从孤立的大鼠心脏的间质渗出物收集的倒置心脏模型
Summary
该方案描述了从分离的灌注大鼠心脏中收集心脏间质液的方法。为了将间质渗出物与冠状静脉流出物灌注液物理分离，Langendorff灌注的心脏被倒置，并且使用软胶乳帽收集在心脏表面上形成的渗出物（间质液体）。
Abstract
本协议描述了一种独特的方法，可以从分离的盐水灌注的大鼠心脏中收集心脏渗出物（CT）。根据Langendorff技术分离和逆行灌注心脏后，将心脏倒置为倒立位置，并通过插入左心室的气囊导管进行机械稳定。然后，预先浇铸以匹配大鼠心脏的平均尺寸的薄胶乳盖被放置在心外膜表面上。胶乳盖的出口连接到硅管，远端开口比心脏底部高10厘米，产生轻微的吸力。在心外膜表面连续生产的CT被收集在冰冷的小瓶中用于进一步分析。对照组和梗死心脏CT值分别为17〜147μL/ min（n = 14），占冠状静脉血液灌注液0.1-1％。蛋白质组学分析和高度信息rmance液相色谱（HPLC）显示收集的CT含有广谱的蛋白质和嘌呤能代谢物。
Introduction
心衰（HF）是世界人类死亡的主要原因1 。 HF经常发生因心肌炎，心肌缺血损伤和左心室重塑，导致心脏收缩功能和患者生活质量逐渐恶化。虽然心脏病学和心脏手术的进步显着降低了HF死亡率，但它们仅仅是一个不可避免的进步性疾病过程的短暂的“延迟者”，具有显着的发病率。因此，目前缺乏有效的治疗强调需要鉴定可预防甚至逆转HF的新型分子靶标。这包括细胞外基质的改变，不受控的心脏免疫反应以及心脏和非心脏细胞之间的相互作用2 。
重要的是要认识到心脏细胞暴露于微环境的微环境tly塑造受伤心脏的免疫和再生反应。在分离的生理盐水灌注的心脏中，在生理和病理生理条件3,4,5下，以来自间质液空间（ 即微环境）的小液滴的形式在心脏表面产生CT。因此，CT（ 即组织间液）的分析可能有助于识别调节心脏代谢和收缩功能的因素6或影响移入受伤心脏后的免疫细胞功能。潜在地，这可能导致开发用于治疗HF的新型治疗策略。
从小鼠心脏收集CT在技术上是具有挑战性的。在常规Langendorff灌注的心脏中，CT的独家收集是困难的，因为CT与冠状动脉的混合静脉流出物灌注液不可预测地稀释从间质空间释放的任何浓度的代谢物/酶。克服这个限制的一个可能的策略是通过插入肺并同时连接肺静脉来排除静脉流出物7 。然而，这种方法面临与肺动脉和静脉的插管和结扎有关的困难，导致静脉流出物潜在渗漏到心脏渗出物中。使用反向心脏模型的概念首先由Kammermeier组引进，Kammermeier将其将分离的灌注心脏倒置到倒置位置，并将薄胶乳帽放置在心外膜表面上，以连续取样CT，而不会导致静脉流出物的污染8，9 。使用该程序，CT显示提供了从心脏9释放的代谢物的非常敏感的测量值，脂肪酸8和病毒颗粒10的毛细管转运。
最近，可能调节局部免疫应答和增加心脏血管生成的旁分泌因子11已经涉及干细胞治疗心脏病的有益作用。逆转心脏中CT的分析可能有助于化学鉴定这些个别旁分泌因子。此外，CT可能有助于确定心脏中免疫细胞体内活化所涉及的因素。
在这里提供的心脏表面CT收集的详细描述对于研究免疫细胞，成纤维细胞，内皮细胞和心肌细胞相对于整体心脏功能的相互作用的研究来说是实验性的。如上所述，间质液携带心脏内细胞间细胞通讯的信息，可以方便地通过收集CT来评估。详细的技术说明，包括如何从反向心脏收集CT的视频协议，应有助于今后应用这种独特的技术。
Protocol
Representative Results
Discussion
逆心模型基于已建立的Langendorff心脏灌注技术12，并且通过简单地将心脏逆转到颠倒位置并使用刚性心室内球囊导管来保持该位置来执行。以这种方式，心脏间质渗出物可以与冠心静脉流出物灌注液物理分离，通过重力从心脏9的底部滴落。 CT可以通过放置在整个心脏表面上的薄而柔软的乳胶帽来连续收集。
该方法易于执行，除了Langendorff设?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本研究由NSFC 81570244，FoKo 23/2013和SFB 1116 / B01以及杜塞尔多夫心血管研究所（CARID）资助。
Materials
| Latex Solution | ProChemie | Z-Latex LA-TZ | http://kautschukgesellschaft.de/%E2%80%A8z-latexla-tz%E2%80%A8 |
| Aluminum Mold | Home made | – | Reverse heart model |
| Universal Ovens | Memmert | UNB 400 | Reverse heart model |
| Latex Balloon | Hugo Sachs | Size 4 | Reverse heart model |
| Milling Machine | Proxxon | MF70 | Reverse heart model |
| Sodium Chloride | Sigma | SZBD0810V | Chemicals |
| Sodium Hydrogen Carbonate | Roth | 68852 | Chemicals |
| Potassium Chloride | Merck | 49361 | Chemicals |
| Magnesium Sulphate Heptahydrate | Merck | 58861 | Chemicals |
| Potassium Dihydrogen Phosphate | Merck | 48731 | Chemicals |
| D(+)-Glucose Anhydrous | Merck | 83371 | Chemicals |
| Calcium Chloride Dihydrate | Fluka | 21097 | Chemicals |
| Balance | VWR | SE 1202 | Weighing chemicals |
| Double Distilled Water | Millpore | – | Disolving chemicals |
| Medical Pressure Transducer | Gold | – | Langendorff apparatus |
| Medical Flow Probe | Transonic | 3PXN | Langendorff apparatus |
| Heating Circulating Bath | Haake | B3 ; DC1 | Langendorff apparatus |
| Laboratory and Vaccum Tubing | Tygon | R-3603 | Langendorff apparatus |
| Animal Research Flowmeters | Transonic | T206 | Langendorff apparatus |
| PowerLab Data Acquisition Device | AD Instruments | Chart 7.1 | Langendorff apparatus |
| LabChart Data Acquisition Software | AD Instruments | Chart 7.1 | Langendorff apparatus |
| Peristaltic Pump | Glison | MINIPULS 3 | Langendorff apparatus |
| Glass Water Column | home made | – | Langendorff apparatus |
| Water Bath Protective Agent | VWR | 462-7000 | Langendorff apparatus |
| Sterile Disposable Filters (0.2µm) | Thermo Scientific | 595-4520 | Langendorff apparatus |
| Blood gas analyzers | Radiometer | ABL90 FLEX PLUS | Gas analyzer |
| 70% ethanol | VWR | UN1170 | Cleaning tubings |
| 100% ethanol | Merck | 64-17-5 | Cleaning tubings |
| Wistar Rats | Janvier | – | Animals |
| Stainless Scissors | AESCULAP | BC702R | Surgical Instruments |
| Stainless Scissors | AESCULAP | BC257R | Surgical Instruments |
| Big Forceps | AESCULAP | – | Surgical Instruments |
| 8m/m Stainless Forceps | F.S.T | 11052-10 | Surgical Instruments |
| superfine (10/0) emery paper | 3M | 051111-11694 | Reverse heart model |
Riferimenti
- Henkel, D. M., Redfield, M. M., Weston, S. A., Gerber, Y., Roger, V. L. Death in heart failure: a community perspective. Circ Heart Fail. 1 (2), 91-97 (2008).
- Limana, F., et al. Myocardial infarction induces embryonic reprogramming of epicardial c-kit(+) cells: role of the pericardial fluid. J Mol Cell Cardiol. 48 (4), 609-618 (2010).
- Brunner, F. Cardiac tissue endothelin-1 levels under basal, stimulated, and ischemic conditions. J Cardiovasc Pharmacol. 26, S44-S46 (1995).
- de Lannoy, L. M., et al. Renin-angiotensin system components in the interstitial fluid of the isolated perfused rat heart. Local production of angiotensin I. Hypertension. 29 (6), 1240-1251 (1997).
- Strupp, M., Kammermeier, H. Interstitial Lactate And Glucose-Concentrations Of the Isolated-Perfused Rat-Heart before, during And after Anoxia. Pflugers Arch. 423 (3-4), 232-237 (1993).
- Wienen, W., Jungling, E., Kammermeier, H. Enzyme-Release into the Interstitial Space of the Isolated Rat-Heart Induced by Changes in Contractile Performance. Cardiovasc Res. 28 (8), 1292-1298 (1994).
- De Deckere, E. A., Ten Hoor, ., P, A modified Langendorff technique for metabolic investigations. Pflugers Arch. 370 (1), 103-105 (1977).
- Tschubar, F., Rose, H., Kammermeier, H. Fatty acid transfer across the myocardial capillary wall. J Mol Cell Cardiol. 25 (4), 355-366 (1993).
- Wienen, W., Kammermeier, H. Intra- and extracellular markers in interstitial transudate of perfused rat hearts. Am J Physiol. 254 (4 Pt 2), H785-H794 (1988).
- Sasse, A., Ding, Z. P., Wallich, M., Godecke, A., Schrader, J. Vascular transfer of adenovirus is augmented by nitric oxide in the rat heart. Am J Physiol Heart Circ Physiol. 287 (3), H1362-H1368 (2004).
- Gnecchi, M., Zhang, Z., Ni, A., Dzau, V. J. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 103 (11), 1204-1219 (2008).
- Herr, D. J., Aune, S. E., Menick, D. R. Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts. J Vis Exp. (101), e52908 (2015).
- Ding, Z., et al. Epicardium-Derived Cells Formed After Myocardial Injury Display Phagocytic Activity Permitting In Vivo Labeling and Tracking. Stem Cells Transl Med. 5 (5), 639-650 (2016).
- Hartwig, S., et al. Secretome profiling of primary human skeletal muscle cells. Biochim Biophys Acta. 1844 (5), 1011-1017 (2014).
- Smolenski, R. T., Lachno, D. R., Ledingham, S. J. M., Yacoub, M. H. Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J Chromatogr. 527 (2), 414-420 (1990).
- Decking, U. K., Juengling, E., Kammermeier, H. Interstitial transudate concentration of adenosine and inosine in rat and guinea pig hearts. Am J Physiol. 254 (6 Pt 2), H1125-H1132 (1988).
- Heller, L. J., Mohrman, D. E. Estimates of interstitial adenosine from surface exudates of isolated rat hearts. J Mol Cell Cardiol. 20 (6), 509-523 (1988).
