该设备在这里描述的实验是由心内科，波士顿儿童医院部和从Haseotes家庭的慈善捐款资助。我们感谢博士。弗兰克·麦高恩和华美他为我们提供了早期经验与这个模型，以及林赛汤姆逊与艺术品的援助。

该协议是根据该机构的动物护理和使用委员会目前的协议下进行的。 1.制备研究<ol><li>打开水浴加热的Krebs-Henseleit缓冲液（KHB）贮存器（设定为42℃）。 </li><li>制备16升含有（以mM计）128 NaCl的，5.7氯化钾KHB的，1.3 硫酸镁 ，25的NaHCO 3，2.7 氯化钙 ，0.53 EDTA，0.54 NAC 3 H 3 O 3，和10.8右旋糖13的基材群众是如下:27.584克氯化钠，1.58克氯化钾，0.578克硫酸镁 ，8.401克碳酸氢钠 ，1.47克CaCl 2，0.744克EDTA，0.22克NAC 3 H 3 O 3和7.208克葡萄糖。 注意:这些组件可以存储在锥形管中以粉末形式，用于在实验的日子。 <ol><li>通过0.22微米过滤器过滤4升的去离子水。 </li><li>加入3.7微升的该水以4升烧杯中。添加的所有组件，除了氯化钙的水。 </li><li>溶解氯化钙中使用单独的烧杯中的剩余的300ml水。 </li><li>在1升/分钟（LPM）5分钟充氧用95％ 的 O 2/5％CO 2的溶液。此校正pH至7.40，并增强的 CaCl 2溶解。 </li><li>在氯化钙加入到的KHB的剩余部分。 </li><li>完成KHB添加到一个容器和循环穿过所有管路30分钟。确保系统无肉眼可见气泡。含氧化合物用95％ 的 O 2/5％CO 2在0.5 LPM。 注:KHB可以存储过夜在冰箱不超过1 - 2天，带回至室温并再过滤使用。不要实验之间重用KHB。 </li><li>准备2×50毫升的清洁烧杯用冰冷KHB，并放置在冰清扫车站附近一斗。确保KHB是冰冷的（而不是冷藏）的前前<html>种植的心脏。 </li><li>将微型压力容积（PV）的导管在10毫升注射器填充有过滤KHB每制造商的说明校准之前30分钟，。 注:KHB温度用来浸泡光伏导管应尽可能接近37℃越好。 </li></ol></li><li>准备动物的麻醉和解剖台。 <ol><li>确保水库异氟醚充足。制定500中的U 1毫升注射器肝素;放在这个注射器26号（1/2"）针头，准备口罩用于麻醉动物。 </li></ol></li><li>设置主动脉块灌注压至80毫米汞柱和左心房（LA）嵌段灌注压至10毫米汞柱。打开两个主动脉块和LA块，让温暖的KHB滴出。当准备解剖动物，打开主动脉区块允许KHB稳步缓慢滴注出来。 </li><li>根据制造商的说明校准光伏导管。 </li></ol>"&gt; 2，动物的制备和解剖注:为达到最佳效果，确保动物是300和500克之间;我们已经发现，425〜450 g的动物重量是理想的我们的系统。 <ol><li>麻醉动物用一个异氟醚室（1 - 2％），直到动物是无意识的。动物转移到解剖台，并将麻醉面罩异氟醚和氧气的动物。执行脚趾捏来评估镇静水平。对眼睛应用兽医药膏，以防止干燥时的麻醉下。 </li><li>注入肝素，在腹腔500单位腹腔。允许10分钟为被吸收的肝素。固定用胶带动物的肢体，以提高胸廓的可视化。 </li><li>心脏的解剖。 <ol><li>一旦确保有一个脚趾捏没有任何反应，从用钳子腹腔提起皮肤远离，然后用剪刀切开腹腔，叔的曲线以下他隔膜返回到肋的后角。 </li><li>一旦隔膜是可见的，使用小剪刀，沿着膜片的前表面切以下现有切口的方向，以允许进入胸腔。延长沿腋中线每次切割为双侧腋窝。 注意:应该执行接下来的步骤有效地，因为一旦隔膜切开通风将受到损害。 </li><li>使用镊子剑突向前退胸腔。切开心包和胸膜。 </li><li>确定下腔静脉（IVC）和主动脉只是膜片上方， 整块前方使用钝钳收回它们。 </li><li>采用大，剪子弯，迅速 ​​使整个IVC和主动脉切口，拉动心脏和肺部出了胸部整块 。切开食管，气管，头臂动脉和静脉头侧从胸部取出心脏和肺。切除你MIC组织与组织的此块。注意不要切升主动脉近端部分。 </li><li>立即浸入心脏和肺在冰冷KHB并如步骤1中所述移动到Langendorff装置上，预先设置。 </li></ol></li></ol> 3.主动脉插管<ol style="margin-left: 40px;"><li>放置在一个平坦的培养皿中心脏 - 肺复杂，并与胸腺和面向实验者和面向表肺部的后方面大血管定向心脏。拉开胸腺两叶，并确定从主动脉头臂动脉的腾飞。 <ol><li>覆盖在盘的边缘主动脉和横切使用小剪刀主动脉瓣约5mm以上，刚好接近右锁骨下动脉的起飞主动脉。 注:切口应产生一个清晰的圆圈 - 截面主动脉。如果是斜角（即广卵形）或不完整的，重复切以达到期望的结果。这将有利于高效主动脉插管。 </li></ol></li><li>在主动脉两侧使用2双弯钳，引导主动脉在主动脉插管（这应该与KHB慢慢滴）。主动脉瓣应该坐1 - 套管的末端低于2毫米。 <ol><li>主动脉插管后，垂直地重新定位钳穿过主动脉保持主动脉到位。另外，放置一个小卡子整个主动脉保持心脏肺复杂的地方，使一个实验者完成这个模型。 </li></ol></li><li>有一个助理刚刚通过镊子低于4-0丝线缝合配合到位，循环围绕套管和两个在前面和后面的心脏搭售多次。完全打开插管开始全面逆行主动脉血流。观察大力敲打心脏。 注:如果心脏没有开始迅速击败（〜200 BPM）大力时，领带或套管可以是闭塞冠状动脉的一个或两个。如对此有怀疑，去掉领带，从冠状动脉重新定位了。如果心脏distends，不打，套管可能是跨主动脉瓣。如果冠状动脉泄漏（从主动脉根KHB喷雾剂），推进套管靠近主动脉瓣（如果一个头臂动脉导管插入到位升主动脉，可能会发生这种现象）。 </li></ol> 4.肺静脉阻塞及插管肺动脉准备注意:该步骤的目的是创建一个封闭的左心房系统以确保所有的体积和压力从左心房块被发送到左心脏结构。未能完全堵塞肺静脉可能会导致预紧力不足，可能会伪造结果或创建一个不稳定的工作心脏的准备。 <ol><li>删除以提高日曝光的胸腺Ë肺动脉（PA）。 </li><li>使心脏的后方面是面向操作者手动旋转主动脉插管。解剖出导致右肺的血管。使用镊子进一步描绘这些船只暂停右肺组织。使用大中型手术夹（或缝线），与封闭一个剪辑的右肺动脉，静脉及支气管。切除右肺远侧夹子。 注:由于解剖肺动脉无困难，我们的做法是阻塞肺静脉扩张肺动脉，使它更容易在没有跳动的心脏模型伤及附近的结构切割。 </li><li>左侧肺重复步骤4.2。 注:潜在的隐患和问题解决:一旦双方的肺动脉被封闭，右心房明显会扩张和心脏可能成为心动过缓。这是因为，右心室变为加压。如果这不发生，则可能是该pulmonary脉没有完全闭塞，并且预压将不足以对工作心脏模式。如果心脏是不是能够保持左心房（LA）插管后的心输出量，并试图过渡到心脏的工作（见下文），考虑将更多的剪辑或周围的肺静脉树桩领带闭塞残留的泄漏。一旦功率放大器被遮挡，但是，第5步，应立即进行，以尽量减少心肌缺血。注意，一些研究者切开肺动脉肺静脉的结扎之前避免右心室的加压。 </li><li>肺动脉切口<ol><li>使心脏的前部方面是面向操作者旋转主动脉插管。识别肺动脉。再次，这动脉可以鼓起。用小剪刀作横切口肺动脉瓣上方约3毫米。 注意:这将立即释放压力和心脏速率可能增加。是造成这种插管很容易去除，导管插入肺动脉后左房插管完成。 </li></ol></li></ol> 5.左房插管<ol><li>使左心房被朝向操作者转动主动脉插管。使用小剪刀，使2 - 3毫米切口在左心房的上身，房室沟大约3mm以上。 <ol><li>左心房套管垂直于二尖瓣平面定位并朝向房间隔指出。 </li><li>打开LA插管，直到KHB流动。确保KHB是温暖的触感，以避免心肌功能障碍是由于低温下转换到工作模式（它在任何非套管坐位时迅速变冷）。插管过程中过渡到滴速。 </li></ol></li><li>使用镊子举行反牵引，插入心房插管进入左心房的身体，注意不要使用excessi已经生效，它可以撕裂心房。 注:LA套管应定位，使得它位于中庭的中间而对心房壁任何紧张。 </li><li>绕过左心房体4-0丝线缝合和打结创建一个围绕套管中庭的印章。小心确保左心房的后方面包括在缝合。添加其他缝合线是必要的。一旦密封，拉回来插管1 - 2毫米，它位于中庭的中间，而不是对房间隔。 注意:最常见的原因，心脏变得过渡到后心脏的工作模式malperfused是洛杉矶套管紧靠房间隔，这闭塞左心房流入。洛杉矶跟踪经常变化表现出适当的波和v波，当套管处于适当的位置（ 见图2E）。 </li><li>打开LA插管阀完全充分预压管理到左心房。监控滴速从心脏（这是从冠脉流出）。保证当LA套管是开放滴速不会改变。如果这样做，热帖围绕插管心房如在步骤6.4中描述，因为这代表了系统中的泄漏。 </li></ol> 6.肺动脉插管并过渡到工作模式的心<ol><li>如果测量心肌耗氧量（或在冠状动脉流出物的其他物质，如药物水平或细胞因子），插入1/32"柔性管到肺动脉现有切口。 注:氧气消耗量为左心房灌流和肺动脉之间污水氧含量的差额2。 <ol><li>对于心肌耗氧量的连续测量，可以使用一种在线氧电极进行比较左心房和冠状窦流出。 </li><li>在毕业缸收集冠状窦污水（来自肺动脉和从心脏滴）因德尔冠状动脉流量的定时定量。 </li><li>如前所述计算心肌耗氧量。2 </li></ol></li><li>过渡到通过关闭逆行主动脉泵工作心脏模式。 注:当这样做时，在洛杉矶压力变得预加载压力和电阻这是以前在的Langendorff模式逆行泵提供阻力现在提供抗心输出量，创造了平均动脉压。如果平均动脉压下降低于〜80毫米汞柱，其原因很可能与任一预压或心肌功能。最有可能的问题是左心房套管，它应重新启动逆行泵后进行调整。 </li></ol> 7.插入左心室压力容积导管的注:光伏导管可以放置任逆行（通过主动脉瓣）或通过心尖穿刺。逆行的好处是是position较为一致，它消除了对心尖穿刺和冠状动脉损伤或预压的损失的伴随风险的需要。然而，逆行安置有时是非常具有挑战性的，所以我们这里介绍两种技术。 <ol><li>附加1.4法式压力容积导管压力容积环系统。根据制造商的说明校准系统在温暖的KHB。确保波形是实时可见。使导管和电缆附近的LV表面以免撞出其以下的位置。 </li><li>对于逆行放置，打开调节阀，并轻轻喂光伏导管穿过主动脉瓣，直到稳定的压力和体积波形被识别。避免过度使用武力，这可能会损坏主动脉瓣或刺穿心室心尖部。 注意:我们已经发现，以最小化导管和该光伏导管必须导航到接近该AV匝数的长度是重要的。它可能有助于降低其自带的系统管道。 </li><li>对于顶位置，用24克血管导管建立在LV心尖穿刺。确保避免左冠状动脉前降支。瞄准朝向从心室心尖主动脉瓣的针。推进压力 - 容积导管插入左心室的主体。停止只要LV压和体积波形识别推进导管。 </li><li>一旦压力容积导管到位，将水套到周围的心脏位置。固定导管到水套的壁用一小片胶带。 </li><li>确保至少在开始进行任何测量或干预前的30分钟内的稳定。 </li></ol> 8.输液用药<ol><li> （可选）使用中注入一个标准药物泵药物（ 如多巴胺）进入左心房块。 注:我们根据whol有剂量的药物因为相当于整个心输出流量e动物体重穿过传导阻滞;仅此一小部分通过冠脉循环的，因为它在体内做。可替代地，第二组灌注液可以用药物治疗的预设浓度被创建和用于灌注心脏。 注意:在我们的协议中，我们注入药物在12分钟内，每次输注的最后10分钟期间收集生理数据并比较它的前一10分钟的基线。 </li></ol> 9.生理手法<ol><li>心率<ol><li> （可选）缝合2起搏导线到右心房壁，并连接到一个临时起搏设备。 注意:这允许心脏速率（天然窦率以上）的精确控制和心脏速率和收缩独立强心药物之间的关系的理解。 </li></ol></li><li>预载<ol><li>改变预紧（定义为左心室舒张末期容积）通过改变列供给左心房块的高度。 </li></ol></li><li>血压<ol><li>操纵上使用的IH-51中的压力阀的血压（后负荷的此模型中的主要决定因素）。 </li></ol></li><li>冠状动脉氧含量<ol><li>通过与灌注KHB的心脏中各种气体混合物饱和实现不同程度的心肌缺氧。通过使用单独的夹套储层（每个都有其自己的气体混合物），以确保气体和KHB之间平衡做到这一点。 </li><li>完成缝合冠状动脉缺血结扎远端冠状动脉。 注:在工作心脏模式近端冠状动脉结扎可导致致命的心肌功能障碍。 </li><li>通过中断或延迟逆行灌注在规定的时间内引起全球冠状动脉缺血。 </li></ol></li></ol>

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<li>Li, J., Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adverse+effects+of+dopamine+on+systemic+hemodynamic+status+and+oxygen+transport+in+neonates+after+the+Norwood+procedure%5BTitle%5D)+AND+%22J+Am+Coll+Cardiol%22%5BJournal%5D)">Adverse effects of dopamine on systemic hemodynamic status and oxygen transport in neonates after the Norwood procedure.</a> J Am Coll Cardiol. 48 (9), 1859-1864 (2006).</li>
<li>Gillis, A. M., Kulisz, E., Mathison, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+electrophysiological+variables+in+blood-perfused+and+buffer-perfused%2C+isolated%2C+working+rabbit+heart%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Cardiac electrophysiological variables in blood-perfused and buffer-perfused, isolated, working rabbit heart.</a> Am J Physiol. 271 (2 Pt 2), H784-H789 (1996).</li>
<li>Asfour, H., Wengrowski, A. M., Jaimes, R., Swift, L. M., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NADH+fluorescence+imaging+of+isolated+biventricular+working+rabbit+hearts%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">NADH fluorescence imaging of isolated biventricular working rabbit hearts.</a> J Vis Exp. (65), (2012).</li>
<li>Demmy, T. L., Magovern, G. J., Kao, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+biventricular+working+rat+heart+preparation%5BTitle%5D)+AND+%22Ann+Thor+Surg%22%5BJournal%5D)">Isolated biventricular working rat heart preparation.</a> Ann Thor Surg. 54 (5), 915-920 (1992).</li>
</ol>

The authors have nothing to disclose.

该工作心脏模型使心室绩效评估与心室前，后负荷，灌注液的氧张力，以及心脏率的完全控制。除其他因素外，它允许的正性肌力药物独立后负荷和前负荷，其中的方式，是不可能使用体内模型的固有心肌影响的评估。因为该模型利用晶体灌洗液，它允许评估心肌而不脱离血红蛋白干扰，从而简化了心肌能态的光谱分析中，例如14在此模型中，右心房不插管作为我们的仪器的一部分，尽管它有可能这样做。我们有意选择不以便利对心肌耗氧量的评估冠状窦流的采样这样做。重要的是，右心脏仍然执行在此模型中的压力和体积的工作，因为它泵助ronary窦流入肺动脉套管。提供了一些右心室预紧提高室间隔定位和增强左心室的性能，并且是该模型的一个重要组成部分。15 有几个实验陷阱就更不用说了。第一个是初始逆行插管，应方便地进行（ 即 ，在不到2分钟），以尽量减少缺血的周期。掌握最重要的技能是升主动脉的有效分离，制备和处理。重要的是，主动脉残端不被切割过短，留下足够的空间用于插管主动脉瓣上方。然而，这也是很重要的主动脉残端不能太长，这会导致围绕所述套管的主动脉torqueing。同样重要的是，该主动脉插管和主动脉根部是适当大小匹配。在一个小套管过大的大动脉也可以导致在插管主动脉根部的torqueing。右锁骨下动脉通常从升主动脉主动脉瓣约为7mm以上起飞。在解剖鉴定臂血管（约1毫米直径），主动脉修剪服务为横向主动脉切口作为重要的里程碑。修剪主动脉只是第一头臂动脉的腾飞以下为宜。此船在修剪主动脉根部纳入一般在过渡到心脏的工作模式导致主动脉根部压力KHB的泄漏和损失。 插管的另一个技术挑战性的方面是左心房插管。虽然这是对导管插入左心耳可行的，我们发现，在插管经常卡住附肢内，并且不容易传递到左心房的主体。因此，我们优选使切口在左心房的主体中，约2毫米优于房室沟。它插入之前定位在适当的平面上的左心房套管，以避免固定在插管时撕裂薄壁心房是重要的。 我们发现，左心房切口的理想大小为约3毫米。创建一个切口过小也可能使左心房套管的安置更加困难，并且可能导致左心房的撕裂。我们用直8毫米，斜面一块左心房块透氧的管（内径2.9毫米）的。我们已发现，使用本，而不是套管带有斜边，导致最稳定的心房插管并有助于确保左心房块的过程。无论所用的管路中，以确保该管道的端部不被房间隔或二尖瓣闭塞（如上面描述的是很重要的，我们发现，在左心房压跟踪在这个REGA有帮助RD），心房插管，因为哪怕是细微的动作可以显著改变左室前负荷并导致血流动力学测量。出于同样的原因，这是重要的，以确保左心房不漏打开左心房块之后以下。无论用于确保该系统中的管道是不透氧以确保有足够的氧气输送到心脏导管的类型是重要的。 该过程的另一技术挑战性的方面是压力 - 容积（PV）导管的放置。我们最初通过主动脉块青睐导管的逆行放置。虽然技术上可行，我们发现这是更简单，方便的通过心尖穿刺放置导管PV。必须小心，以监测整个实验期间导管的位置，如在倍导管可以或缩小左心室的移动。这可以通过监测pressu进行再随着时间的推移体积描记。 最后，应注意，以确保KHB溶液创建新鲜每个实验。这是可能称出KHB的组分，并将它们存储在时间提前粉末形式的锥​​形管中。关于实验的当天，这些可以与无菌的，过滤的水，二氧化碳/氧气，然后钙加入到该混合物进行混合。同样重要的是洗系统酶的活性粉状洗涤剂如Tergazyme（或类似），并定期更换灌注液过滤器。 这个实验准备的几个限制，应注意。首先，类似于晶体全部灌注的Langendorff准备，科华生物等asanguinous灌流液有显著减少携氧相对于血液的能力。虽然这部分是通过冠状动脉血管扩张和超生理冠脉流量补偿，编制不完全physiologic表示这个原因。第二，因为在这个仪器中使用的的Windkessel室的几乎无限遵守的，在收缩压和舒张压都仅最低限度地分开（参见图2A），从而使冠状动脉灌注压是非生理。这可能会在未来的模型通过将一种顺应性元件到负荷块来克服。第三，与所有离体心脏的准备，心脏经过一段时间之后 - 热缺血（2 3分钟），这可能造成心肌损伤或功能障碍。通过该技术的做法最大限度地减少这种损伤是最重要的代表性成果。此外，虽然有必要对动物福利，吸入麻醉药可以用作心肌抑制剂在再灌注过程的早期，虽然预计由于心脏与KHB灌注该效果迅速废除。 所描述的工作心脏系统允许多种生理学的有关病人护理，研究和教学逻辑调查。与一些附加的修改，该系统还可以用来模拟相关的先天性心脏疾病的重要生理，包括肺动脉高压和单心室生理学。限制包括这是离体的制备，该心脏是由一个缓冲器，而不是一个高氧含量的血液灌注。

离体器官的研究允许超出了体内可能的生理条件控制。 体外心脏的准备工作首先由奥托的Langendorff，1谁与逆行灌注描述一个孤立的模型来描述。随后，其他所描述的"工作心脏"模型，其中，所述心肌进行压力和体积的工作。2这样的制剂已在阐明4-6心肌的作用机制，3心肌代谢，以及强心药物的效果被工具。7- 9 增强心肌收缩药物的使用是危重病人常见。然而，很少有数据可比较这些药物对收缩力和心肌耗氧量，数据可在术后的设置是在护理病人的心脏衰竭的临床症状有用的相对影响10然而，由于大多数强心剂药物不仅影响心肌，而且动脉阻力，静脉电容11，和患者的代谢率，12 离体分离的心脏模型仍然由以研究对这些药物的效果的最佳手段心肌正确。 我们描述了使用对心肌功能和氧耗性肌力药物的负载独立研究的体外模型。从SD大鼠心脏采用左心室工作心脏模型插管，采用改良的Krebs亨氏灌流液灌注。主动脉和左心房压力得到控制。压力 - 体积阻抗导管通过心尖穿刺放入左心室用于连续监测收缩和舒张功能。氧消耗在氧含量左心房perfus之间的索引差连续测定吃和肺动脉污水。待测试的药物被注入左心房块，并分别测量和比较在心脏性能和氧代谢的变化与前一基线。

<table><tbody><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>8.401 g/4 L</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6758</td><td>0.744 g/4 L</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td>1.580 g/4 L</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td>0.578 g/4 L</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td>0.220 g/ 4 L</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td>27.584 g/4 L</td></tr><tr><td>Dextrose</td><td>Sigma-Aldrich</td><td>D9434</td><td>7.208 g/4 L</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>1.470 g/4 L</td></tr><tr><td>Biventricular working heart model</td><td>Harvard Apparatus</td><td>IH-51</td><td></td></tr><tr><td>Pressure volume catheter</td><td>Millar, Inc</td><td>SPR-944-1</td><td>6 mm spacing catheter used</td></tr><tr><td>LabChart Pro 8</td><td>AD Instruments</td><td>Version 8.1</td><td></td></tr></tbody></table>

rodent working heart model for the study of myocardial performance and oxygen consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

在逆行灌注（ 图1A）和左心室工作心脏（ 图1B）完全仪表心脏示意图。典型的主动脉，左心房和左心室压力和体积描记显示在图2A - D的典型的最终舒张压是约3 - 5毫米汞柱在此模型中，与峰收缩压大约为100毫米汞柱图2E说明了变化。在离开时放置和套管的定位过程中的LA套管移离房间隔远心房跟踪。在这些实验中，主动脉压力设定为90毫米汞柱，和LA压力为10毫米汞柱。 为了测试儿茶酚胺的作用，各生理参数（主要来自压力容积导管和相关的软件得到）进行比较，以紧接在前面<html>基准期。在所示的例子中，多巴胺输注在15微克/千克/分钟进入左心房块。虽然最终舒张压是在两个条件相同（考虑此模型中的固定心房压力），左心室舒张末期容积2.5％降低，左心室收缩末期容积由4.9％下降，产生一个增加心搏量（ 图3A）。与安慰剂输注相比，左心室冲程工作，确定为压力-容积曲线内的区域，与多巴胺治疗期间增加了32％（ 图3B，P &lt;0.001，t检验，每组n = 10）。将其用在相对 ​​于安慰剂输注（ 图3C）心肌耗氧量更大的增加相关联。以这种方式，不同的强心剂药物和剂量的相对效力和能源成本可以比作一个其上负载条件影响另一个独立的。 内容"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54149/54149fig1.jpg" /> 图1: 流程示意图在逆行灌注和工作心脏模式 （ 面板A 完全仪器心 :的Langendorff模式; B组:。工作心脏模式在逆行模式，科华生物在一组灌注压入主动脉根部注入。这种模式被用来恢复心肌缺血性时间和仪器仪表中，在工作心脏模式下，灌流液灌注冠状动脉循环前流经左侧心脏。在这种模式下，心肌必须产生自己的灌注压。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig1large.jpg" target="_blank">请点击这里查看一个更大的版本这个数字。</a> <img alt="图2" src="/files/ftp_upload/54149/54149fig2.jpg" /> Figure 2: 在基准衡量代表压力和体积描记获得。 （A）主动脉根部的压力，（B）左心房压，（C），左心室压力和（D）显示基线测量时左心室容积描记。行程容积，行程工作，心输出量，tau蛋白和其它参数，可以自动计算并通过软件实时显示。在工作模式下心脏心输出量较差迟钝的左心房跟踪（E）可以是一个线索，套管在左心房错位。请注意，在精心布置的左心房压力跟踪突出的v波是常见的，可能是由于在完全仪器动物减少的左心房达标。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图 3: 多巴胺对在PV曲线（A）中 ，包括增加的行程容积左移的压力-体积曲线多巴胺输液的结果的影响 ，降低收缩末期容积，与基线测量结果进行比较。请注意，这些光伏曲线的一些部件的形状不同于通常在体内测定不同（ 见图4）由于没有动脉和静脉顺应性的。（B）的相对的前一基线，冲程工作输注期间增加显著更多巴胺比安慰剂的（**，P = 0.0017，t检验），象心肌耗氧量（*，P = 0.013，t检验，C）。利用该模型，在基线的平均心肌耗氧量为0.22±0.02毫摩尔O 2 /克组织/分钟，使用的估计dissolv在40°C 165微摩尔/升盐水ED含氧量这种测量可以用来比较各种药物的心肌耗氧量。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig3large.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/54149/54149fig4.jpg" /> 图4: 压力容量环的分析。图中所示的理论压力-体积环介绍普通心动周期继主动脉瓣（AV） 的封闭件（1），等容收缩发生时（1 - 2）。如心室压力低于心房压力降低。该阶段的持续时间由头表示。二尖瓣（MV），然后用心房收缩期同时地打开，填充心室（2 - 3）。收缩然后用等容contracti开始关于（3 - 4），直到左心室的压力超过舒张动脉压中，AV打开时的时间。每搏量线的区别1 - 2和3 - 4冲程工作是1内的区域- 2 - 3 - 4曲线<a href="https://www.jove.com/files/ftp_upload/54149/54149fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption at JoVE.com

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

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20.1K 次观看.  Boston Children’s Hospital. Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

视频: Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

科研

医学

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

一个孤立的工作心脏系统的大型动物模型

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

前维沃·兰根多夫注入心脏的心电生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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 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 ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

心肌缺血再灌注损伤的改进啮齿动物模型

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

灭鼠工作心脏模型心肌的性能和氧耗的研究

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前维沃·兰根多夫注入心脏的心电生理学研究

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大鼠心肌梗死后心力衰竭的新模型

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为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法