我们感谢五齐默尔曼优秀的技术援助和鱼照顾。这项工作是由瑞士国家科学基金会，授予数量:310000_120611支持。

1。设备设置<ol><li>主要工具用于执行cryoinjury的是冷刀，笔状的仪器，由不锈钢制成（ 图1A）。圆柱柄长40毫米，有一个直径为8毫米。涂药的一角是6毫米长，直径0.8毫米。尖和手柄之间的交界处，是4毫米长的锥形。此外，冷刀的手柄应隔离用塑料管和磁带，以避免冻伤的手指，同时在手术过程中的工具。 </li><li>为冷冻所需的其他材料，立体显微镜，锋利的镊子，解剖微弹簧剪刀和一个塑料移液管。此外，准备麻醉转让的鱼的鱼和一个塑料勺子烧杯。 </li><li>在手术过程中，鱼放在潮湿的海绵上与它腹朝上（ 图1B）。要保持一个稳定的过程中的动物，适当的凹槽，应手动切断海绵。 </li><li>准备坦克与系统水转移，手术后的鱼。 </li><li>成立了第一双定时器10秒倒计时，然后自动24秒倒计时。 </li></ol> 2。 Cryoinjury程序<ol><li>降温超低温液态氮在3-5厘米的一角，至少有3分钟的浸泡。 </li><li>麻醉成年斑马鱼浸入0.02％tricaine直到鱼变成在它的后面，其鳃几乎停止不动（约90秒）。 </li><li>转让持有鱼倒挂（ 图1B），已被切割到一个潮湿的海绵用塑料勺子鱼。保持稳定使用非惯用手钳鱼。视觉定位后内侧缘的心，并直接使用虹膜切除术剪刀穿破皮肤。 （ 图1C）。 </li><li>上述由铜的心脏，使一个小切口（约2毫米）tting直前方通过皮肤，肌肉（ 图1D）。切不要通过骨鳃仪器，因为这会杀死动物。 </li><li>用剪刀尖轻轻撕的皮下组织银上皮细胞层（ 图1E）有直接访问跳动的心室。不要插入剪刀更深的体腔，这一意志刺穿心脏。跳动的心脏应该是可见的，并没有广泛出血发生在开胸手术（ 图1F）。 </li><li>开始编程定时器已被设定为24秒10秒。在10秒内从液氮中取出冷冻探针。确保有没有更多的超低温的氮轻轻晃动。传播的切口，横向用钳子打开胸部（ 图1F）。 </li><li>一旦定时器环10秒，但轻轻触摸立即心室尖的PR电子冷却的超低温（ 图1G）。 </li><li>一旦定时器铃响24秒，系统的水倒入2-3毫升的胸口上用塑料吸管从组织释放的超低温，并转移到系统水箱的鱼。 </li><li>鱼应该重新启动后几秒钟的呼吸，那么它应该继续游泳。如果没有呼吸约45秒后，用塑料吸管喷出水，直到它开始进入鳃呼吸本身刺激动物。在我们的经验，95％的鱼类生存的手术，手术当天发生的所有死亡。这不会是必要的缝合切口。 </li></ol> 3。心脏的收集和固定<ol><li>准备在离心管1毫升2％的福尔马林固定的心脏，两钳，微解剖剪刀和开槽的潮湿海绵。 </li><li>在选择cryoinjury后的一天，安乐死鱼在0.1％tricaine 5分钟。 </li><li>放置鱼腹部朝上成潮湿的海绵，开槽。做一个心通过与微观解剖剪刀鳃软骨以上大（约4毫米）切口。打开广泛的切口，用血管钳（ 图1H）。 </li><li>掐了bulbus动脉，心室（ 图1，HI）前一个白色结构，并从拉心腔。一整个心脏解剖图1J和图2A所示。 </li><li>到离心管中最多放置3心1毫升2％福尔马林。轻轻转动管数次，并保持在一夜之间在4°C。 </li></ol> 4。心脏安装<ol><li>在PBS冲洗5分钟的心中。转入10毫升30％的蔗糖，应在4°C预冷的心，轻轻混匀。标本应首先浮在表面的蔗糖溶液。继续在4°C孵育1小时20分钟后这时候，心中会下沉到试管底部。 </li><li>准备一个盒子，用干冰。采取嵌入模具，倒在模具的底部1华侨城安装介质5毫米的层。 </li><li>用钳子把安装在模具的中型到OC牛逼的心。体视显微镜下，调整心室心尖部，模具底部和bulbus动脉标本，以实现对顶部的方向。 </li><li>干冰放置试样模具。当安装介质开始冻结，填补其余的模具与华侨城中等，让它完全冻结。切片前至少1小时保持在-80℃的模具。冰冻标本可存储多月，在-80°C。 </li></ol> 5。心切片<ol><li>成立有恒冷切片机用切割16微米大小，-24℃的室内温度和试样温度在-22°C。 </li><li>将冻块样品在低温恒温器和解决的方向，开始切割块底部平行。 </li><li>准备每一个块六个Superfrost加幻灯片，，且具他们从1到6。开始切割，直到达到组织，修剪周围用剃刀刃的标本块。 </li><li>一心要获得6重复，首节幻灯片1，幻灯片2的第二部分，第三张幻灯片3，一旦你放在第六部分幻灯片6，重新启动幻灯片1并继续下去，直到整个器官被切断。收集两部分幻灯片上8点左右行（ 图3）。 </li><li>让幻灯片干燥，在室温下1小时。他们在密闭的箱子存储多达1年内，在-20°C </li></ol> 6。代表结果 DPCI（4几天后cryoinjury的代表心肌损伤后，该协议是在整个心脏解剖图2D - F）。到V在体内 isualize心肌组织中，我们使用转基因鱼心脏特定cmlc-2启动子11,12的控制下表达绿色荧光蛋白和核DsRed2。 EGFP和DsRed2荧光信号的情况下，划定一个圆盘形的梗塞区，沿心尖侧脑室壁。 执行组织的细胞和分子生物学分析，心中是固定的切片，并与恒温器。与横向的心脏部分的连续系列的一个代表性的幻灯片如图3所示。 的部分，可以分析各种方法（ 图4）。心脏再生与疤痕的程度来确定的，我们进行病理分析，使用酸性品红橙- G（AFOG）染色，心脏和肝纤维化组织差异标签9。根据原位杂交过程中取得了基因表达分析<suP&gt; 13。要检测标志蛋白的分布，我们采用9特异性抗体免疫荧光法。结合不同的染色程序导致的细胞和分子机制参与心肌再生以下cryoinjury鉴定。 <img alt="图1" src="/files/ftp_upload/3666/3666fig1.jpg" /> 图1。诱导的成年斑马鱼心室cryoinjury。 （A）的超低温照片。 （二）成年斑马鱼被放置在海绵的缝隙，其腹侧的一面朝上。 （CF）胸部切口访问的心。 （C）通过皮肤和肌肉的小切口，两者之间的胸鳍（红线）。 （四）剪刀插入切口，切开皮肤后的红色箭头。 （五）下的皮肤，精致的银色皮下组织层（蓝色虚线包围）轻轻打开访问的心。 （六）切口与传播镊子和跳动的心室（绿色箭头）是为cryoinjury访问。 （七）冷超低温轻轻插入胸部触及心脏。 "（HJ）心的集合。 （H）深又长的切口，通过胸部的鳃弓，进入心包腔。可见两个心脏的结构是:bulbus动脉（白色箭头）和心室（绿色箭头）。 （一）bulbus动脉钳和拉从体腔。 （十）:整个的心离身体。 <img alt="图2" src="/files/ftp_upload/3666/3666fig2.jpg" /> 图2。代表控制和表达EGFP和心肌细胞中的核DsRed2成年转基因斑马鱼解剖cryoinjured心中。 （交流）没有受伤的心。 （一）暗场照明显示心室（V）bulbus动脉（BA，白）和阀门（VA），心房与心室。 T（B和C）的荧光图像<html>他完整的心室显示EGFP和DsRed2表达在心肌细胞。 （DF）的心在后4天cryoinjury（4 DPCI）（四）暗场照明，揭示了一个圆盘形的心肌梗死（黄色虚线）。 （英法），心肌细胞标记EGFP和DsRed2被检测不到的梗死面积，表明受损心肌（虚线包围）。 <img alt="图3" src="/files/ftp_upload/3666/3666fig3.jpg" /> 图3。心脏的切片。 （一）所有的心从心对心脏的顶部（1和2，在红）（绿3和4）的底部开始的横向部分的图纸。 （二）与一系列与AFOG（酸性品红橙 - G）染色的连续横向部分的幻灯片库。第一部分（1和2，红色正方形）包含心室心尖部，心（3和4，绿色方块）的最后部分，包括bulbus动脉。 iles/ftp_upload/3666/3666fig4.jpg"/&gt; 分析图4。进行心脏部分7天cryoinjury。 （一）AFOG（酸性品红橙 - G）染色标签橙色健康的肌肉细胞，蓝色和红色纤维中含有胶原蛋白的疤痕组织。 （B） 在心室肌球蛋白重链（vmhc）原位杂交mRNA的可视化的完整的心肌（蓝染色）。受伤的组织是没有心脏基因转录（红色虚线）。 （三）原肌球蛋白Immunoassaying（绿色）标签完整的心肌细胞和未受损的组织（虚线包围）。的DAPI（蓝色）的污渍所有细胞的细胞核。 （四）基因标记的心肌表达DsRed2（红色）在其细胞核。的DAPI（蓝色）标签所有的核。梗死区不包含的DsRed2阳性细胞（白色虚线）。

<ol>
	<li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration.">Heart regeneration.</a> Nature. 473, 326-335 (2011).</li><li>Singh, B. N., Koyano-Nakagawa, N., Garry, J. P., Weaver, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+of+newt:+a+recipe+for+regeneration.">Heart of newt: a recipe for regeneration.</a> J. Cardiovasc. Transl. Res. 3, 397-409 (2010).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Semin. Cell Dev. Biol. 18, 36-45 (2007).</li><li>Ausoni, S., Sartore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fish+to+amphibians+to+mammals:+in+search+of+novel+strategies+to+optimize+cardiac+regeneration.">From fish to amphibians to mammals: in search of novel strategies to optimize cardiac regeneration.</a> J. Cell Biol. 184, 357-364 (2009).</li><li>Borchardt, T., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+regeneration+in+non-mammalian+model+systems:+what+are+the+differences+between+newts+and+man.">Cardiovascular regeneration in non-mammalian model systems: what are the differences between newts and man.</a> Thrombosis and haemostasis. 98, 311-318 (2007).</li><li>Bos, E. J. v. a. n. d. e. n., Mees, B. M., de Waard, M. C., de Crom, R., Duncker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+cryoinjury-induced+myocardial+infarction+in+the+mouse:+a+comparison+with+coronary+artery+ligation.">A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation.</a> Am. J. Physiol. Heart. Circ. Physiol. 289, H1291-H1300 (2005).</li><li>van Amerongen, M. J., Harmsen, M. C., Petersen, A. H., Popa, E. R., van Luyn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoinjury:+a+model+of+myocardial+regeneration.">Cryoinjury: a model of myocardial regeneration.</a> Cardiovasc. Pathol. 17, 23-31 (2008).</li><li>Schnabel, K., Wu, C. C., Kurth, T., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+cryoinjury+induced+necrotic+heart+lesions+in+zebrafish+is+associated+with+epicardial+activation+and+cardiomyocyte+proliferation.">Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation.</a> PLoS ONE. 6, (2011).</li><li>Chablais, F., Veit, J., Rainer, G., Jazwinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+heart+regenerates+after+cryoinjury-induced+myocardial+infarction.">The zebrafish heart regenerates after cryoinjury-induced myocardial infarction.</a> BMC Dev. Biol. 11, 21 (2011).</li><li>Gonz&#225;lez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li><li>Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reptin+and+pontin+antagonistically+regulate+heart+growth+in+zebrafish+embryos.">Reptin and pontin antagonistically regulate heart growth in zebrafish embryos.</a> Cell. 111, 661-672 (2002).</li><li>Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Chablais, F., Jazwinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF+signaling+between+blastema+and+wound+epidermis+is+required+for+fin+regeneration.">IGF signaling between blastema and wound epidermis is required for fin regeneration.</a> Development. 137, 871-879 (2010).</li><li>Baust, J. G., Gage, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+cryosurgery.">The molecular basis of cryosurgery.</a> BJU Int. 95, 1187-1191 (2005).</li><li>Whelan, R. S., Kaplinskiy, V., Kitsis, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+the+pathogenesis+of+heart+disease:+mechanisms+and+significance.">Cell death in the pathogenesis of heart disease: mechanisms and significance.</a> Annual review of physiology. 72, 19-44 (2010).</li></ol>

已通过弗里堡州兽医办公室在动物实验研究。

精确应用的极端寒冷的14 cryoinjury被定义为控制组织损伤。直接热冲击破坏细胞蛋白质的破坏和细胞内液冰晶体的形成，破裂的细胞膜。因此，受伤的细胞死亡过程细胞凋亡和坏死14。这些细胞死亡的机制都已经被证明有助于心肌梗死后15到组织的损失。因此，cryoinjury诱发心脏病心肌梗死的一个合适的模型。一些研究报告很好地证明了这在各种哺乳类动物，包括小鼠，大鼠，兔和猪6,7方法。适应的在斑马cryoinjury协议使梗死响应在不同物种之间的比较讨论。 其他两个小组独立开发的成年斑马鱼心脏cryoinjury程序。主要区别是在河畔的盖瑞方法，工具的类型和应用冻结时间。在由冈萨雷斯罗莎等。的研究，心包被打开了被撕裂的组织，而不是削减。他们用0.3毫米的铜灯丝连接到聚酰胺管在液氮冻结，直到几秒钟的心融化，可观察到10预冷。在研究施纳贝尔等，其直径为2毫米的尖头锥形件的长度为20毫米的干冰用于触及10秒8的心。这两个群体获得通过心室损伤的心肌死亡。我们的方法的优点是富含胶原蛋白的疤痕，从而更好地模仿梗死早期愈合在哺乳动物系统中观察到的反应更重现的沉积。 期间斑马鱼cryoinjury的过程中关键的一步是通过皮肤和心包无切口，刺穿心脏底层。正确performeð手术导致出血少。大出血表​​明无意用剪刀脑室穿刺。在这种情况下，应收回动物实验。为了避免刺穿心脏，剪点应着眼于皮肤浅角度。 创建重复性损伤的另一个重要方面是最佳的精确时间脑室冷超低温的精确定位。冻结时间过短会导致炎症与小细胞死亡。长期冻结，另一方面，可能会造成广泛的损害心脏，这可能会导致以增强动物的死亡率。我们确定的最佳时间保持在23-26秒之间的冻结状态范围为2厘米长的成年斑马鱼心室，它可能会有所不同动物的大小依赖性。 因为我们的协议是非常简单和快速的，有没有实验限制ations在受到足够数量的动物，以获得足够的生物复制。氩氦刀治疗是很好的耐受性动物。在不同的时间点后cryoinjury心中的集合允许在再生过程中的后续阶段的详细描述。实验分析的cryoinjured的心灵可能包括:1）可视化原位杂交基因转录，2）检测免疫荧光染色，3）的蛋白质分布不同结构的组织学成像。了解在斑马鱼心肌梗死后的愈合过程会影响再生生物学领域。此外，它可能是有益的设计在再生医学的新的治疗方法。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>微解剖弹簧剪刀</td><td> roboz手术仪器有限责任公司</td><td> RS-5602 </td></tr><tr><td> tricaine </td><td> Sigma-Aldrich公司</td><td> E10521 </td></tr><tr><td>甲醛〜36％ </td><td> Sigma-Aldrich公司</td><td> 47630 </td></tr><tr><td>蔗糖</td><td> Sigma-Aldrich公司</td><td> 84100 </td></tr><tr><td>组织TEK华侨城复合</td><td>樱花Finetek </td><td> 4583 </td></tr><tr><td>幻灯片Superfrost加</td><td> Fisher Scientific则</td><td> 12-550-15 </td></tr><tr><td>剥离一个单向一次性嵌入模具 - 截断模具T8 </td><td> polyscience，公司</td><td> 18985 </td></tr></tbody></table>

induction of myocardial infarction in adult zebrafish using cryoinjury

哺乳动物心脏不能再生的重大急性损伤，如心肌梗死1。相比之下，urodele两栖类和硬骨鱼保留了很少或没有疤痕整个生命2,3心肌再生的非凡能力。它不知道为什么只有一些非哺乳类脊椎动物可以重新创建一个完整的器官，从4,5残余组织。为了了解在不同物种之间的再生反应的细胞和分子的差异，我们需要使用类似的方法诱导急性损伤。 在哺乳动物中，最常用的模型来研究心脏修复一直是急性心肌缺血后结扎冠状动脉组织的破坏或后cryoinjury 6,7。已在蝾螈和斑马鱼的心脏再生主要心尖2,3部分切除后的研究。近日，几组有establ在成年斑马鱼8-10 ished的cryoinjury技术。这种方法具有很大的潜力，因为它允许从哺乳动物和非哺乳动物物种得到的结果进行比较讨论。 在这里，我们提出了一个方法来诱导重现圆盘形的斑马鱼cryoinjury的心室梗死。此损伤模型的基础上迅速组织冻融，导致大规模的约20％的室壁心​​肌细胞死亡。首先，一个小切口，通过胸部与虹膜切除术剪刀访问的心。室壁直接冻结申请不锈钢超低温液态氮预冷23-25​​秒。停止冻结的心，鱼水在室温下的冷冻探针的尖端下降。程序是很好的耐受动物，成活率为95％。 表征的再生过程，心中收集并固定在不同天cryoinjury后。随后，标本嵌入的cryosectioning。节的幻灯片在原位杂交和免疫组织学分析，处理。这项承诺，增强了我们理解的因素，都需要在斑马鱼再生的可塑性，与心肌再生的机制提供了新的见解。在斑马鱼心脏再生的概念和分子生物学的理解会影响发育生物学和再生医学。

Watch this Scientific Journal Video about Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury at JoVE.com

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

斑马鱼代表一个有价值的模型来研究脊椎动物的心脏再生的机制。在这里，我们提出了一个在成人使用cryoinjury斑马鱼的心脏梗死诱导的协议。这种方法在大量的细胞死亡的结果，20％的室壁内，类似的观察哺乳动物梗死。

在成年斑马鱼心肌梗死的感应使用Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

induction-of-myocardial-infarction-in-adult-zebrafish-using-cryoinjury

Research

JoVE Journal

Medicine

22.1K 次观看.  University of Fribourg, Fribourg, Switzerland. 斑马鱼代表一个有价值的模型来研究脊椎动物的心脏再生的机制。在这里，我们提出了一个在成人使用cryoinjury斑马鱼的心脏梗死诱导的协议。这种方法在大量的细胞死亡的结果，20％的室壁内，类似的观察哺乳动物梗死。

视频: Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

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 ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

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 regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

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 ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

小鼠急性心肌梗死的实时压力容积分析

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

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 ...