Name: optical mapping of langendorff-perfused rat hearts
Uploaded: 2009-08-11T19:00:00
Description: Watch this Scientific Journal Video about Optical Mapping of Langendorff-perfused Rat Hearts at JoVE.com

Эта работа проводится при поддержке грантов от Национального института здоровья (HL068915; HL088206) и взносов в Фонд сердечной проводимости при Детской больнице Бостона.

Часть 1: Подготовка решений и изолированной перфузии система сердца <ol><li> В первой половине дня эксперимента, 4,0 л Кребса Хенселейта готовят, как описано ранее [5, 6]. </li><li> 11 ммоль / л 2,3-бутандион monoxime (BDM) растворяют в 1,0 л Кребса Хенселейта сливают из перфузат подготовлен в шаге 1.1. </li>...

<ol>
	<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 (1), 21-33 (2004).</li><li>Hucker, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Images+in+cardiovascular+medicine.+Optical+mapping+of+the+human+atrioventricular+junction.">Images in cardiovascular medicine. Optical mapping of the human atrioventricular junction.</a> Circulation. 117 (11), 1474-147 (2008).</li><li>Entcheva, E., Bien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+optical+mapping+of+excitation+in+cardiac+cell+networks+with+ultra-high+spatiotemporal+resolution.">Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.</a> Prog Biophys Mol Biol. 92 (2), 232-257 (2006).</li><li>Entcheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+electrical+activity+in+cardiac+cells+using+an+all-solid-state+system.">Fluorescence imaging of electrical activity in cardiac cells using an all-solid-state system.</a> IEEE Trans Biomed Eng. 51 (2), 333-341 (2004).</li><li>Stamm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+endotoxin-induced+alterations+in+myocardial+calcium+handling:+obligatory+role+of+cardiac+TNF-alpha.">Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.</a> Anesthesiology. 95 (6), 1396-1405 (2001).</li><li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Skrzypiec-Spring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Fedorov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart Rhythm. 4 (5), 619-626 (2007).</li></ol>

Удаление сердца от анестезии крыс должна быть выполнена быстро, чтобы избежать ишемии миокарда. Если ишемия или недостаточное коронарной перфузии происходит, сердце, скорее всего, развиваться аритмий и может стать инфаркта. Кроме того, эти сердца покажет недостаточной флуоресценции в...

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<td>RedShirt Imaging, Decatur, GA 30030 USA </td>
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<td>CardioPlex Software</td>
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<td>RedShirt Imaging, Decatur, GA 30030 USA</td>
<td>&nbsp;</td>
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<td>LUXEON LED Light Source 460-490 nm </td>
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<td>Lumileds Lighting, US, LLC, San Jose, CA 95131 USA</td>
<td>LXHL-PB02</td>
<td>&nbsp;</td>
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<td>ECG Amplifier Type 689 Hugo Sachs Elektronik</td>
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<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>730149</td>
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<td>Dichroic Mirror 505 nm</td>
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<td>Semrock, Rochester, NY 14624 USA</td>
<td>FF505-SDi01-25x36</td>
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<td>Emission Filter 605 nm Long Pass</td>
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<td>SciMedia, Costa Mesa, CA 92626 USA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>THT Sideways</td>
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<td>SciMedia, Costa Mesa, CA 92626 USA</td>
<td>25 BM-8</td>
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<td>Mini Ball Joint Holder</td>
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<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0177</td>
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<td>Small Stimulation Electrode Set</td>
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<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0160</td>
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<td>BM-6 Benchtop Vibration Isolation Platform</td>
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<td>Technology Inc., Inglewood, CA 90301</td>
<td>25 BM-6</td>
<td>&nbsp;</td>
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<td>Monopolar ECG Electrode</td>
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<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0200</td>
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<td>Roller Pump SCI 400</td>
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<td>Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA</td>
<td>401U/D1</td>
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<td>Roller Pump MasterFlex Easy Load II</td>
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<td>Cole Parmer, Vernon Hills, Illinois 60061 USA</td>
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<td>Tubing Marprene #14</td>
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<td>Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA</td>
<td>902.0016.016</td>
<td>&nbsp;</td>
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<td>MasterFlex Tubing</td>
<td>Equipment</td>
<td>PharMed, Westlake, OH 44145 USA</td>
<td>06485-25</td>
<td>&nbsp;</td>
<tr>
<td>S48 Square Pulse Stimulator</td>
<td>Equipment</td>
<td>Grass Technologies, West Warwick, RI 02893 USA</td>
<td>Model S48</td>
<td>&nbsp;</td>
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<td>SIU5 RF TRANSFORMER ISOLATION UNIT</td>
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<td>Grass Technologies, West Warwick, RI 02893 USA</td>
<td>Model SIU5</td>
<td>&nbsp;</td>
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<td>5 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-5</td>
<td>&nbsp;</td>
<tr>
<td>2 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-2</td>
<td>&nbsp;</td>
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<td>0.5 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-0</td>
<td>&nbsp;</td>
<tr>
<td>0.25 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA </td>
<td>120142-025</td>
<td>&nbsp;</td>
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<td>10 ml Heating Coil</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>158822</td>
<td>&nbsp;</td>
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<td>Compliance Bubble Trap</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>130149</td>
<td>&nbsp;</td>
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<td>Luer Disconnect Cannula</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>72-1444</td>
<td>&nbsp;</td>
<tr>
<td>3-Way stopcock, FLL to MLT, No Port Covers</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 72-2630</td>
<td>&nbsp;</td>
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<td>Thermocouple Thermometer</td>
<td>Equipment</td>
<td>Cole Parmer, Vernon Hills, Illinois 60061 USA</td>
<td>WU-91100-40</td>
<td>&nbsp;</td>
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<td>Ultra Fine IT-Series Flexible Microprobe </td>
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<td>PhysiTemp Instruments Inc., Clifton, NJ 07013 USA</td>
<td>IT-24P</td>
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<td>Oscilloscope Tektronix TDS 1002</td>
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<td>Tektronix Inc., Beaverton, OR 97005 USA</td>
<td>TDS 1002B</td>
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<td>2,3-Butanedione monoxime </td>
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<td>Sigma, St. Louis, MO 63132 USA</td>
<td>B0753</td>
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<td>Ketamine HCl</td>
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<td>Hospira Inc., Lake Forest, IL 60045 USA</td>
<td>RL-0065</td>
<td>&nbsp;</td>
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<td>Xylazine</td>
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<td>Lloyd Inc., Iowa 51601 USA</td>
<td>LB15705A</td>
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<td>E-TOXA-CLEAN&reg;</td>
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<td>Sigma, St. Louis, MO 63132 USA </td>
<td>E9029</td>
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<td>Di-8-ANEPPS</td>
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<td>Invitrogen, Carlsbad, CA 92008 USA</td>
<td>D-3167</td>
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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.

Watch this Scientific Journal Video about Optical Mapping of Langendorff-perfused Rat Hearts at JoVE.com

Optical Mapping of Langendorff-perfused Rat Hearts

В этой статье описываются высоким временным и пространственным разрешением технику оптически образ потенциала действия движения на поверхности Langendorff-перфузии сердца крысы использованием потенциометрического красителя (ди-8-ANEPPS).

Оптический Картирование Langendorff-перфузии сердца крысы

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

Research

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Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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

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Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...