心肌顿抑相关的小鼠离体心脏模型心脏停搏
Summary
The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.
Abstract
下面的协议是用来评估心脏功能受损或心肌顿抑以下中度缺血性侮辱。该技术是模拟与众多临床相关现象，包括心脏手术心脏停搏和体外循环，非体外循环冠状动脉搭桥术，移植，心绞痛，缺血短暂， 等相关的缺血性损伤。该协议提出了模拟低温高钾心脏停搏及再灌注啮齿动物的心专注于心肌收缩功能测定的一般方法。简言之，小鼠心脏灌注在Langendorff离模式下，仪表用脑室气球，和基线心脏功能参数被记录下来。稳定之后，心脏随后经历介绍输液的心脏停跳低温解决方案，以启动舒张逮捕。停跳间歇交付了2小时。心脏是再灌注和WÄRMEd可正常体温的温度和心肌功能的恢复进行监控。使用此协议的结果可靠郁闷心脏收缩功能无心肌组织损伤总值啮齿动物。
Introduction
心肌顿抑定义为缺血或缺血性侮辱与心脏1,2,3,4,5的时间延长了短暂的可逆降低收缩活动，尽管恢复充足的血流量。提出的方法是专门用于模拟临床上相关的缺血性损伤可导致收缩功能可逆损伤（ 即与心脏手术利用心脏停搏，局部缺血，咽峡炎， 等等的短时间相关缺血性损伤）。在对比严重缺血的研究（心肌梗死，坏死）此协议的开发是为了评估心肌功能恢复和心肌无组织损伤，重塑，和细胞死亡。大多数纸的讨论类似，使用低温和间歇性停搏交付心脏手术元素的标准心脏停搏协议。
心肌PROT大多数心脏手术期间挠度依靠停跳和体外循环。虽然停搏（CP）解决方案和策略差异很大（血液，晶体，冷，暖等 ）的最常见的元素是：1）高钾血症和/或其他战略逮捕的心脏舒张，从而限制了能源利用率从心肌收缩产生的， 2）低温减缓新陈代谢，帮助维持ATP和其他能源储备而被逮捕。目前心麻痹液提供保护心脏缺血对侮辱，否则将被证明是致命的。然而，在手术缺血损伤的心脏保护的策略是不完美的，并且所得到的轻度缺血性损伤可导致可逆的心脏收缩功能障碍，尽管足够的血液流量（心肌顿抑），酸中毒，心肌损伤，血管作用包括减少冠状动脉灌注和血管痉挛。
此协议不同从标准的离体心脏缺血模型评估心肌梗死和，它的计算结果较温和的缺血性损伤严重缺血可导致心脏功能受损下面简要缺血或心脏停搏相关缺血性侮辱。 （审查上的Langendorff灌流技术和I / R研究看6 – 8）。对于一般准则，与小鼠离体心脏相关的实验参数进行透彻的分析看萨瑟兰（E T） 人，2003年9这里介绍的方法详细介绍了必要的设备，试剂，步骤，策略和技巧，以可靠诱发惊人的小鼠心脏。小的修改是必要的，该技术适用于老鼠。
简要地离体小鼠心脏被的Langendorff经由递送高钾hypotherm的灌注与生理的Krebs-Henseleit缓冲液（KHB）约30分钟，随后冷保护心脏骤停IC停搏液。被捕之后，心功能恢复复温，并与科华生物的心脏再灌注期间监测。变化的心脏收缩功能的恢复程度可以评价以评估心脏剂和不同的心脏保护策略。
Protocol
Representative Results
Discussion
前款协议细节的方法来评价心肌顿抑中学与心脏停搏有关全球缺血。在我们手中这个协议产生心脏功能近似〜减少40％（LVDP +/- DP / dt）的同在30分钟再灌注后的时间点最小的变化在心脏速率。由于心脏再灌注时复温及心功能的所有参数20分钟至30稳定之前减少在一个大大缩减的心脏速率初始时间点。冠脉流量一般在很大早期再灌注因充血增加，然后下降到小于〜20-30％，比下面的30分钟再灌注控制水…
Divulgazioni
The authors have nothing to disclose.
Materials
| Cardioplegia Solution (St Thomas II) | Symbol / Concentrations (mM) | ||
| Sodium Chloride | NaCl; 110 | ||
| Potassium chloride | KCl; 16 | ||
| Calcium Chloride | CaCL2; 1.5 | ||
| Magnesium Chloride | MgCL2; 16 | ||
| Sodium Bicarbonate | NaHCO3; 10 | ||
| Krebs-Heinslet Buffer | |||
| Sodium Chloride | NaCl; 118 | ||
| Potassium Chloride | KCl; 4.8 | ||
| Magnesium Sulfate | MgSO4; 1.7 | ||
| Sodium Bicarbonate | NaHCO3; 24.9 | ||
| Potassium Phosphate (monobasic) | KH2PO4; 1.2 | ||
| Calcium Chloride | CaCL2; 1.4 | ||
| Sodium Pyruvate | Na pyruvate; 2 | ||
| Glucose | C6H12O6; 6 | ||
| Balloon reagents | |||
| Corn Syrup | |||
| Spaghetti | |||
| Silicon Dispersion Gel | |||
| styrofoam block | |||
| lab oven/incubator ( 50C) | |||
| Langendorff Perfusion equipment | |||
| Isolated perfused heart sytem (IH-SR (Hugo-Sachs) or equivalent) | |||
| Data acquisition system (DSI, ADinstruments or equivalent) | |||
| Heated water circulator | |||
| Cooling water circulator | |||
| Perfusion pump capable of 2-30 ml/min | |||
| Inline perfusion filters – 1 um glass fiber | |||
| Pressure sensors and amplifiers for LVP and perfusion pressure | |||
| Small graduated cylinder (~10 mL) | |||
| Small temperature probe and thermometer (Werner or equivalent) | |||
| perfusion resevoir (1L) | |||
| cardioplegia resevoir (~200 mL) | |||
| gas bubbler | |||
| 95/5 O2/CO2 mix | |||
| Surgical tools and reagents | |||
| Metzenbaum and Potz surgical scissors | |||
| two Dumont size 5 forceps | |||
| ketamine | |||
| xylazine | |||
| heparin | |||
| small clamp with soft sides to hold aorta (i.e. terminal clamp with taped ends) | |||
| Silk 2-0 and 4-0 sutures |
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