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://www.jove.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

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

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

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心肌内细胞交货:观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

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 ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

生理条件下猪心的切片和培育

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

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 ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

在健康和受伤的老鼠心脏中为三维纤维细胞组织进行精炼的清晰组织清除

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

兔模型中的孤立肺灌注系统

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...