光学测绘的Langendorff灌注大鼠心脏
Summary
本文介绍了高时空分辨率技术的光学图像行动的Langendorff灌注大鼠的心，用一个电位染料（DI – 8 – ANEPPS）表面上的潜在流动。
Abstract
电压敏感的荧光染料的心脏表面的光的映射已成为一个重要的工具，以调查在实验模型电励磁，在量程范围从细胞培养到整个器官<sup> [1，2]</sup>。几乎立即可使用状态的最先进的的光学成像系统，在正常心律或心律失常的启动和维护整个动作电位的产生和传播的可视化<sup“[1]</sup>。最新的市售系统可以提供过高的时空分辨率的信息和最初开发定制的设备为基础，以克服由更传统的电方法施加的障碍<sup“[1]</sup>。在高分辨率和高速互补金属氧化物半导体（CMOS）摄像头和强烈的光亮，发光二极管（LED）以及电压敏感染料，光学，和过滤器的进步已经开始使电气信号采集心血管疾病的细胞生物学家谁更习惯于用显微镜工作的实际。虽然新一代的CMOS摄像头，可以获取10000帧每秒16,384像素阵列上，根据类型的样品制备，长期形成的荧光采集技术，如光电二极管阵列，激光扫描系统，冷却电荷耦合器件（CCD ）相机仍然有一些明显的优势方面的动态范围，信号的信噪比和量子效率<sup> [1，3]</sup>。在本研究中，Lewis大鼠心脏灌流<em>体外</em>与一个晶体灌流（克雷布斯Henseleit溶液）在37 °上修改的Langendorff器具彗星。经过20分钟的稳定期，心中间歇灌注11 mmol / L的2,3 – 丁二酮肟，以消除收缩在图像采集相关的议案。对于光的映射，我们满载着心中的快速反应电位探头DI – 8 – ANEPPS<sup“[4]</sup>（5μmol/ L的），并简要475 ± 15 nm的激发光照亮的准备。在一个典型的2第二期照明，高速CMOS摄像头连接到一个水平macroscope>从心脏编制发出的605纳米的光成像。对于本演示，心中节奏在每分钟300次同轴电极连接到一个孤立的电刺激单元。同时双极电图的录音被收购，分析与使用现成可用的软件的电压信号。在这种方式下，可以可视化的Langendorff灌流大鼠心脏表面上的动作电位和电图信号注册。
Protocol
第1部分：准备的解决方案和离体心脏系统在上午的实验中，4.0 L是克雷布斯- Henseleit溶液准备如前所述[5，6] 。 11 mmol / L的2,3 – 丁二酮肟（BDM），溶解在1.0步骤1.1准备灌流转自克雷布斯Henseleit解决方案的大号。 从在步骤1.1准备灌流克雷布斯- Henseleit一个额外的150毫升和5 mol / L的DI – 8 – ANEPPS（10毫摩尔/升，溶解在二甲基亚砜（DMSO）的股票稀释）混合。 这3个方案?…
Discussion
麻醉大鼠的心去除，必须快速进行，以避免心肌缺血。如果发生缺血或冠状动脉灌注不足，心脏可能会发展性心律失常，并可能成为心肌梗死。此外，这些心会显示信息录音和后续分析的荧光发射不足。加载染料之前，需要充分克雷布斯- Henseleit溶液灌注心肌细胞，建立和保持一个稳定的电脉冲的生理电解质的环境。准确的灌流准备也是必要的，以保持适当的的器官的活力和功能。电解质浓度或?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从美国国立卫生研究院的研究补助金（HL068915 HL088206）和在波士顿儿童医院的心脏传导基金捐款支持。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| CardioCMOS-SM128f | Equipment | RedShirt Imaging, Decatur, GA 30030 USA | ||
| CardioPlex Software | Equipment | RedShirt Imaging, Decatur, GA 30030 USA | ||
| LUXEON LED Light Source 460-490 nm | Equipment | Lumileds Lighting, US, LLC, San Jose, CA 95131 USA | LXHL-PB02 | |
| ECG Amplifier Type 689 Hugo Sachs Elektronik | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | 730149 | |
| Dichroic Mirror 505 nm | Equipment | Semrock, Rochester, NY 14624 USA | FF505-SDi01-25×36 | |
| Emission Filter 605 nm Long Pass | Equipment | SciMedia, Costa Mesa, CA 92626 USA | ||
| THT Sideways | Equipment | SciMedia, Costa Mesa, CA 92626 USA | 25 BM-8 | |
| Mini Ball Joint Holder | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0177 | |
| Small Stimulation Electrode Set | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0160 | |
| BM-6 Benchtop Vibration Isolation Platform | Equipment | Technology Inc., Inglewood, CA 90301 | 25 BM-6 | |
| Monopolar ECG Electrode | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0200 | |
| Roller Pump SCI 400 | Equipment | Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA | 401U/D1 | |
| Roller Pump MasterFlex Easy Load II | Equipment | Cole Parmer, Vernon Hills, Illinois 60061 USA | Model 77201-60 | |
| Tubing Marprene #14 | Equipment | Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA | 902.0016.016 | |
| MasterFlex Tubing | Equipment | PharMed, Westlake, OH 44145 USA | 06485-25 | |
| S48 Square Pulse Stimulator | Equipment | Grass Technologies, West Warwick, RI 02893 USA | Model S48 | |
| SIU5 RF TRANSFORMER ISOLATION UNIT | Equipment | Grass Technologies, West Warwick, RI 02893 USA | Model SIU5 | |
| 5 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-5 | |
| 2 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-2 | |
| 0.5 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-0 | |
| 0.25 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-025 | |
| 10 ml Heating Coil | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 158822 | |
| Compliance Bubble Trap | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 130149 | |
| Luer Disconnect Cannula | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | 72-1444 | |
| 3-Way stopcock, FLL to MLT, No Port Covers | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 72-2630 | |
| Thermocouple Thermometer | Equipment | Cole Parmer, Vernon Hills, Illinois 60061 USA | WU-91100-40 | |
| Ultra Fine IT-Series Flexible Microprobe | Equipment | PhysiTemp Instruments Inc., Clifton, NJ 07013 USA | IT-24P | |
| Oscilloscope Tektronix TDS 1002 | Equipment | Tektronix Inc., Beaverton, OR 97005 USA | TDS 1002B | |
| 2,3-Butanedione monoxime | Reagent | Sigma, St. Louis, MO 63132 USA | B0753 | |
| Ketamine HCl | Reagent | Hospira Inc., Lake Forest, IL 60045 USA | RL-0065 | |
| Xylazine | Reagent | Lloyd Inc., Iowa 51601 USA | LB15705A | |
| E-TOXA-CLEAN® | Reagent | Sigma, St. Louis, MO 63132 USA | E9029 | |
| Di-8-ANEPPS | Reagent | Invitrogen, Carlsbad, CA 92008 USA | D-3167 |
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Tags
Optical MappingLangendorff-perfused Rat HeartsVoltage-sensitive Fluorescent DyesElectrical ExcitationExperimental ModelsAction PotentialsArrhythmiasSpatiotemporal ResolutionsCustom-built EquipmentElectrophysiological MethodsComplementary Metal-oxide-semiconductor (CMOS) CamerasLight-emitting Diodes (LEDs)Voltage-sensitive DyesOpticsFiltersCardiovascular Cell BiologistsMicroscopesFluorescence Acquisition TechnologiesPhotodiode ArraysLaser Scanning SystemsCooled Charged-coupled Device (CCD) Cameras
Cite This Article
Sill, B., Hammer, P. E., Cowan, D. B. Optical Mapping of Langendorff-perfused Rat Hearts. J. Vis. Exp. (30), e1138, doi:10.3791/1138 (2009).