这个工作是由自然科学和加拿大金吉尔伯特工程研究理事会资助项目拥有全宗德追忆魁北克一个助学金-桑特。

动物被遵守处理与当地动物护理委员会规定，按照加拿大委员会关于动物保护的准则。大鼠收容单独在恒定条件下（21-22ºC温度和湿度的40-50％），其中包括一个12小时黑暗的光周期，开始于上午08:00。周小球和自来水在整个研究可随意。的输送由供后3天的环境适应期，实验前是允许的。雄性SD大鼠间300-800克体重已经经常使用该协议。 1. MI手术<ol><li>诱导麻醉的氯胺酮60 mg / kg和赛拉嗪10mg / kg的腹膜内注射。通过无反射确认适当麻醉时，爪子被挤压。 </li><li>剃手术部位。插管大鼠气管内管:导管（16G1.77英寸），然后将动物侧卧位。放置在加热垫上动物保持温度37℃左右。连接气管内管异氟醚2％。 </li><li>准备手术部位与葡萄糖酸洗必泰和异丙醇。在手术过程中，使用无菌手套和消毒器械。应用眼药膏双眼防止干燥，而在麻醉下。把无菌区对动物和旁边的动物另一个无菌区的仪器。 </li><li>切开皮肤，手术刀片＃10。剥离肌肉组织用止血钳夹。发生在腹卧位动物。打开胸壁用止血钳和定位胸部拉钩。打开心包用止血钳夹。 </li><li>以产生冠状动脉阻塞，循环中的下行冠状动脉周围360毫米长的4-0丝线缝合，通过连续的心肌组织。插入丝线的两端成14G，1.25厘米长的塑料管。 </li><li>拉动丝线缝合及脓液的两端小时管向下靠着动脉以阻塞它，进行40分钟。安全闭塞夹紧塑料管与止血钳夹。 </li><li> 40分钟后，通过释放止血钳，接着除去软管和丝线释放闭塞。 注意:深水对照大鼠经历相同的手术过程减去冠状动脉的闭塞。 </li><li>前闭合胸壁，放置一个柔性导管（18G，4.5厘米）至胸腔将空气抽出胸部用5-ml注射器，以防止气胸。 </li><li>关闭胸腔腔2-0缝合，缝合肌肉用4-0丝线缝合，用3-0丝线缝合皮肤。 </li><li>前关闭最后一个皮肤点，再通过导管在胸部空腔先前放置抽离空气，用5-ml注射器的胸部。 </li><li>最后缝合皮肤点。停止异氟醚通气。 </li><li>监测大鼠的觉醒。不要让动物无人看管，直到它有倍健ð足够的意识，以保持胸骨斜卧。 </li><li>治疗的大鼠，每8小时24小时用镇痛药（丁丙诺啡，0.05毫克/千克IP）中，用抗生素（duplocillin的单次剂量，0.2毫克/千克IP）。 </li><li>手术后，房子只单独直到时间的牺牲， 即后3天心肌梗死。 </li><li>梗塞大小，主要坏死，表示为梗塞组织的该区域在左心室的风险比例。这可以通过使用三苯基着色11测量。 </li></ol> 2.杏仁核隔离<ol><li>首先将动物头锥形袋，用它的鼻子伸出的窄端光圈。安全的大鼠，以避免任何运动。上断头台正确地定位它的头，剪刀的刀片之间。斩首的动物。 </li><li>在培养皿中的老鼠的贴装头保持在碎冰。上执行碎冰（4℃）中组织分离的所有过程。 </li><li>剖开SKULL用剪刀，小心地拉起，避免下毁损的组织分离骨鞘骨咬骨钳。 </li><li>放置头骨底部和脑的后腹侧表面之间的刮刀的平头刀刃，并仔细分离大脑从颅骨轻轻沿头骨底部向前推动刀片直到大脑可以慢慢举起出的头骨。丢弃的头骨。 </li><li>将大脑在它的背部表面。确定下丘脑（结构小脑前），减少大脑冠在其前端的和后端的前后;丢弃大脑的前部和后部部分，如果没有必要用于其他用途。 </li><li>可视化的杏仁核小球颞叶下方，两侧，毗邻下丘脑。 </li><li>翻转大脑平在其正面年底，背水面离实验者。 </li><li>开始与脑的左侧或右侧。分开半球，然后从相邻的杏仁核皮层。切方式约8mm这种松散皮质以允许扁桃体的隔离。切掉扁桃体与手术刀。 </li><li>通过沿暗缝线横跨它运行时，几乎各半切割隔离从其centromedial部扁桃体的基底部分。这种结构并不总是容易辨认。 </li><li>将杏仁核子部分单独确定的小瓶中，并保持在碎冰。 </li><li>重复解剖过程与其他半球。 </li><li>浸入液氮小瓶1分钟。保持小瓶在-80℃的冰箱中待用。 </li></ol> 3，caspase-3活性<ol><li>制备裂解缓冲液与蔗糖的0.32M，1％的Triton-X-100的Tris-HCl，pH值8，5mM的乙二胺四乙酸，二硫苏糖醇的2毫米，苯甲基磺酰氟1mM的，10微克/微升亮肽素的10毫和10微克/微升胃蛋白酶抑制素A的10微克/μL抑肽酶。 </li><li>添加150微升裂解缓冲液至每个样品（5-10毫克）。置于冰上。超声处理在冰上每个样品进行5秒，最大强度（40瓦）。孵育组织在裂解缓冲液在冰上30分钟。涡旋每个样品5分钟。 </li><li>通过将样本交替地在液氮中，并在恒温控制的加热板设定为37℃，进行3次冷冻/解冻循环。在13000克离心机组织（4 ℃）10分钟。小心取出上清液，保持管冰。 </li><li>制备在0和10微克/毫升牛血清白蛋白标准品的蛋白浓度。准备空白，只有缓冲区。 </li><li>加入200μlBradford试剂到每个标准。阅读在595nm处的标准。对于蛋白水平，加入5微升样品上清液至795微升蒸馏水和200μl的Bradford试剂的。读每个样品在595nm处并量化蛋白标准曲线。 </li><li>准备反应缓冲液，用50mM的Tris-HCl，pH值7，5mM的MgCl 2的的&lt;/次&gt;，乙二醇四乙酸为1mM，3-胆酰胺丙基0.1％）二甲基铵基] -1-丙磺酸盐和二硫苏糖醇1mM的。 </li><li>补充蛋白质的25微克至反应缓冲液和caspase-3底物，以获得正和负反应性样品。对于阴性样品中，添加反应缓冲液，25微克蛋白质，0.4微升AC-DEVD-CHO 800μM和0.8微升AC-DEVD-AMC的10mM的。对于阳性样品，加25微克蛋白质反应缓冲液和0​​.8微升AC-DEVD-AMC的10mM的。处理所有阴性和阳性样品一式三份。通过添加反应缓冲液完成每个样品的最终体积达到200微升。 </li><li>通过加入188.7微升反应缓冲液0.5微升AC-DEVD-CHO 800微米的，0.8微升AC-DEVD-AMC的10mM和10μl的裂解缓冲液产生阴性对照。对于阳性对照，加入189.2微升反应缓冲液，0.8微升AC-DEVD-AMC的10mM和10μl的裂解缓冲液。 </li><li>孵育样品和在37℃。在黑暗中3小时对照</li><li>停止与600微升甘氨酸0.4 M和氢氧化钠0.4 M（pH为10），每个样品和控制反应。 </li><li>荧光定量荧光分光光度法在365 nm，发射465纳米波长的激发波长。加入2毫升蒸馏水，以在玻璃试管反应。阅读对照和样品10秒，1分每一秒。 </li><li>量化具体活动每个样品中:（（阴性样品减去阳性样品的荧光）2.8毫升×容积荧光）由（的蛋白质25毫克孵化3小时×数量时）划分。 </li></ol>

<ol>
	<li>Denault, J. B., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases:+keys+in+the+ignition+of+cell+death.">Caspases: keys in the ignition of cell death.</a> Chemical Reviews. 102, 4489-4500 (2002).</li><li>Boucher, 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=Post-ischemic+cardioprotection+by+A2A+adenosine+receptors:+dependent+of+phosphatidylinositol+3-kinase+pathway.">Post-ischemic cardioprotection by A2A adenosine receptors: dependent of phosphatidylinositol 3-kinase pathway.</a> Journal of Cardiovascular Pharmacology. 43, 416-422 (2004).</li><li>Kaloustian, S., 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=Celecoxib+after+the+onset+of+reperfusion+reduces+apoptosis+in+the+amygdala.">Celecoxib after the onset of reperfusion reduces apoptosis in the amygdala.</a> Apoptosis. 12, 1945-1951 (2007).</li><li>Kaloustian, S., 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=Apoptosis+time+course+in+the+limbic+system+after+myocardial+infarction+in+the+rat.">Apoptosis time course in the limbic system after myocardial infarction in the rat.</a> Brain Research. 1216, 87-91 (2008).</li><li>Rousseau, G., Bah, T. M., Godbout, R., Lakshmanadoss, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19,+Post-Myocardial+Infarction+Depression.">Chapter 19, Post-Myocardial Infarction Depression.</a> Novel Strategies in Ischemic Heart Disease . , 333-362 (2012).</li><li>Wann, B. P., 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=Apoptosis+detected+in+the+amygdala+following+myocardial+infarction+in+the+rat.">Apoptosis detected in the amygdala following myocardial infarction in the rat.</a> Biological Psychiatry. 59, 430-433 (2006).</li><li>Arseneault-Breard, J., 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=Combination+of+Lactobacillus+helveticus+R0052+and+Bifidobacterium+longum+R0175+reduces+post-myocardial+infarction+depression+symptoms+and+restores+intestinal+permeability+in+a+rat+model.">Combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 reduces post-myocardial infarction depression symptoms and restores intestinal permeability in a rat model.</a> British Journal of Nutrition. 107, 1793-1799 (2012).</li><li>Bah, T. 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=Escitalopram+reduces+circulating+pro-inflammatory+cytokines+and+improves+depressive+behavior+without+affecting+sleep+in+a+rat+model+of+post-cardiac+infarct+depression.">Escitalopram reduces circulating pro-inflammatory cytokines and improves depressive behavior without affecting sleep in a rat model of post-cardiac infarct depression.</a> Behavioural Brain Research. 225, 243-251 (2011).</li><li>Bah, T. M., Kaloustian, S., Rousseau, G., Godbout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+with+pentoxifylline+has+antidepressant-like+effects+in+a+rat+model+of+acute+myocardial+infarction.">Pretreatment with pentoxifylline has antidepressant-like effects in a rat model of acute myocardial infarction.</a> Behavioural Pharmacology. 22, 779-784 (2011).</li><li>Gilbert, K., 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=Attenuation+of+post-myocardial+infarction+depression+in+rats+by+n-3+fatty+acids+or+probiotics+starting+after+the+onset+of+reperfusion.">Attenuation of post-myocardial infarction depression in rats by n-3 fatty acids or probiotics starting after the onset of reperfusion.</a> British Journal of Nutrition. 109, 50-56 (2013).</li><li>Gilbert, K., Bernier, J., Godbout, R., Rousseau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolvin+D1,+a+metabolite+of+omega-3+polyunsaturated+fatty+acid,+decreases+post-myocardial+infarct+depression.">Resolvin D1, a metabolite of omega-3 polyunsaturated fatty acid, decreases post-myocardial infarct depression.</a> Marine Drugs. 12, 5396-5407 (2014).</li><li>Wann, B. P., 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=Behavioural+signs+of+depression+and+apoptosis+in+the+limbic+system+following+myocardial+infarction:+effects+of+sertraline.">Behavioural signs of depression and apoptosis in the limbic system following myocardial infarction: effects of sertraline.</a> Journal of Psychopharmacology. 23, 451-459 (2009).</li><li>Gorr, T. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+re-visited:+critical+perusal+of+under-appreciated+technical+issues.">Western blotting re-visited: critical perusal of under-appreciated technical issues.</a> Proteomics: Clinical Applications. , (2015).</li><li>McKernan, D. P., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Killing+the+blues':+a+role+for+cellular+suicide+(apoptosis)+in+depression+and+the+antidepressant+response.">'Killing the blues': a role for cellular suicide (apoptosis) in depression and the antidepressant response.</a> Progress in Neurobiology. 88, 246-263 (2009).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways:+paper+wraps+stone+blunts+scissors.">Apoptotic pathways: paper wraps stone blunts scissors.</a> Cell. 102, 1-4 (2000).</li></ol>

作者什么都没有透露。

我们与此协议在过去10年的经验导致了关键的方法问题的鉴定。首先，在碎冰上培养皿快速大脑解剖重要的是要保持制剂的质量。第二，必须认识到，组织超声处理较短的脑杏仁核相对于其他类型的组织，如心脏（10秒）或肾脏（20秒）。第三，大应注意在添加到样品之前仔细混合基板。我们遇到变量信号当混合顺序是不充分的。第四，读取样品时无气泡应该存在于石英池中。否则，该信号可能被改变。 我们提出在过去的几年中的变化，以增加可重复性的结果:增加衬底体积，以避免体积小于1微升。仍然，小变化可以连续测定之间发生，和至少3个对照样品应当用于控制的变化。这些控件的荧光措施被平均，平均为100％。实验样品在荧光措施转化在对照样品的百分比。 该技术的另一个限制是，组织只有一部分使用，因此，caspase-3的活性可能被低估。事实上，细胞凋亡的时间窗口短于任何给定的细胞，即使它出现在相邻区域处的采样1,4,15的时间可能被错过。反过来也是可能的:细胞凋亡可能是在组织中的某些部分特别强烈，导致胱天蛋白酶-3活性的高估。我们建议使用剩余的组织（杏仁核）的互补性技术，如TUNEL法或Bax蛋白/ Bcl2的比例（参见导言）的。 荧光分析法具有至少两个优点Western印迹。首先，它直接生成的量化数据。第二，它可以测量酶促一个ctivity本身而不是蛋白质或RNA的表达，这可能是不相关的，并且已知的是蛋白表达可以在没有中酶活性的任何变化增加。此外，荧光分光光度法可以在其它组织或用不同的动物物种中进行，并且可以为其它蛋白酶亚型进行调节，以不同的底物，从而使安全可以用比较的。 总之，本文件说明了如何使用荧光分光光度法来测量胱天蛋白酶-3活性在杏仁核，提供凋亡发生在边缘系统的这个区域的MI后3天的证据。

胱天蛋白酶或半胱氨酸依赖性天冬氨酸定向蛋白酶是一个家族参与各种稳态processess 1中的酶。胱天蛋白酶可以根据其在细胞凋亡或炎症中的作用被广义地分类。胱天蛋白酶-1，-4，-5和-12参与炎症而那些从事凋亡可以分划为引发剂胱天蛋白酶（胱天蛋白酶-8，-9）和胱天蛋白酶刽子手（胱天蛋白酶-3，-6和-7 ）。 胱天蛋白酶在许多病理发挥显著的作用，主要表现在疾病，其中细胞凋亡能量过大，心肌梗死（MI）或脑缺血后观察到。 在我们的实验中使用的MI后综合征的大鼠模型中，经确定的caspase-3不仅在心肌2而且在其中牵涉情绪和情感3-6的控制边缘系统激活。它还可以看出，caspase-3的不同区域被激活边缘系统，例如杏仁核和海马，以及局部缺血损伤后4约3天内，活化的峰。有趣的是，caspase-3的活性与心肌梗死后的行为障碍，并通过药理和营养干预其衰减减少这些伤害，提示caspase-3和心梗后抑郁7-12之间可能存在的联系。 Caspase-3的活化可通过各种技术来测量。 Western印迹台确定胱天蛋白酶的酶活性，可检测活性酶以及它们的酶原形式。 Western印迹，但是，是半定量，和小的变化可以通过微弱的信号-噪声比13丢失。更好的方法是基于在基板（DEVD-AMC）的由胱天蛋白酶-3，释放一个荧光化合物，并且可以通过荧光分光光度法测量的裂解。 Caspase-3的活化是凋亡的可靠指标。细胞凋亡钙的测定n为不稳定的，因为发生的时间窗口短，和邻近细胞可以吞噬凋亡小体或细胞14。凋亡可以通过其它技术，如末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定法来进一步证实，其检测从凋亡信号传导级联6，或亲/抗凋亡蛋白的比率，如Bax的/得到的DNA片段BCL2 6。

<table><tbody><tr><td>Spectroflurometer</td><td>Photon technology Instrument (now Horiba)</td><td>Ratiomaster M-40</td><td>with DeltaRAM&amp;nbsp; Random Access Monochromator&amp;nbsp;</td></tr><tr><td>Respirator</td><td>Harvard apparatus</td><td>683</td><td>small animal ventilation</td></tr><tr><td>Glass cuvette</td><td>NSG precision cells</td><td>3G10</td><td>Optical Glass (340-2,500 nm) 10 mm path</td></tr><tr><td>Sonicator</td><td>Cole Parmer</td><td>4710&amp;nbsp; series</td><td></td></tr><tr><td>Spectrometer</td><td>Varian</td><td>Cary 50 Bio</td><td></td></tr></tbody></table>

caspase-3 activity in the rat amygdala measured by spectrofluorometry after myocardial infarction

心肌梗死（MI）具有在生理和行为水平显着中期和长期的后果，但所涉及的机制仍不清楚。我们的实验室已开发出心肌梗死后综合征，显示受损心脏的功能，在边缘系统的神经元丧失，认知障碍和抑郁的行为迹象的大鼠模型。在神经元水平，胱天蛋白酶-3的活化介导的MI后凋亡不同边缘区域，如杏仁核 - 3天达到峰值后心肌梗塞。认知和行为障碍出现2-3周后MI和这些与caspase-3的活性的措施相关统计学。这里描述的协议被用来诱发心肌梗死，收集杏仁核组织和使用荧光分光光度法测定caspase-3活性。为了诱导心肌梗死，所述下行冠状动脉被遮挡40分钟。该协议为caspase-3的活性评价心梗开始后第3天:被处死老鼠和杏仁核中分离RAPI从大脑DLY。样品迅速在液氮中冷冻并保持在-80℃直至实际分析。以评估的caspase-3的激活的技术是基于一个衬底（DEVD-AMC）由胱天蛋白酶-3，释放可通过荧光分光光度法来测量一个荧光化合物的裂解。该方法是定量的和可再现的，但所需要的设备是昂贵的，用于量化样品的过程是耗时的。这一技术可以应用到其他组织中，如心脏和肾脏。 DEVD-AMC可以通过其它基片代替，以测量其他胱天蛋白酶的活性。

扁桃体分离出8假（对照）和8 MI大鼠缺血/再灌注协议诱导后三天。使用可以由活性的caspase-3被裂解并通过荧光分光光度法检测出的荧光caspase-3的活性测定在这个组织上。正和负的测量（见步3.11，3.12和3.16），一式三份在10秒进行测量和，用1点在每个秒。阳性和阴性对照之间的差异，计算和的差异为这个实验期间进行的所有假组织的平均设定在100％。结果从MI大鼠根据本参考文献被缩放的， 图1示出了最后的结果:它表明相比假（P &lt;0.05）中的MI大鼠显著更高的活性。 <img alt="图1" src="/files/ftp_upload/53207/53207fig1.jpg" /> 图1. Caspase-3的在心肌梗死大鼠杏仁核活性，表示为平均活性的假对照百分比，设定为100％（8只大鼠每组）。 *表示显著的组间差异（P &lt;0.05）。数据表示为平均值±SEM表示。

Watch this Scientific Journal Video about Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction at JoVE.com

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Caspase-3活性在鼠杏仁核测量用荧光分析法心肌梗死后

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

caspase-3-activity-rat-amygdala-measured-spectrofluorometry-after

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.7K 次观看.  Hospital du Sacre-Coeur de Montreal. Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

视频: Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

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

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

生物化学

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

大鼠慢性心肌梗塞的组织学定量分析

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

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 New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

<meta charset="utf8"></head><body>大鼠心肌梗死后心力衰竭的新模型

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

大鼠心肌梗死后心力衰竭的新模型

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

完整的心包缺血性啮齿动物模型

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

<meta charset="utf8"></head><body>用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

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.

<meta charset="utf8"></head><body>用于研究基因敲除小鼠心脏修复和重塑的心肌梗死实验模型

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.

用于研究基因敲除小鼠心脏修复和重塑的心肌梗死实验模型

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

Caspase-3活性在鼠杏仁核测量用荧光分析法心肌梗死后

摘要

探索更多视频

此视频中的章节

大鼠急性心肌梗死

使用荧光测定法或流式细胞术测量半胱天冬酶活性

大鼠慢性心肌梗塞的组织学定量分析

使用左前部降冠动脉的永久结扎的月心肌梗死模型

大鼠心肌梗死后心力衰竭的新模型

大鼠心肌梗死后心力衰竭的新模型

完整的心包缺血性啮齿动物模型

用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

用于研究基因敲除小鼠心脏修复和重塑的心肌梗死实验模型