这项工作是由来自美国国立卫生研究院的拨款（以R01-HL095828兆瓦凯）支持。

1。设立研究<ol><li>准备四个改性克雷布斯Henseleit溶液16升（MM: 氯化钙 ，1.20，2.00，3.30氯化钾118氯化钠，24.0 硫酸镁 1.20碳酸氢钠3，KH 2 PO 4计 ，10.0葡萄糖，2.00 NaPyruvate，和20.0毫克/ L，白蛋白）。应准备开始实验尽可能接近的解决方案。 （孔径:22微米，康宁）无菌过滤后，pH值应调整至7.4。溶液渗透压，应该是275和295 mOsm /公斤之间。 </li><li>用纯化水冲洗管和商会的工作心脏系统。从系统中删除，直到所有的水已经运行泵。 </li><li>添加纤维素膜过滤器（孔径5微米，Advantec）在用灌注泵（Langendorff灌流泵，左心灌注泵，右心灌注泵）。 </li><li>执行每个压力传感器的两点校准（0和60毫米汞柱）。 </li><li>打开水洗澡。一个加热循环水浴（科尔帕尔默）用于地暖的水套式管和热交换器。灌流预温在一个单独的水浴（Oakton仪器）。这两个浴场保持溶液温度37℃ </li><li>打开泵在一个闭环循环灌流。灌流通过毒气95％O 2和5％的CO 2在80千帕的超细纤维氧合（hemofilters）的传递。含氧灌流然后流入维持在温度为37°C，进入心脏插管前，通过热交换器。 </li></ol> 2。心脏切除<ol><li>首先，设置工作心脏系统的工作在恒压的Langendorff模式。将主动脉块范围内的50至60毫米汞柱的压力。 </li><li>麻醉兔子肌肉注射氯胺酮（44毫克/公斤）和甲苯噻嗪（10毫克/千克）。后兔是镇静剂，巴比妥（50毫克/ K的g）和静脉注射肝素（2000美）通过耳缘静脉或后肢内侧的横向的大隐静脉。 </li><li>当兔子是完全不响应，缺乏对疼痛反射确定，胸腔被迅速拉开，心包切片，被夹住主动脉，心脏和肺被切除。肺部此时应留在心脏，以帮助隔离肺静脉。 </li><li>隔离和cannulate主动脉直径5毫米的套管附加到洋溢着60毫升灌流和200单位肝素注射器。确保主动脉零号丝线缝合的套管，慢慢压下注射器冲洗心脏的血液。 </li></ol> 3。双心室插管<ol><li>连接心脏的工作心脏系统的主动脉块。防止空气进入主动脉，可引起冠状动脉栓塞。这是最好的导管主动脉BL玉珠由接近在斜角主动脉的连接和允许灌流轻轻滴入套管连接器，而它是连接。 </li><li>虽然心灌注在恒压的Langendorff模式，去除脂肪和结缔组织，并找到以下的船只:劣质和上腔静脉，奇静脉，肺动脉，肺静脉。 </li><li>结扎，上腔静脉。肺动脉略低于削减地方分支机构的左，右肺动脉。 </li><li>集团余下的所有船只之间的心脏和肺部，并使用1缝合结扎他们（肺静脉）。取出的肺部。 </li><li>切左心耳在角落的小孔。确保洛杉矶灌流充满。同时确保完全充满灌流套管，当它被插入Cannulate洛杉矶。缝合左心耳插管。 </li><li>打开泵（泵＃2）左侧提供流动到T他左心房。设置2 - 6毫米汞柱，并调整±2毫米汞柱，确定心房扩张之​​间的预压力。 </li><li>关闭的Langendorff泵（泵＃1）心脏的工作模式切换心。 </li><li>一度减少到10毫米汞柱的主动脉压力，然后慢慢增加80至100毫米汞柱的范围内。这将允许打开和运作，因为它会在正常生理条件下的主动脉瓣。最终的负荷压力将取决于的LV收缩。它应设置一个值，比高峰期左室压力减少了约20毫米汞柱。 </li><li>测定左室心输出量可以通过测量流量的灌流，退出的主动脉块（毫升/分钟）。正常心输出量是14.77和16.43毫升/每分钟100体重17克和平均340毫升/分钟为2.2公斤的兔子。主动脉压力应类似于图1所示的压力信号。 </li><li>通过I Cannulate的RAnferior腔静脉。确保RA和套管完全充满灌流，并插入导管，同时防止形成气泡。缝合静脉插管。 </li><li>打开右侧泵（泵＃3）提供流至右心房。设置约3毫米汞柱的压力。 </li><li>确保房车充满灌流，并cannulate肺动脉。确保套管完全充满灌流，同时它插入，以防止气泡。缝合肺动脉插管。 </li></ol> 4。信号采集:压力，单相动作电位，fNADH <ol><li>双心室插管完成后，小心地插入主动脉的主动脉插管通过压力传感器导管（米勒）。轻轻地浏览过去的主动脉瓣和进LV。监察LV压力信号，以确保导管尖端的正确定位。例如LV的压力在图1。 </li><li>轻轻按压对心室外膜单相动作电位电极。监测的信号，以实现相应的动作电位测量。在信号轻微的运动伪影是正常的。 </li><li>一个双极刺激电极放在右心房步伐心。在我们的协议，心中节奏的周期长度在300和150毫秒之间，相应的，分别以200和400 BPM。 </li><li>测量左室心外膜表面的温度。如果研究的需要，温度保持在37°C，然后放置心内的水套的心腔或淹没在加热灌流浴心保持一个恒定的温度，整个心。 </li><li>定位的CCD相机（安道尔IXON DV860 128x128像素）和聚焦镜头等观察认为适当的领域。将相机连接到工作站和图像以2 fps收购使用安道尔SOLIS softwa重。 </li><li>打开汞灯灯前开始的成像。通过激发滤光片（350±25纳米，色度技术）和光纤光导（HORIBA Jobin Yvon的模型1950-1M）的光被照亮的心脏表面。紫外线光通过光导衰减小。紫外线照射也可提供高功率LED LED射灯组成的系统（Mightex PLS-0365-030-S的）和一个控制单元（SLC-SA04美Mightex）。 </li><li>关闭室内灯光，并尽量减少任何环境照明。着眼于心脏的光导（或LED射灯）套圈，以实现统一的外膜照明。发出的NADH的荧光（fNADH）通过排放过滤器（460±20 nm的色度技术），是由CCD相机的成像。 </li><li>监测使用的成像软件的兴趣选择一个地区一段时间fNADH改变。选择生活的更新模式，以监测区域内的直径平均像素强度f利益。 </li><li>心应在双心室工作模式运作，以产生适当的压力。 fNADH水平应该是在外膜表面低而稳定，以确认足够的冠状动脉灌注。在这点在研究一个具体的实验协议应实施测试假说。 </li><li>当研究完成后，从系统中删除的心脏，并排出所有的灌流。纯净水冲洗系统管和商会。对于日常​​维护，系统应定期清洗Mucasol解决方案或稀释的双氧水，需要。 </li></ol> 5。脱机处理fNADH图片<ol><li>一种方式比较NADH的数据集（fNADH（I，J，T））之间的实验是每个荧光图像，参考图像（正常化fNADH（I，J，T 0））从9数据集，如以下公式所示。 NADH的荧光正常化的另一种方法是对PL王牌小块的铀玻璃在实验前9，18，19场的观点。铀玻璃将荧光紫外灯提供一个可以用来作为一个稳定的参考信号亮起时（450 - 550纳米）。 </li></ol><img alt="式（1）" src="/files/ftp_upload/4115/4115eq1.jpg" /> 6。代表结果 如图1所示的双心室兔心脏的准备前，基底意见。左心室压力测量航行过去的主动脉瓣进入左心室压力传感器导管（米勒SPR-407）。主动脉，肺动脉，左心室压力压（LVP）在图1C所示。通常是介于0和10毫米汞柱，舒张压LVP的。最低的舒张主动脉压力是约60毫米汞柱。收缩期峰值LVP是依赖于充盈压（预置或洛杉矶压力）和收缩，优化，应该是80和100毫米汞柱之间。最大的主动脉压力和LVP的最大应密切配合，如在图1C所示。 单相动作电位快速去极化相和复极阶段，是典型的离体兔心（图） 如图1D所示。地图可以相对容易地记录从承包的心，但通常会在舒张小运动伪影， 如图1D所示。地图是用于心脏起搏时（捕获）确认成功夹带，也可以用来衡量当地因缺血或其他急性扰动的电生理改变。心电图也可以测量淹没在浴温暖灌流心脏和电极放置在心脏的左，右两侧的浴。第三无动于衷电极被放置在洗澡，远离心脏，或连接到主动脉。心电图将提供全球激励和复极过程，这是评估整体的电气功能和揭示存在缺血的有用信息。 fNADH成像揭示了心脏的线粒体氧化还原状态的变化，它可以用来测量缺血或缺氧地区的时空进展。在这项研究中，心外膜fNADH测量监控的氧化还原状态的变化，在三个周期长度（CLS），300，200，150毫秒的起搏率。平均fNADH值从感兴趣的区域（红色框， 图2）显示，基线fNADH水平提高，为缩短周期长。当起搏率接近窦性心律（CL = 300毫秒）基线fNADH水平相对恒定。随着周期长度缩短低于300毫秒，"基线fNADH水平的提高，在最短的CL（150毫秒）的增幅最大。高的决议fNADH成像充分的前表面在200和400 BPM是如图3所示。 200 BPM fNADH水平不变和空间均匀。 BPM在400，fNADH水平大幅增加，整个心外膜。显着的空间异质性，观察RV和LV隔地区内发生的最大升幅。 收缩（运动伪影）fNADH信号振荡，振荡频率对应的心跳率（ 图2）。在双心室插管，心脏的基地举行4套管，这有助于防止心脏收缩时的摆动。因此，振荡幅度总是比任何更长的时间尺度（5-10秒）在fNADH的趋势，缺血或缺氧引起的。 <img alt="图1" src="/files/ftp_upload/4115/4115fig1.jpg" /> 图1。典型的压力，从一个孤立的双心室单相动作电位工作RABBIT心脏。显示四个套管心脏基底观点:2，肺动脉，主动脉，左心房，右心房B.前视图显示心脏的左心室（LV）和右心室。 （右）。C.代表的压力。热门:左心室压力（实线）和主动脉压力（虚线）。底部:D.肺动脉高压的代表单相动作电位。在面板C. <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4115/4115fig1large.jpg" target="_blank">点击这里查看大图</a>所示的压力信号对齐。 <img alt="图2" src="/files/ftp_upload/4115/4115fig2.jpg" /> 图2。离体兔心脏双心室fNADH成像。顶部:一个视图（左）和三个fNADH图像领域的卡通显示。每个图像上相应的起搏周期长度（CL）表示。红色框表示地区的利益为底部面板fNADH的信号。单相动作电位电极的尖端被看作是在该地区的利益的权利。心外膜汞灯和光导照明使用， 如图5所示。只底:心外膜表面被照亮了周围地区的利益。平均fNADH由该地区的利益，在顶部面板中的红色框表示。缩短生产周期长的的平均fNADH增加。 <img alt="图3" src="/files/ftp_upload/4115/4115fig3.jpg" /> 图3。完整的离体兔心脏双心室前壁表面fNADH图像。心脏是从RA在200 bpm和400 BPM的节奏。 fNADH被拍摄（2 fps的128×128像素分辨率0.4毫米），同时使用两个高功率LED（Mightex PLS-0365-030-S 365纳米，4％，照亮了整个前外膜我ntensity，最大50毫瓦）。

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+ischemia+in+hypertrophic+langendorff-perfused+rat+hearts.">Regional ischemia in hypertrophic langendorff-perfused rat hearts.</a> Am. J. Physiol. 277, H1532-H1539 (1999).</li><li>Ashruf, J. F., Coremans, J. M., Bruining, H. A., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+of+cardiac+work+is+associated+with+decrease+of+mitochondrial+NADH.">Increase of cardiac work is associated with decrease of mitochondrial NADH.</a> Am. J. Physiol. 269, 856-862 (1995).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ. Res. 95, 21-33 (2004).</li><li>Rogers, J. M., Walcott, G. P., Gladden, J. D., Melnick, S. B., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Panoramic+optical+mapping+reveals+continuous+epicardial+reentry+during+ventricular+fibrillation+in+the+isolated+swine+heart.">Panoramic optical mapping reveals continuous epicardial reentry during ventricular fibrillation in the isolated swine heart.</a> Biophys. J. 92, 1090-1095 (2007).</li><li>Qu, F., Ripplinger, C. M., Nikolski, V. P., Grimm, C., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+panoramic+imaging+of+cardiac+arrhythmias+in+rabbit+heart.">Three-dimensional panoramic imaging of cardiac arrhythmias in rabbit heart.</a> J. Biomed. Opt. 12, 044019 (2007).</li><li>Chinchoy, E., Soule, C. L., Houlton, A. J., Gallagher, W. J., Hjelle, M. A., Laske, T. G., Morissette, J., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+four-chamber+working+swine+heart+model.">Isolated four-chamber working swine heart model.</a> Ann. Thorac. Surg. 70, 1607-1614 (2000).</li><li>Hill, A. J., Laske, T. G., Coles, J. A., Sigg, D. C., Skadsberg, N. D., Vincent, S. A., Soule, C. L., Gallagher, W. J., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+human+hearts.">In vitro studies of human hearts.</a> Ann. Thorac. Surg. 79, 168-177 (2005).</li><li>Schenkman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+performance+as+a+function+of+intracellular+oxygen+tension+in+buffer-perfused+hearts.">Cardiac performance as a function of intracellular oxygen tension in buffer-perfused hearts.</a> Am. J. Physiol. Heart Circ. Physiol. 281, H2463-H2472 (2001).</li><li>Pijl, A. J., Pfaffendorf, M., Mathy, M., Van Zwieten, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+by+nifedipine+in+isolated+working+hearts:+A+comparative+study+on+three+different+types+of+experimental+ischemia.">Cardioprotection by nifedipine in isolated working hearts: A comparative study on three different types of experimental ischemia.</a> J. Cardiovasc. Pharmacol. 21, 70-76 (1993).</li><li>Khatib, S. Y., Boyett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+glyburide+(glibenclamide)+on+myocardial+function+in+langendorff+perfused+rabbit+heart+and+on+myocardial+contractility+and+slow+calcium+current+in+guinea-pig+single+myocytes.">Effects of glyburide (glibenclamide) on myocardial function in langendorff perfused rabbit heart and on myocardial contractility and slow calcium current in guinea-pig single myocytes.</a> Mol. Cell Biochem. 242, 81-87 (2003).</li><li>Kates, R. E., Yee, Y. G., Hill, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+albumin+on+the+electrophysiologic+stability+of+isolated+perfused+rabbit+hearts.">Effect of albumin on the electrophysiologic stability of isolated perfused rabbit hearts.</a> J. Cardiovasc. Pharmacol. 13, 168-172 (1989).</li><li>Combs, C. A., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+imaging+of+dehydrogenase+activity+within+living+cells+using+enzyme-dependent+fluorescence+recovery+after+photobleaching+(ED-FRAP).">Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP).</a> Biophys. J. 80, 2018-2028 (2001).</li><li>Coremans, J. M., Ince, C., Bruining, H. A., Puppels, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(Semi-)quantitative+analysis+of+reduced+nicotinamide+adenine+dinucleotide+fluorescence+images+of+blood-perfused+rat+heart.">(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.</a> Biophys J. 72, 1849-1860 (1997).</li><li>Scholz, T. D., Laughlin, M. R., Balaban, R. S., Kupriyanov, V. V., Heineman, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+substrate+on+mitochondrial+NADH,+cytosolic+redox+state,+and+phosphorylated+compounds+in+isolated+hearts.">Effect of substrate on mitochondrial NADH, cytosolic redox state, and phosphorylated compounds in isolated hearts.</a> Am. J. Physiol. 268, 82-91 (1995).</li><li>Holcomb, M. R., Woods, M. C., Uzelac, I., Wikswo, J. P., Gilligan, J. M., Sidorov, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+dual+camera+systems+for+multimodal+imaging+of+cardiac+electrophysiology+and+metabolism.">The potential of dual camera systems for multimodal imaging of cardiac electrophysiology and metabolism.</a> Exp. Biol. Med. (Maywood). 234, 1355-1373 (2009).</li></ol>

没有利益冲突的声明。

孤立的Langendorff灌流心脏仍然是一个突出的工具，学习心脏生理2。尤其是心律失常，尤其是那些使用荧光成像跨膜电位20研究有用。一个优势是整个离体心脏的外膜，可观察到21，22。另一个优点是，在血液中，有一个明确的晶体缓冲溶液灌注不干扰荧光信号。限制的Langendorff技术是没有良好的心脏的代谢，这往往需要在生理前，后负荷压力的情况下执行工作的心脏研究适合。 为了提高离体心脏的准备工作相关的代谢研究，尼利介绍修改的Langendorff技术，建立适当的左心室（LV）的前，后负荷压力3。模型被称为孤立的LV工作心脏模型，并已被广泛用于研究LV的性能和代谢4-6。 LV的工作心脏模型是优于功能评价Langendorff模型，但它并没有提供正确加载的右心室（RV）。 demmy 等人 。首次报道了修改LV的工作心脏模型7，8 1双心室模型（低压及RV）。他们发现，每搏输出量，心输出量和压力，发展，提高在工作的LV模式转换为双心室工作模式8心。一个正确加载的房车，也提高整个鼻中隔的异常压力梯度递减的间隔功能。双心室工作的心已被证明保持长达3小时8输出主动脉，肺血流量，平均动脉压，平均肺动脉高压，心脏率和心肌ATP，磷酸肌酸水平。双心室心脏研究工作通常使用心FROM小动物，如大鼠和家兔，由于心输出量和的灌流所需量是远远高于大型动物的心。然而，双心室心脏研究工作已进行了从猪，犬，甚至人类23，24的心中。 双心室工作模式的离体心脏的代谢需求大大高于Langendorff灌流。重要的是，灌流的解决方案提供足够的氧气和代谢底物，以支持双心室心脏功能。标准晶体缓冲的解决方案，如克雷布斯-Henseleit 16，17，25或Tyrodes 26，27，有氧气的溶解度高达5.6毫克/ L。当这些解决方案毒气carbogen（95％O 2和5％的CO 2气体混合），并包含合适的代谢底物（葡萄糖，葡萄糖，和/或丙酮酸钠），它们是适用于双心室工作在规范跳动的心人窦率（兔子约180 BPM）。 快节奏的代谢需求增加和标准灌流液中溶解氧量可能不足以充分支持双心室工作的心，在高利率承包。晶体缓冲区含有红细胞或全血混合的​​解决方案已用于心脏的准备工作，以确保足够的氧气供应。以往的研究表明，添加到的克雷布斯-Henseleit溶液的红细胞，改善工作心脏功能，并在严格的起搏协议第16心室颤动的发生率也减少了。一个限制使用全血红细胞或混合物，是血红蛋白的干扰与光的波长，用于荧光成像13。其他基板，如白蛋白，也可添加到灌注液解决方案，延长心脏活力，减少水肿28。 在荧光成像激发光的强度高，光线分布应均匀。实现均匀的照明，是不是总是很容易，由于外膜表面的曲率。在我们的研究，我们形象fNADH过滤从汞灯的光（350±25纳米）。一个岔光纤导光板用于直接到心外膜表面的紫外线灯。两个输出套管通过适当的定位，可以实现均匀照明。紫外线LED光源，也可以使用，正如我们在图3。 LED光源是相对便宜，因此可分为成像系统中的多个来源。 LED也可以骑自行车在高利率和关闭同步激发光图像采集。 NADH的漂白，应尽量减少组织光照时间29。这可以通过骑自行车上的照明和关闭使用电子IC快门和一盏灯或LED照明系统和控制器。如果照明与心动周期同步，然后fNADH图像采集局限于舒张，这会减少运动伪影的荧光信号。 trigging照明和图像采集，使用，如LV的压力，压力信号，将是一个办法做到这一点。 在我们的研究中，我们观察到，在fNADH单位时间的变化，可以5倍以上400 BPM高于200 BPM。这表明，快节奏，提高心脏的氧化还原状态。这是否造成缺氧或无力的心肌细胞氧化辅酶为NAD +速度不够快，以避免对NADH的积累仍然是一个悬而未决的问题。 的双心室工作心脏准备的性能取决于多种因素。其中最重要的是设置适当的前，后负荷的压力，模仿生理条件正在调查中。必须调整，特别是LV后负荷（主动脉压）代表全身的压力。如果实在是太高了，LV将无法克服的压力，导致在返流。压力过低会影响冠状动脉灌注。 LV的预压力（左心房压力）也应调整，以提供舒张末容积，是适当的实验协议。 fNADH活组织成像荧光成像13的既定模式。其心脏组织中的应用说明了巴洛和机会时，他们醒目海拔地区缺血组织内的fNADH后结扎冠状动脉血管14。用一个Fairchild的示波器相机和UV闪光摄影，电影，他们fNADH图像记录上。 coremans 等人 。扩大后使用的NADH荧光/紫外反射率来衡量的概念E的离体心脏血液灌流大鼠心脏30外膜的代谢状态。一个videofluorimeter用于成像，并用录像机录得的数据。后来，肖尔茨等 。用摄谱仪和光电二极管阵列测量从一个LV的大面积平均fNADH。这种方法减少外膜荧光非均质性和流通中的局部变化的影响，同时又揭示fNADH 31宏观与工作有关的变化。这种方法计算的利息跨越一个fNADH成像数据集， 如图2所示，所有帧地区的的平均fNADH水平相似。正如我们在这篇文章中提出，今天的技术，提供高速CCD相机和数字控制的高功率紫外线射灯。这些技术使的fNADH和心脏代谢的时空动态研究许多新的观点。相对低成本的光学和光源使FNADH的成像有用的配件为传统的心脏光学标测系统。9，32

<table border="1"><tbody><tr><td> 化工 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯化钠</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td>的S-3014 </td></tr><tr><td>氯化钾</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> P3911-500G </td></tr><tr><td> 氯化钙 </td><td>费希尔科学，公平的草坪，新泽西州</td><td> C77-500 </td></tr><tr><td> 硫酸镁 </td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td>的M-7506 </td></tr><tr><td> 碳酸氢钠 </td><td>费希尔科学，公平的草坪，新泽西州</td><td>的S-233 </td></tr><tr><td> KH 2 PO 4 </td><td>费希尔科学，公平的草坪，新泽西州</td><td> 423-316 </td></tr><tr><td>葡萄糖</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 158968-500G </td></tr><tr><td> NaPyruvate </td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> P2256-25G </td></tr><tr><td>白蛋白</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> A9418-100G </td></tr></tbody></table>

nadh fluorescence imaging of isolated biventricular working rabbit hearts

离体心脏的Langendorff 1成立以来，仍然是一个突出的工具，学习心脏生理2。然而，它不能很好地适合心脏代谢的研究，这需要在生理前，后负荷压力的情况下执行工作的心脏。尼利介绍修改的Langendorff技术，建立适当的左心室（LV）的前，后负荷压力3。模型被称为孤立的LV工作心脏模型，并已被广泛用于研究LV的性能和代谢4-6。然而，这种模式下，不提供正确加载的右心室（RV）。 demmy 等人 。首次报道了修改LV的工作心脏模型7，8 1双心室模型。他们发现，每搏输出量，心输出量和压力，发展，提高在工作的LV模式转换为双心室工作模式8心</sup&gt;。正确加载的房车，也减少了跨隔改善室间隔功能异常的压力梯度。双心室工作心已维持长达3小时8输出主动脉，肺血流量，平均动脉压，心脏率，心肌ATP水平。 研究，如缺血，心肌损伤的代谢作用时，它往往是必要的，以确定受影响的组织位置。这可以通过成像的NADH（烟酰胺腺嘌呤二核苷酸的简化形式）9-11荧光，辅酶大量的发现在线粒体。 NADH的荧光（fNADH）的显示与当地的氧气浓度12附近的线性反比关系，并提供了线粒体氧化还原状态的措施13。 fNADH成像在缺氧和缺血性条件已被用作染料的方法，以确定缺氧地区14，15和监察进展随着时间的推移10缺氧的条件。 该方法的目的是监测期间的协议，改变心肌细胞的新陈代谢率，或引起缺氧或创建一个组合两个双心室工作心线粒体氧化还原状态。从新西兰白兔的心被连接到1双心室工作心脏系统（雨果萨克斯Elektronik公司）和改性的克雷布斯-Henseleit溶液16灌注在37°C。记录主动脉，左心室，肺动脉，左，右心房压力。使用单相动作电位电极的电活动。到图像fNADH，从汞灯的光过滤（350±25纳米），用来照亮的外膜。发出的光被过滤（460±20纳米）和CCD相机成像使用。在双心室心外膜fNADH的变化，在不同的起搏率。心脏模型和fNADH成像的结合为研究内的急性心肌病理生理条件下现实的情况下提供了一个新的，有价值的实验工具。

Watch this Scientific Journal Video about NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts at JoVE.com

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

目标是在生理前，后负荷压力的情况下离体心脏的线粒体氧化还原状态监测。提出了一个双心室工作兔心脏模型。高时空分辨率的NADH荧光成像是用来监测线粒体外膜组织的氧化还原状态。

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 ...

nadh-fluorescence-imaging-isolated-biventricular-working-rabbit

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

17.9K 次观看.  The George Washington University. 目标是在生理前，后负荷压力的情况下离体心脏的线粒体氧化还原状态监测。提出了一个双心室工作兔心脏模型。高时空分辨率的NADH荧光成像是用来监测线粒体外膜组织的氧化还原状态。

视频: NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

生物学

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

医学

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

兔子模型中的跨胸超声心动图检查

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

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 ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

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).

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 ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

NADH的荧光成像隔离双心室工作离体兔心

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