There are no acknowledgments.

1，构建Langendorff装置上（ 见图1） <ol><li>使用3/8“管，心脏水库连接到储血槽。 <ol><li>确保该管道经过滚压泵。注意：这可能需要使用两个3/8“至1/4”管连接器来建立一块1/4“的管子要经过滚子泵。 </li><li>储血槽连接到加热器/氧合器带有3/8“管。 </li><li>使用3/8“油管加热器/氧合器连接到一个Y型连接器。 </li><li> Y形连接器的一个臂连接到离心泵，然后离心泵连接到第二Y形连接器（全用3/8“管）。 </li><li>附上一张3/8“油管固定止血阀的朝上的手臂，这将作为两个泡沫陷阱和插入压力传感器的手段。 </li><li>附上一张3/8“油管向下手臂。这部分将附加到AOR<html>抽动套管（ 即后负荷线）。 </li><li>连接Y形连接件的另一臂的方法采用3/8“管预装载室的流入。确保该管道穿过第二轮泵。 </li><li>连接多余的3/8“油管这个室的流出。该部分将要连接到左心房（ 即预置线）。 </li></ol></li><li>氧气罐和加热装置连接到加热器/氧合器。 </li><li>夹紧线从Y连接器将要预装载室，因为这条线将不会被使用，直到心脏被置于工作模式。 </li></ol> 2，压力 - 体积导管准备<ol><li>在37℃水浴，温一瓶生理盐水。 </li><li>浸泡光伏电导导管及压力传感器的温生理盐水至少30分钟。 </li><li>打开的数据采集系统，允许两个热身为至少30分钟。 </li></ol>TLE“&gt; 3。准备Langendorff装置上<ol><li>打开氧气罐，加热装置，辊子泵连接的两个容器，以及离心泵。加热装置应设置为动物的体温（〜36℃）。 </li><li>根据制造商的说明冲洗血液。慢冲洗速度被推荐用于从血液中（ 如 ，过剩的电解质，溶解细胞物质）更彻底清除废物。 </li><li>一旦血液清洗，检查血细胞比容水平前，血液稀释。 </li><li>每瓶洗涤红细胞用生理盐水所需的血球浓度（推荐：20％-25％），并添加到Langendorff装置上。 </li><li>调整两个泵的速度通过​​该系统（不包括预压腔）开始血流量。 </li><li>检查血液的混合物的pH和电解质，并调整至生理所使用的物种。注意：为了防止有害的再灌注后的钙通量，在Langendorff装置上的钙离子水平最初应保持在较低水平（0.3-0.5毫摩尔/升）。 <ol><li>如果有一个减少的血细胞比容与钾同时增加，检查乳酸脱氢酶和血浆中游离血红蛋白，以排除溶血。 </li><li>在溶血情况确实发生，确保所有连接牢固，并没有明显的区域的切向的。 </li></ol></li><li>附上米勒导管插入的PowerLab系统的二次压力槽。 </li><li>根据制造商的说明进行校准的压力传感器。 </li></ol> 4，准备为心，以附件的Langendorff装置上注意：正确逮捕心脏应该用于涉及心脏孤立系统中的任何大型动物实验。缺乏心脏停搏的可能损害心脏，这样就不会产生可测量的工作。 Celsior液，或低钾UNIVER威斯康星SITY（UW）溶液被推荐的，因为不仅是这些解决方案类似于临床上所使用的，但该溶液的低钾有助于防止高钾血症，而电路上。的心脏停搏液量将取决于心脏的大小，用1升水足够的猪心。 <ol><li>从储存容器中迅速取出心脏，倒出来的任何存储解决方案在心室，吸干并称重。 </li><li>为了帮助维持心肌冷温度，直到心脏是准备好了的Langendorff，心脏恢复到储存容器和方向，使主动脉朝上。 </li><li>插入一个3/8“的套管进入主动脉，并与一个拉链结固定。 </li></ol> 5，装上心脏的Langendorff离<ol><li>降低离心泵的缓慢滴入。 </li><li>滴入血液进入主动脉，直到它被充满血液并完全去除空气。 </li><li>仔细附上主动脉çannula到上的Langendorff主动脉导管。使附着时注意。 </li><li>将校准的压力传感器通过止血阀[DS1]到本机主动脉。 </li><li>开始压力测量和调节离心泵的速度，直到所需的灌注压的实现。注：压力可能转变为冠脉阻力的变化。因此，监测动脉压密切，特别是在最初的再灌注。 </li><li>提高温度对加热单元心肌内温度的测量是在37°C。注意：有将是加热单元和变化，心肌内的温度进行了调整之间的延迟。因此，温度变化应逐步进行。 </li><li>得到基线（T = 0）的样品从静脉储血槽，以测量pH，电解质，和其它生化测定。 </li><li>将温度探头插入隔和监视心肌温度。减小加热单元的温度如果心肌温度超过39°C。 </li><li>采取血液样本，每15分钟，调整为所需的实验生理学参数。 <ol><li>约1毫摩尔的钙添加到血液溶液，每隔5分钟，以确保离子钙是&gt; 0.8 mmol / L的工作模式开始前的。 </li></ol></li></ol> 6，把心入工作模式<ol><li>插入一个适当大小的套管插入左心房/肺静脉。这可以与任何一个荷包缝合或拉链领带（如适用）来完成。 </li><li>根据需要关闭在左心房的任何漏洞，可能会泄漏，如其他肺静脉起源与缝合或钉书钉。 </li><li>调整预压室的高度，使得所述柱的高度得到所需的预加载压力。注意：假设血液/晶体混合物的密度等于水的密度，1毫米汞柱=1.36厘米从主动脉瓣到顶部的距离的血液水平在预置储存器（ 例如 ，15毫米汞柱20.4厘米）。 </li><li>松开油管将预载室，慢慢地开始预压辊泵，使预紧室和预紧油管用血完全填满。 </li><li>一旦预压管被完全去除空气，慢慢地填补了左心房和套管用血。 </li><li>而不让任何空气进入系统，连接预紧油管左心房插管。 </li></ol> 7，获取心室压力 - 体积（PV）录音<ol><li>按照制造商的压力和Rho比色皿校正的数据采集系统的使用说明。 </li><li>用3-0聚丙烯缝线在左心室（LV）的顶点放置一个荷包缝合。 </li><li>使用16号针头，使荷包内的刺切口。 </li><li>插入光伏电导导管进入心尖切口。注：理想导管放置将视端上具有LV内的所有感测电极和对LV以外两个激励电极。确保一个适当大小的动物和导管已选定（见讨论）。 </li><li>按下右上角开始记录数据，并确定有多少容积段是活跃的“开始”按钮。 <ol><li>如果所有段不活跃，调整导管位置，直到所有段都是活动的。注：在导管的轻微扭曲可能是必要的，以优化循环形态</li><li>如果无法获得信号在各阶层，调整激励电极，并按照制造商的指示感应电极的位置。 </li></ol></li><li>一旦获得所需的配置，请按照制造商的数量和alpha校准说明。 </li><li>使用正确校准导管，获得至少30秒基线压力容积数据。注：这些压力容积环将提供体积依赖新生削弱心脏功能（ 如 ，心输出量，每搏输出量）的测量。 <ol><li>一旦有足够的循环获得，继续到下一个步骤而无需停止数据的记录，以便获得咬合压力 - 容积数据。 </li></ol></li><li>闭塞预紧管慢慢地用管钳。注：压力 - 容积环应该开始变得更小和向下移动，并在左侧。这就是所谓的“走”。 <ol><li>获得10-15秒的走了下来，然后松开管夹，以便预紧重新进入左心房。 注：这些压力容积环将提供心脏功能的音量独立测量（ 例如 ，预装搏出功，收缩末期压力-容积关系）。 </li><li>通过点击“停止”按钮，在屏幕的右上角停止记录数据。 </li><li>至少等待5分钟重复前闭塞。 </li></ol></li><li>重复步骤7.7和7.8，以获得重复测量。 </li></ol>

<ol>
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The authors have nothing to disclose.

在Langendorff离体心脏灌流装置和工作心脏模型，导致一些心脏生理学，病理学，药理学和最根本的发现。该模型的多功能性允许其在各种正常和病理条件下1-18具有各种物种的利用。然而，分离的心脏模型不常用于大型哺乳动物，特别是人的心脏，部分由于增加了两个装置的设计和数据收集的复杂性。因此，本文中所呈现的示范协议试图改善这些复杂性，结果在研究分离的猪心脏的相对重现的手段。 我们设置的一个关键组成部分是动脉顺应性/后负荷室更换为离心泵。这种交换允许冠脉灌注压和后负荷中的Langendorff增强控制和心脏的工作模式，respectively，允许这种设置可以很容易地适应不同大小和种类的心。例如，在本设计中，猪心都灌注在40-45毫米汞柱，而人类的心灵都灌注在60-65毫米汞柱。这种变化的压力由调节离心泵的设置简单地实现;该系统的任何组件需要被物理地调整。此外，放置一个压力传感器主动脉根部内监视的根压使期间的Langendorff模式恒定流量和恒定的压力之间的轻松过渡。虽然这种变化消除了经典的柔性小室，离心式泵，通过允许双向流动发生基于该压力梯度，可作为柔性小室。随着收缩和退出每搏输出量，整个泵逆行流的作用是减少后负荷压力，复制主动脉弹性。 此装置的开放式设计也很重要。具有心脏挂在一开一而不是一个半封闭室或漏斗意图，允许对压力 - 容积测量更简单仪器。开放式设计允许使用的心尖切口LV导管放置，避免了跨瓣方法。该跨瓣的方法是技术上更困难的，通常需要透视为妥善安置。此外，这种方法还可以诱发瓣膜关闭不全。通过使用心尖方法，我们轻松，安全地将导管左心室内，同时消除了额外的成本和透视的不便。开放式设计也能提供轻松访问超声心动图和污水的收集，进一步扩大了可以在该系统上进行评估的功能和生化参数。 开放式设计，同时促进数据收集，并做心肌温度调节更加困难。保持生理温度的已知问题一的Langendorff 1或工作心脏系统1,3,11,13。 Langendorff灌流系统通常包含一个热腔，有助于保持适当的温度，但这个房间也使得插入心室压力容积导管更加困难。为了解决这个开放式的设计的下温度调节，氧合器/热交换器放置在水库后。热交换器和主动脉插管之间的最小空间减少热损失，而心肌温度探头确保正常体温。使用夹套管道或外部加热源也可以用来帮助温度控制。 该协议的另一个独特的元素被洗下，研究猪的自体血和生理盐水重构它。虽然，使用增强与晶体缓冲区无论是全血灌流或红细胞的情况并不少见，但它与目前的问题。前者通常需要供体动物，它增加了SUBSTantial成本的实验，而后者可以具有免疫原性的问题，因为它通常是从牛血1,11-13而得。通过洗涤原猪自身的血液，该协议只需要一个单一的动物和免疫原性问题进行消融。此外，在洗涤过程中除去大部分的电解质，这意味着它们可以每实验参数很容易地操纵。最后，通过使用血液保护单元除去大部分蛋白质在血液中，这是该过程的两个优点和缺点。的优点是，任何凝固和免疫/感染性蛋白质除去，减少血块或污染的可能性。的缺点是，这种混合物具有低的渗透压，从而导致心肌水肿和可能损失的心脏功能的一段时间。这个问题可以解决但是，通过添加白蛋白或其它胶体。 确保一个适当大小的进制和导管已被选定作为是使用适当的工作心脏设备一样重要。理想情况下，该导管将被放置的心室空间内所有的感测电极，有两个激励电极（ 即最邻近电极）心室空间之外。如果动物的心室腔太小，或电极之间的间距过大，则所有的段不会在低压空间内完全。同时激励电极的位置可以调节，一个小的LV腔也可导致导管弯曲或曲线，使得数据收集困难。因此，对于大型动物心脏功能分析，建议至少为60千克动物的大小。用这样大小的动物时，为7毫米的电极间距通常允许在导管完全插入。 总之，这份手稿描述了一个孤立的工作心脏系统，简化灌注压调节，数据山坳经文，和整体设计，同时使温度控制只是稍微更加困难。这些修改对离体工作心脏会希望允许其与大型哺乳动物的心脏，包括人类在内的使用量增加，促进我们的心肌病理改变的认识，使更多的临床相关的治疗方案被发现。

我们的许多基本心脏生物学和生理学的认识已经从使用的隔离，逆行灌注豚鼠心脏和离体工作心脏系统实验。这些实验系统仍在广泛应用的今天，以扩大我们的心脑血管的重要议题，包括缺血再灌注损伤知识2，预处理4，基于细胞治疗受损心肌5,7，药物6,9，和心脏移植保护心脏的作用技术8,15-18。 尽管这两个分离的心脏系统可用于任何哺乳动物物种，它们主要用于在小哺乳动物，如豚鼠，大鼠或兔3,12,13。较大的动物模型，如猪和人，提供更多的临床相关数据，但不经常使用，由于成本较高，更大的生物变异，较大的血液灌注液量，并BIGG呃设备1,12-15件。此外，数据收集是比较困难的，特别是对于孤立的工作心中1,3,12-15。由于这些复杂的结果，临床相关的离体心脏模型都很少使用，严重阻碍心血管转化研究进展。 在试图解决这些复杂性，隔离工作心脏制备进行了修改，以创建一个可以很容易地适应不同品种的心灵，包括人类，无论是在恒压或恒流量的Langendorff条件的系统。后负荷遵守腔置换为离心泵简化调节的Langendorff模式和后负荷灌注压在工作模式的过程。而不是封闭的，套水库含有的心脏，这个系统采用开放腔，使数据采集更加容易，通过使利用电导导管经心尖途径。 Moreov呃，这个开放式的设计允许访问的心脏超声心动图评价，进一步拓宽，可以在这些实验中测得的生理参数。这些改进将有希望鼓励其他人使用这个系统对于大型动物转化研究。

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerLab 16/35 with LabChart Pro</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">PL3516/P</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MPVS Ultra Pressure-Volume Unit</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">880-0168</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ventri-Cath Catheter (5F, 12E, 7mm, DField, Straight, 122cm)</td>
			<td style="width:147px;">Millar</td>
			<td style="width:128px;">VENTRI-CATH-507s</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pressure Catheter (3.5F, Single, Straight, 100cm, Ny, Non Repairable)</td>
			<td style="width:147px;">Millar</td>
			<td style="width:128px;">SPR-524</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PV Extension Cable (10ft)</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">CEC-10PV</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catheter Interface Cable (10ft)</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">PEC-10D</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rho Calibration Cuvette</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">910-1060</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MPVS Ultra BNC Cable Pack</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">880-0172</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autotransfusion system</td>
			<td style="width:147px;">Sorin</td>
			<td style="width:128px;">7320000</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bowl Set with Low Volume (135 ml) Centrifuge Bowl</td>
			<td style="width:147px;">Sorin</td>
			<td style="width:128px;">7135100</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygenator/Heat Exchanger</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:128px;">3CXSX18RX</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Perivascular flow probe</td>
			<td style="width:147px;">Transonic Systems</td>
			<td style="width:128px;">PAU Series</td>
			<td style="width:319px;">Size of flow probe will depend on animal size; for 60 kg pig, recommend 20 or 24 mm probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Perivascular flowmeter module</td>
			<td style="width:147px;">Transonic Systems</td>
			<td style="width:128px;">TS420</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Myocardial temerpature sensor</td>
			<td style="width:147px;">Smiths Medical</td>
			<td style="width:128px;">MTS-40015</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16 G 1&#34; Regular needle</td>
			<td style="width:147px;">BD Inc.</td>
			<td style="width:128px;">305197</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-0 polypropylene suture (double-arm)</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:128px;">8526H</td>
			<td style="width:319px;">For purse-string stitches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-0 polypropylene suture (single-arm)</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:128px;">8833H</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable ties</td>
			<td style="width:147px;">ULINE</td>
			<td style="width:128px;">S-1021</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable tie gun</td>
			<td style="width:147px;">ULINE</td>
			<td style="width:128px;">H-241</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Clear, Flexible PVC Tubing</td>
			<td style="width:147px;">VWR International</td>
			<td style="width:128px;">89068</td>
			<td style="width:319px;">Inner diameter depends on cannulas, pumps and other equipment used; most commonly use 1/4&#34;, 3/8&#34; tubing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight Tubing Connectors</td>
			<td style="width:147px;">VWR International</td>
			<td>46600</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Y-Shaped Tubing Connectors</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td style="width:128px;">6152</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jacketed Bubble Trap</td>
			<td style="width:147px;">Radnoti</td>
			<td style="width:128px;">14040</td>
			<td style="width:319px;">For preload chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal pump</td>
			<td style="width:147px;">Maquet</td>
			<td style="width:128px;">70105</td>
			<td style="width:319px;">The centrifugal pump and roller pumps were obtained used from perfusion department after clinical use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roller pumps</td>
			<td style="width:147px;">Maquet</td>
			<td style="width:128px;">HL-20</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemostasis Valve</td>
			<td style="width:147px;">Merit Medical</td>
			<td style="width:128px;">MAP150</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blood gas analyzer</td>
			<td style="width:147px;">Instrumentation Laboratory</td>
			<td style="width:128px;">570001000</td>
			<td style="width:319px;"></td>
		</tr>
	</tbody>
</table>

an isolated working heart system for large animal models

由于其在19 世纪后期推出的的Langendorff离体心脏灌流装置及工作心脏模型的后续发展，已经非常宝贵的工具，用于研究心血管功能和抗病1-15。虽然在豚鼠心脏制剂可以用于任何哺乳动物的心脏，大多数涉及该装置的研究使用小动物模型（ 例如 ，小鼠，大鼠和兔）由于增加的对于较大的哺乳动物1,3,11系统的复杂性。一个主要的困难是，确保在一个范围内的不同心脏大小的恒定冠状动脉灌注压-利用该装置1,11任何实验的一个重要组成部分。通过用离心泵替代经典静压负荷柱，下面描述的工作的Langendorff心脏装置可以很容易地调整和灌注压的严密调节，这意味着相同的设置可以被用于各种硬币s或心脏大小。此外，这种配置也可以无缝地恒定压力或恒定的流量之间的再灌注期间切换，这取决于用户的喜好。此设置的开放性，尽管使温度调节比其他设计更为困难，可以很容易地收集污水和心室压力容积数据。

图1是该电路的原理图，包含所建议的导管放置。该设备的重要组成部分包括以下内容：使用离心泵控制后负荷;压力导管（深蓝色线）在主动脉根部监测灌注压的位置;和压力容积（PV）的导管（淡蓝色线）transapically的放置。虽然在图中的连接似乎是直的连接，“Y”连接器的建议，特别是相对于预压线。 图2示出来自压力传感器灌注的电路，这是一贯之间40-42毫米汞柱，在20分钟的过程中被放置在猪心脏的主动脉根部获得的数据。变化在冠状动脉阻力可以使灌注的压力波动（ 图3）。这些变化可能是次要的和渐进的，纠正他们<html>自我随着时间的推移（ 图3a）。然而，在某些情况下，这些变化可以是突然的，并且需要调整的流量通过离心式泵，以维持所需的灌注压力（ 图3b）。因为变化可能发生，需要监测再灌注期间，主动脉根部的压力。 通过利用经心尖刺切口，压力 - 容积数据可以容易地获得分离的心脏系统上。在该实验中，已被存储在2小时冷（4℃）保存液猪心脏被使用。经初步引进光伏导管，该循环是质量差（ 图4a），与交叉和没有明显的心动周期组件的多个领域。然而，随着左心室内导管的最小操作，环路形态显着提高（ 图4b），从而允许将获得的测量结果。 ve_content“&gt;尽管导管位置的优化，获得的离体电路（ 图5中最上面一行）上的线圈可以具有不同的形态比在体内的循环（ 图5，底行），这些变化，以循环的形态可能是由于对心脏的电路上不同的方向相比，在仰卧的动物，以及缺乏在活的动物中发现的解剖附件（如心包）的。此外，使用起搏导线，以帮助调节心脏速率（推荐的附着位点：室间隔）引入外部电流，导致出现在体外回路的右下部分中的尖峰然而，只要这些循环仍设有心动周期的组件，它们仍能产生可解释的数据。 表1列出了从使用PV导管这些压力-容积环得到的多个功能参数。<html>冷静态存储可能引起心脏，这有助于解释一些相比， 在体内测量的变化，所得到的电路上的值的一些固有的损伤。有些负载因变量内的变化也是由于电路和活的动物之间的预紧力可能不同。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig1highres.jpg" width="500" /> 图的装置1。框图 ​​。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig2highres.jpg" width="500" /> 图再灌注2。代表主动脉根部的压力测量。 <html> g3highres.jpg“宽度=”500“/&gt; 图3。改变再灌注期间可能发生的主动脉根部压力的例子 ， 这些变化可能是渐进的，自我修正（A），或突然并要求对离心泵（B）更改设置。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig4highres.jpg" width="500" /> 在导管的初始插入transapically（A）和次要导管操作（B）之后获得图4。压力-容积环。注循环形态，由此循环交叉被消除并在心动周期中的元素的改善是可识别的。在这两组循环的右下部分中的尖峰是由于使用了一个起搏器，它引入了一个非本征电信号。 <img alt="&lt;/ HTML">“图5”FO：内容宽度=“5英寸”SRC =“/ files/ftp_upload/51671/51671fig5highres.jpg”宽度=“500”/&gt; 图5。采取离体电路（顶行）的代表性的压力-体积的测量，与体内测量（底行）进行比较，同样，起搏器的尖峰可以看出，在这两组体外回路的右下方。 <img alt="表1" fo:content-width="5in" src="/files/ftp_upload/51671/51671table1.jpg" width="500" /> 表1 2小时的冷库（右栏）的CO 后得到的猪心脏的体内 （左列）和对工作心脏设备功能参数：心输出量; E A：动脉弹性; EDP​​VR：舒张末期压力 - 容积关系; EDV：舒张末期容积; ESPVR：收缩末期压力 - 容积关系; PRSW：预载，搏出功;聚乙烯醇：压力 - 容积面积; SV：每搏输出量;南方周末：工作行程。

Watch this Scientific Journal Video about An Isolated Working Heart System for Large Animal Models at JoVE.com

An Isolated Working Heart System for Large Animal Models

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 ...

an-isolated-working-heart-system-for-large-animal-models

Research

JoVE Journal

Medicine

30.5K 次观看.  Duke University Medical Center. 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.

视频: An Isolated Working Heart System for Large Animal Models

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

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 ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

从孤立的大鼠心脏的间质渗出物收集的倒置心脏模型

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

大动物模型肝组织血浆凝固的评价

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

慢性血栓栓塞性肺动脉高压大型动物模型中急性右心衰竭的诱导和表型分析

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

在缺血性心脏病的猪模型中部署3D打印冠状动脉狭窄植入物的新颖的皮下方法

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

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 ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

用于评估基因治疗缺血性心脏疗效的大型动物模型

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

用于慢性病大型哺乳动物心脏模型对比增强显影显微计算机断层扫描的组织制备技术

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...