剂，以减少梗塞扩展和减少疤痕形成。然而最近，一个范式转变发生在心脏周围的愈合和许多研究工作思路已被重定向把重点放在心脏再生4的潜力。 直到最近， 在体内心脏再生的研究仅限于非脊椎动物模型，如那些在urodele两栖动物和硬骨鱼5-7。然而，在新生小鼠的能力用于心脏再生的发现已经导致的哺乳动物心脏再生的两种手术模型的开发:心尖和冠状动脉闭塞的切除以诱导心肌梗塞8,9。在2011年，一个鼠标顶点切除模型来证明完整心脏再生有可能在1日龄（P1）。然而，这种力量的初步新生儿期后迅速下降。哺乳动物的心脏在P7出生祖细胞数量下降后不久就失去了它的再生潜能，并成为心肌双核，输其增殖能力，并永久退出细胞周期10,11。了解新生和成年哺乳动物心脏之间的根本分歧可能会导致新的见解心脏再生。 而先端切除确实提供洞察收缩组织的再生长，该模型不模拟典型的人心脏损伤，因此不借给本身以及对治疗剂的开发。冠状动脉闭塞模型，然而，更直接地模拟MI病状的病理生理方面，并且因此可以提供更有用的见解可能适用于供人类使用的治疗进步的机制。 外科冠状动脉结扎已被用作在许多动物模型12-14的有用实验技术。在成人冠状动脉结扎模型中，将动物麻醉并插管以允许胸腔的开口，同时维持respirati上。该心脏继续定期击败，允许冠状血管的可视化，并允许精确缝合位置。此外，心脏仍然是粉红色的灌注继续，结扎后缺血心肌出现面色苍白，表示成功的冠状动脉结扎术。对新生小鼠中描述的协议，但是，是较不可靠的冠状动脉不可视和外科医生必须估计何处放置缝合线15。虽然冠状动脉脉管系统的一般解剖的方向是相同的，动物个体变异和在LAD的分支中存在16。因此，当"盲目去"的动脉很容易错过。然后如超声心动图的其它技术是必需的，以确认心肌梗塞的成功诱导，并确保所有的手术导致类似梗塞大小。这里描述的改善在最近发表的方法15，其中，在LAD的位置可以ESTABlished，因此LAD可连接可重复诱发心肌梗死。 这种技术不需要在新生小鼠在低温状态气管插管或机械通气，如开胸不会导致肺萎陷。然而，在前面描述的方法，严重低温必须被诱导到两个完整呼吸暂停和心律15停止的点。这种方法的主要限制是冠状动脉不再灌注和心脏甚至LAD结扎前出现面色苍白。在这里所描述的方法中，冠状动脉可视化是可能在深低温和心律停止，与手术后的新生小鼠的完全恢复之前的一个点麻木的。此方法提供100％的再现性的一大优势。

<table><tbody><tr><td>8-0 Nylon Suture</td><td>Microsurgery Instruments</td><td>8-0 Nylon</td><td></td></tr><tr><td>11-0 Nylon Suture</td><td>Shanghai Pudong Medical Products Co Ltd</td><td>H1101</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Small forceps</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>Micro Needle Holder</td><td>Fine Science Tools</td><td>12060-02</td><td></td></tr><tr><td>Zeiss Opmi 6s/S3 Microscope</td><td>Zeiss</td><td>300002</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>CA2L9100</td><td></td></tr><tr><td>Isoflurane Chamber</td><td>Made in Feng laboratory</td><td></td><td></td></tr><tr><td>Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>2,3,5-Triphenyltetraolium chloride (TTC)</td><td>Sigma</td><td>T8877</td><td></td></tr><tr><td>Stereomicroscope SteREO Discovery. V8</td><td>Zeiss</td><td>435400</td><td></td></tr><tr><td>AxioVision 8.0</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Axiocam Icc5</td><td>Zeiss</td><td>426554</td><td></td></tr><tr><td>Heat pad</td><td>Sunbeam&amp;nbsp;</td><td>731A0-CN</td><td></td></tr><tr><td>Sterile Gloves</td><td>VWR</td><td>414004-430</td><td></td></tr><tr><td>Gauze Sponges</td><td>Ducare</td><td>90212</td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Ultrasound imaging system, Vevo2100</td><td>Visual Sonics</td><td>VEVO2100</td><td></td></tr><tr><td>Ultrasound transducer, 40 MHz</td><td>Visual Sonics</td><td>MS400</td><td></td></tr></tbody></table>

myocardial infarction in neonatal mice, a model of cardiac regeneration

冠状动脉结扎诱发心肌梗塞已在许多动物模型被用来作为一种工具来研究心脏修复和再生机制，并定义治疗的新目标。几十年来，完整的心脏再生的模型，两栖类和鱼类存在，但哺乳动物对口不可用。近期A产后窗口发现其间的小鼠具有再生能力，导致建立心肌再生的哺乳动物模型。哺乳动物心脏再生的新生小鼠的手术模型在本文中描述。简言之，将日龄1（P1）的小鼠通过异氟烷麻醉，并放置在冰垫以诱导体温过低。胸部被打开后，与左冠状动脉前降支（LAD）被可视化，缝线周围放置在LAD以造成心肌缺血左心室。手术过程需要10-15分钟。可视化的冠状动脉被准确缝合位置和重现性是至关重要的。心肌梗死和心功能不全是由三苯基氯化四氮唑（TTC）染色和超声心动图检查确诊分别。完全再生后21天心肌梗死是由组织学验证。该协议可以用于心肌梗塞后阐明哺乳动物心脏再生的机制的工具。

在P1的心肌梗死程序可以在10内完成 - 15分钟并拥有死亡率为7.8％（5出64个幼崽）。手术后，老鼠从低温麻醉未来5内恢复 - 20分钟（恢复时间取决于体温外科医生的麻醉和速度期间达成）。当使用P7幼仔（用于与非再生心肌比较），需要冷却的一个较长时期达到麻木。 P7幼仔大得多，并有更多的困难来自心脏损伤和低温回收，导致死亡率的26.9％（14选自52幼仔）的高得多。 ...

Watch this Scientific Journal Video about Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration at JoVE.com

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

myocardial-infarction-in-neonatal-mice-a-model-of-cardiac-regeneration

Research

JoVE Journal

Medicine

18.3K 次观看.  Western University. This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

视频: Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

将新生儿小鼠心脏巨噬细胞移植到成年小鼠体内

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

科研

生物化学

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

发育生物学

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

根尖切除小鼠模型来研究早期哺乳动物心脏再生

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

医学

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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 ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

新生儿鼠心在朗根多夫制剂中的应用改良技术

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

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. ...