心内神经元对体外 Langendorff 系统心脏电生理和心脏交感神经的影响
1Department of Cardiology-Electrophysiology, cNEP (cardiac Neuro- and Electrophysiology research group), University Heart Center,University Hospital Hamburg-Eppendorf, 2DZHK (German Center for Cardiovascular Research), 3Institute of Experimental Cardiovascular Research,University Medical Center Hamburg-Eppendorf
Summary
在这里, 我们提出了一个调节心内自主神经系统的协议, 并评估其对基本的电生理, 心脏交感神经和阵营动力学的影响使用前体 Langendorff 设置.
Abstract
自19晚期th世纪的发明以来, Langendorff前体心脏灌注系统继续是研究广泛的生理、生物化学、形态学和药理学参数的相关工具。中枢失神心。在这里, 我们描述了调节心脏内自主神经系统的设置和评估其对基本电生理, 心脏交感神经和循环腺苷 (阵营) 动力学的影响。心内自主神经系统由心房脂肪垫的机械解剖调节–小鼠神经节主要位于–或使用全球以及靶向的药理干预。将 octapolar 电生理导管引入右心房和右心室, 用心外膜放置的多电极阵列进行高分辨率映射, 以确定心脏电生理和心脏交感神经。Förster 共振能量转移 (焦虑) 成像进行实时监测的营地水平在不同的心脏地区。采用神经标记法对 Neuromorphology 的全心抗体染色方法进行研究, 以指导心脏自主神经系统特定靶点的识别和调制。前体 Langendorff 设置允许在短时间内进行大量的可重现实验。然而, 设置的部分开放性质 (e. g) 使恒温控制变得困难, 应保持在最低限度。该方法可以对分散心内心脏自主神经系统进行分析和调节。
Introduction
Langendorff 体外心脏灌注系统继续是一个相关的工具, 以执行广泛的生理, 生物化学, 形态学和药理学研究中枢失神经心脏 1,2 ,3,4,5自其发明在后期 19th世纪6。迄今为止, 该系统仍然广泛用于各种主题 (e. g, 缺血再灌注) 或研究心脏药理作用7,8, 是心血管研究的基本工具。这种方法的长寿源于几个优点 (e. g), 测量结果不受中枢神经系统或其他器官、系统循环或循环激素的影响。如有需要, 可以以控制方式将药物添加到灌注缓冲器或直接应用于特定结构。实验是可重现的, 在很短的时间内可以进行比较高的实验。(部分) 开放性质的设置可以使温度调节困难, 需要考虑。虽然 Langendorff 系统也用于较大的物种9, 但较小的动物主要用于实验性的设置不太复杂, 更大的生物变异性 (g., 转基因小鼠模型) 可以使用。
在本协议的实验设置中, 心内自主神经系统对基本电生理参数、心室心脏交感神经、心外膜传导、环腺苷 (阵营) 动力学的影响是评价。大量的心内神经节, 主要位于心房脂肪垫, 现在众所周知的控制心脏电生理独立于中枢神经控制, 要么保持完好或手动删除与仔细机械夹层。对自主神经系统的药理调节, 要么通过向灌注缓冲器添加药物, 要么在局部通过心房神经节的靶向调节来进行。实验后, 心脏是非常适合的免疫组织化学评估, 因为所有的血细胞已被删除由于连续灌注, 这可以提高染色质量。
所述技术的总体目标是为详细研究自主神经系统对心脏电生理和心脏交感神经的影响提供新的视角。使用这种技术的一个原因是, 在没有中枢神经系统影响的情况下, 可以研究和改变自主神经系统。一个主要的好处是容易的就业的药理实验, 其中潜在的亲或抗心律失常性质的新旧的药物可以测试。此外, 各种心脏疾病的转基因和击倒老鼠模型可以用来研究心律失常、心力衰竭或代谢性疾病的机制。这种方法增强了我们对心房的自主神经系统对心室心脏电生理的影响以及心律失常的诱导的理解。
Protocol
所有涉及动物的程序均由汉堡州、汉堡大学动物保育和使用委员会的地方当局批准。 1. Langendorff 装置的研制 注: 可使用商用 Langendorff 灌注系统。 制备改性克雷布斯-Henseleit 溶液 (119 毫米氯化钠、25毫米碳酸氢钠、4.6 毫米氯化钾、1.2 毫米磷酸钾磷酸钙、1.1 毫米硫酸镁、2.5 毫米氯化钙、8.3 毫米葡萄糖和2毫米钠)丙酮酸)。将 95% oxygen/5rbon 的混合物?…
Representative Results
图 1显示了 Langendorff 设置的映像, 其中包括2个多电极阵列 (多边环境协定)。实验前, 心内导管定位在套管附近, 方便快速方便地插入右心房/右心室, 确保在平衡开始之前有很短的时间。房间的下部可以提高 (请参见图 1中的箭头), 以使会议厅完全闭合并保证稳定的温度。 <strong class="xfi…
Discussion
本论文通过不同的制图和刺激技术, 提出了 Langendorff 的体外心脏灌注系统作为研究心内神经元对心脏电生理和心脏交感神经的影响的工具. 包括心内膜和心外膜的方法。
协议的几个部分对于安装来说是至关重要的。首先, 重要的是使用一种制备技术, 其中心房脂肪垫保持完好或迅速删除, 而不伤害心肌。第二, 适当大小的开放必须削减在右心房, 以方便插入 octapolar 导管进…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 Hartwig Wieboldt 出色的技术援助, 以及汉堡-离心大学医学中心的 UKE 显微成像设施 (Umif) 提供显微镜和支持。这项研究是由 Förderverein des Universitären Herzzentrums 汉堡 e 和 DZHK (德国心血管研究中心) [FKZ 81Z4710141] 资助的。
Materials
| Sodium chloride | Sigma-Aldrich | S3014 | Modified Krebs-Henleit solution |
| Sodium hydrogencarbonate | Sigma-Aldrich | 401676 | Modified Krebs-Henleit solution |
| Potassium chloride | Sigma-Aldrich | P5405 | Modified Krebs-Henleit solution |
| Potassium phosphate monobasic | Sigma-Aldrich | P5655 | Modified Krebs-Henleit solution |
| Magnesium sulfate heptahydrate | Sigma-Aldrich | M1880 | Modified Krebs-Henleit solution |
| Calcium chloride dihydrate | Sigma-Aldrich | C7902 | Modified Krebs-Henleit solution |
| Glucose | Sigma-Aldrich | G5767 | Modified Krebs-Henleit solution |
| Sodium pyruvate bioXtra | Sigma-Aldrich | P8574 | Modified Krebs-Henleit solution |
| Carbogen (95% O2 / 5% CO2) | SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany | Modified Krebs-Henleit solution | |
| Sterile filter steritop-GP 0.22 | EMD Millipore | SCGPT05RE | Modified Krebs-Henleit solution |
| Atropine sulfate | Sigma-Aldrich | A0257 | Neuromodulation |
| Hexamethonium chloride | Sigma-Aldrich | H2138 | Neuromodulation |
| Nicotine free base 98-100% | Sigma-Aldrich | N3876 | Neuromodulation |
| Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | Whole mount staining |
| Tris(hydroxymethyl)aminomethane | Sigma-Aldrich | 252859 | Whole mount staining |
| Methanol | Sigma-Aldrich | 34860 | Whole mount staining |
| Hydrogen peroxide solution 30% (w/w) in H2O | Merck, KGA, Darmstadt, Germany | H1009 | Whole mount staining |
| Dimethyl sulfoxide | Merck, KGA, Darmstadt, Germany | D8418 | Whole mount staining |
| Phosphate-buffered saline tablets | Gibco / Invitrogen | 18912-014 | Whole mount staining |
| Triton-x-100 | Sigma-Aldrich | T8787 | Whole mount staining |
| Albumin bovine fraction V | Biomol, Hamburg, Germany | 11924.03 | Whole mount staining |
| Chicken anti neurofilament | EMD Millipore | AB5539 | Whole mount staining |
| Rabbit anti tyrosine hydroxylase | EMD Millipore | AB152 | Whole mount staining |
| Goat anti choline acetyltransferase | EMD Millipore | AP144P | Whole mount staining |
| Donkey α rabbit IgG Alexa 488 | Thermo Fisher Scientific | A21206 | Whole mount staining |
| Donkey α goat IgG Alexa 568 | Thermo Fisher Scientific | A11057 | Whole mount staining |
| Donkey α chicken IgY Alexa 647 | Merck, KGA, Darmstadt, Germany | AP194SA6 | Whole mount staining |
| Biotin-conjugated donkey α rabbit igG | R&D Systems | AP182B | Whole mount staining |
| Biotin-conjugated donkey α goat igG | R&D Systems | AP192P | Whole mount staining |
| Biotin-conjugated goat α chicken igY | R&D Systems | BAD010 | Whole mount staining |
| Vectashield mounting medium | Vector laboratories, Burlingame, CA, USA | H-1000 | Immunohistochemistry |
| Vectastain ABC kit | Vector laboratories, Burlingame, CA, USA | PK-4000 | Immunohistochemistry |
| Steady DAB/Plus | Abcam plc, Cambridge, UK | ab103723 | Whole mount staining |
| HistoClear | DiaTec, Bamberg, Germany | HS2002 | Immunohistochemistry |
| BisBenzimide H33342 trihydrochloride (Hoechst) | Sigma-Aldrich, St. Louis, MO, USA | B2261 | Immunohistochemistry |
| Vectashield HardSet mounting medium | Vector laboratories, Burlingame, CA, USA | VEC-H-1400 | Immunohistochemistry |
| Perfusion system | HUGO SACHS ELEKTRONIK – HARVARD APPARATUS GmbH, March-Hugstetten, Germany | 73-4343 | Langendorff apparatus |
| Data acquisition system and corresponding software for catheter and physiological parameter | Powerlab 8/30 & Labchart, ADInstruments, Dunedin, New Zealand | PL3508 PowerLab 8/35 | Langendorff setup |
| Octapolar catheter | CIB’ER Mouse, NuMed Inc., Hopkinton, NY, USA | custom | Langendorff setup |
| Stimulus generator | STG4002, Multi Channel Systems, Reutlingen, Germany | STG4002-160µA | Stimulation setup |
| Stimulation software | Multi Channel Systems, Reutlingen, Germany | MC_Stimulus II | Stimulation setup |
| Data acquisition system and corresponding software for epicardial electrograms | ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany | USB-ME128-System | MEA setup |
| Multi-electrode array | MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany | EcoFlexMEA36 | MEA setup |
| Multi-electrode array recording software | Multi Channel Systems, Reutlingen, Germany | MC_Rack | MEA setup |
| Spring scissors | Fine Science Tools GmbH, Heidelberg, Germany | 15003-08 | Heart Preparation |
| Strabismus Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14575-09 | Heart Preparation |
| Mayo Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14110-15 | Heart Preparation |
| Dumont SS Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11203-25 | Heart Preparation |
| London Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11080-02 | Heart Preparation |
| Narrow Pattern Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11003-13 | Heart Preparation |
| Plastic Wrap | Parafilm M, Bemis NA, based in Neenah, WI, United States | Consumable Materials | |
| Stereomicroscope | Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany | FRET | |
| LED | CoolLED, Andover, UK | pE-100 | FRET |
| DualView | Photometrics, Tucson, AZ, USA | DV2-SYS | FRET |
| DualView filter set | Photometrics, Tucson, AZ, USA | 05-EM | FRET |
| optiMOS scientific CMOS camera | Qimaging, Surrey, BC, Canada | 01-OPTIMOS-R-M-16-C | FRET |
| Imaging software | Micro-Manager; Vale Lab, University of California San Francisco, CA, USA | FRET | |
| Analysis Software | Image J software; Public Domain, NIH, USA | FRET | |
Referências
- Bell, R. M., Mocanu, M. M., Yellon, D. M. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. Journal of Molecular and Cellular Cardiology. 50 (6), 940-950 (2011).
- Sutherland, F. J., Hearse, D. J. The isolated blood and perfusion fluid perfused heart. Pharmacological Research. 41 (6), 613-627 (2000).
- Hearse, D. J., Sutherland, F. J. Experimental models for the study of cardiovascular function and disease. Pharmacological Research. 41 (6), 597-603 (2000).
- Valentin, J. P., Hoffmann, P., De Clerck, F., Hammond, T. G., Hondeghem, L. Review of the predictive value of the Langendorff heart model (Screenit system) in assessing the proarrhythmic potential of drugs. Journal of Pharmacological and Toxicological Methods. 49 (3), 171-181 (2004).
- Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. Isolated heart perfusion according to Langendorff-still viable in the new millennium. Journal of Pharmacological and Toxicological Methods. 55 (2), 113-126 (2007).
- Langendorff, O. Investigation of the living mammalian heart. Pflügers Archiv. 61, 291-332 (1895).
- Matsumoto-Ida, M., Akao, M., Takeda, T., Kato, M., Kita, T. Real-time 2-photon imaging of mitochondrial function in perfused rat hearts subjected to ischemia/reperfusion. Circulation. 114 (14), 1497-1503 (2006).
- Rassaf, T., Totzeck, M., Hendgen-Cotta, U. B., Shiva, S., Heusch, G., Kelm, M. Circulating nitrite contributes to cardioprotection by remote ischemic preconditioning. Circulation Research. 114 (10), 1601-1610 (2014).
- Schechter, M. A., et al. An isolated working heart system for large animal models. Journal of Visualized Experiments. 88 (88), 51671 (2014).
- Stockigt, F., et al. Total beta-adrenoceptor knockout slows conduction and reduces inducible arrhythmias in the mouse heart. PLoS One. 7 (11), e49203 (2012).
- Berul, C. I. Electrophysiological phenotyping in genetically engineered mice. Physiological Genomics. 13 (3), 207-216 (2003).
- Curtis, M. J., et al. The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias. Pharmacology & Therapeutics. 139 (2), 213-248 (2013).
- Schrickel, J. W., et al. Enhanced heterogeneity of myocardial conduction and severe cardiac electrical instability in annexin A7-deficient mice. Cardiovascular Research. 76 (2), 257-268 (2007).
- Rudolph, V., et al. Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation. Nature Medicine. 16 (4), 470-474 (2010).
- Jungen, C., et al. Disruption of cardiac cholinergic neurons enhances susceptibility to ventricular arrhythmias. Nature Communications. 8, 14155 (2017).
- Calebiro, D., et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biology. 7 (8), e1000172 (2009).
- Sprenger, J. U., Perera, R. K., Götz, K. R., Nikolaev, V. O. FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors. Journal of Visualized Experiments. (66), e4081 (2012).
- Alanentalo, T., et al. Tomographic molecular imaging and 3D quantification within adult mouse organs. Nature Methods. 4 (1), 31-33 (2007).
- Whittington, N. C., Wray, S. Suppression of red blood cell autofluorescence for immunocytochemistry on fixed embryonic mouse tissue. Current Protocols in Neuroscience. 81, 2.28.1-2.28.12 (2017).
- Fukuda, K., Kanazawa, H., Aizawa, Y., Ardell, J. L., Shivkumar, K. Cardiac innervation and sudden cardiac death. Circulation Research. 116 (12), 2005-2019 (2015).
- Wengrowski, A. M., Wang, X., Tapa, S., Posnack, N. G., Mendelowitz, D., Kay, M. W. Optogenetic release of norepinephrine from cardiac sympathetic neurons alters mechanical and electrical function. Cardiovascular Research. 105 (2), 143-150 (2015).
- Rivinius, R., et al. Control of cardiac chronotropic function in patients after heart transplantation: effects of ivabradine and metoprolol succinate on resting heart rate in the denervated heart. Clinical Research in Cardiology. , (2017).
- Ajijola, O. A., et al. Augmentation of cardiac sympathetic tone by percutaneous low-level stellate ganglion stimulation in humans: a feasibility study. Physiological Reports. 3 (3), e12328 (2015).
Citar este artigo
Jungen, C., Scherschel, K., Bork, N. I., Kuklik, P., Eickholt, C., Kniep, H., Klatt, N., Willems, S., Nikolaev, V. O., Meyer, C. Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System. J. Vis. Exp. (135), e57617, doi:10.3791/57617 (2018).