鼠短轴心脏心脏切片用于电生理研究
Summary
在这里，我们描述了成年小鼠可行心室切片的制备及其对尖锐电极动作电位记录的应用。这些多细胞制剂提供了保留在体内的类似组织结构，这使得它们成为体外电生理和药理学研究的有价值的模型。
Abstract
鼠心肌细胞已广泛用于心脏生理学和新治疗策略的体外研究。然而，解离的心肌细胞的多细胞制剂并不代表心肌细胞，非肌细胞和细胞外基质的复合体内结构，其影响心脏的机械和电生理学性质。在这里，我们描述了一种技术，用于制备具有保留的体内类似组织结构的成年小鼠心脏的活动性心室切片，并且证明其适用于电生理记录。切除心脏后，将心室与心房分离，用含有2,3-丁二酮一肟的无Ca 2+溶液灌注，并包埋在4％ 低熔点琼脂糖块中。将块放置在具有振动叶片的切片机上，并且制备厚度为150-400μm的组织切片，保持振动频率60-70 Hz的刀片速度，并尽可能缓慢地向前移动刀片。切片的厚度取决于进一步的应用。切片储存在冰冷的Tyrode溶液中，用0.9mM Ca 2+和2,3-丁二酮一肟（BDM）处理30分钟。之后，将切片转移到37℃的DMEM中30分钟以洗出BDM。切片可用于具有尖锐电极或微电极阵列的电生理研究，用于力测量分析收缩功能或调查移植的干细胞衍生的心肌细胞和宿主组织的相互作用。对于尖锐的电极记录，将切片置于倒置显微镜的加热板上的3cm细胞培养皿中。用单极电极刺激切片，用锋利的玻璃电极记录切片内心肌细胞的细胞内动作电位。
Introduction
由于Yamamot和Mcllwain于1966年在体外证实了脑片的电活动，所以在基础科学中经常使用薄片切片1 。此后，从脑2 ，肝3 ，肺4和心肌组织5,6,7的切片进行了电生理和药理学研究。 1990年8月描述了来自新生大鼠心脏的心室切片中的第一次膜片钳记录，但是这种技术被遗忘了一段时间。十多年后，我们组织制定了一种新的方法来准备鼠胚胎9 ，新生儿10和成人11心脏切片。这些活组织切片可用于急性实验（成人切片）s可以培养数小时）或短期培养实验（胚胎和新生儿切片可以培养几天）。切片在体内显示如电生理特征和均匀的激发扩散，如通过尖锐电极动作电位和微电极阵列记录所评估的。由于其“二维”形态，它们允许记录电极直接进入心室的所有区域，这使得它们成为电生理调查的有趣工具，并且与Langendorff灌注的整个心脏相比提出了新的实验选择。离子通道阻滞剂如维拉帕米（L型Ca 2+通道阻断剂），利多卡因（Na +通道阻断剂），4-氨基吡啶（非选择性电压依赖性K +通道阻滞剂）和亚麻酸（KCNQ K +通道阻塞） 9，11 </sup>对应于已解离的心肌细胞的已知作用。等轴力测量显示正的力频率关系，强烈建议完整的收缩功能10 。这些研究结果表明，鼠心室切片适合作为生理和药理学研究的体外组织模型。此外，受体心脏的心室切片与尖锐的电极记录结合已经被证明是一个非常有用的工具，用于表征移植胎儿12,13,14和干细胞衍生的15个心肌细胞的电和机械整合以及成熟。
总之，心室切片是一种有价值且成熟的多细胞组织模型，应被认为与分离的心肌细胞和Langendorff灌注心脏互补在心血管研究中，提供体内类似组织结构（与分离的细胞相反）的主要优点，以及直接访问诸如尖锐电极记录到心脏的所有区域的测量技术（与全心脏制剂相反）。
Protocol
Representative Results
Discussion
心室切片使电生理，药理和机械研究具有体内保留的组织结构，并可将测量技术直接进入心脏的所有区域。已经在胚胎，新生儿和成人切片9,10,11中证明了生理作用潜力。生物活性染色证实切片的活力除了切片程序直接损伤的表层外，其余的活性染色9 。使用尖锐玻璃电极的切片内的动作电位记录可用于研究如本文所述的移植心肌细胞的整合和成熟，或加入心脏活性?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们承认研讨会和神经生理学研究所的动物设施提供的支持。这项工作得到了Walter und Marga Boll-Stiftung，KölnFortune和Dezscher Stfung Herzforschung的支持。
Materials
| Leica VT 1000s | Leica Microsystems, Wetzlar, Germany | Microtome with vibrating blade. | |
| Stainless Steel Blades | Campden Instruments, Loughborough, England | 7550-1-SS | |
| Pasteur pipettes | Sigma-Aldrich, St. Louise, USA | Z627992 | |
| Fine brush, e.g. size 6 (4/32") | VWR, International, Radnor, USA | 149-2125 | |
| Preparation table | self made | ||
| Molt for embedding ventricles in agarose | self made | ||
| 1 ml Syringe | Becton, Dickinson; Franklin Lakes, USA | 300013 | |
| 27Gx3/4“ Needles | Braun, Melsungen, Germany | 4657705 | |
| 20G 11/2“ Needles | 4657519 | ||
| Small scissor | WPI, Sarasota, USA | 501263 | |
| Tweezers #5, 0.1 x 0.06 mm tip | WPI, Sarasota, USA | 500342 | |
| Oxygen gas (medical grade O2) | Linde, Munich, Germany | ||
| Carbogen gas (95 % O2, 5 % CO2) | Linde, Munich, Germany | ||
| NaCL | Sigma-Aldrich, St. Louise, USA | 7647-14-5 | |
| KCL | Sigma-Aldrich, St. Louise, USA | 746436 | |
| CaCl2 | Sigma-Aldrich, St. Louise, USA | 746495 | |
| KH2PO4 | Sigma-Aldrich, St. Louise, USA | NIST200B | |
| HEPES | Sigma-Aldrich, St. Louise, USA | 51558 | |
| NaHCO3 | Sigma-Aldrich, St. Louise, USA | S5761 | |
| D(+)-Glucose | Sigma-Aldrich, St. Louise, USA | G8270 | |
| MgSO4 | Sigma-Aldrich, St. Louise, USA | M7506 | |
| NaOH | Sigma-Aldrich, St. Louise, USA | S8045 | |
| Cyanoacrylate glue | Henkel, Düsseldorf, Germany | ||
| Low-melt Agarose | Roth, Karlsruhe, Germany | 6351.2 | |
| Heparin-sodium-25000 I.E./5mL | Ratiopharm, Ulm, Germany | ||
| Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX | ThermoScientific, Waltham, USA | 10566016 | |
| SEC-10LX Amplifier | npi electronic GmbH, Tamm, Germany | SEC-10LX | |
| EPC 9 | HEKA Elektronik GmbH, Lambrecht, Germany | ||
| Zeiss Axiovert 200 | Zeiss, Oberkochen, Germany | ||
| Low magnification Micromanipulator | Narashige, Tokyo, Japan | Nm-3 | |
| High magnification, three-axis micromanipulator | Narashige, Tokyo, Japan | MHW-3 | |
| Peristaltic perfusion pump | Multi Channel Systems, Reutlingen, Germany | PPS2 | |
| 2-channel temperature controller | Multi Channel Systems, Reutlingen, Germany | TCO02 | |
| Square pulse stimulator | Natus Europe GmbH, Planegg, Germany | Grass SD9 | |
| Glass capillaries | WPI, Sarasota, USA | 1B150F-1 |
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