This study was funded by NSFC 81570244, FoKo 23/2013, and SFB 1116/B01 and by the Cardiovascular Research Institute D&#252;sseldorf (CARID).

All experiments were approved by the local regulatory agency (LANUV of Nordrhein-Westfalen, Germany) and were performed according to the guidelines of animal use. Animals were fed with a standard chow diet and received tap water ad libitum. All equipment and chemicals necessary to each step of the experiment are available in the Table of Materials.
1. Preparation of the Latex Cap and Intraventricular Balloon
<ol>
	<li>Make an aluminum mold using a milling m...

<ol>
	<li>Henkel, D. M., Redfield, M. M., Weston, S. A., Gerber, Y., Roger, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+in+heart+failure:+a+community+perspective.">Death in heart failure: a community perspective.</a> Circ Heart Fail. 1 (2), 91-97 (2008).</li><li>Limana, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+induces+embryonic+reprogramming+of+epicardial+c-kit(+)+cells:+role+of+the+pericardial+fluid.">Myocardial infarction induces embryonic reprogramming of epicardial c-kit(+) cells: role of the pericardial fluid.</a> J Mol Cell Cardiol. 48 (4), 609-618 (2010).</li><li>Brunner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+endothelin-1+levels+under+basal,+stimulated,+and+ischemic+conditions.">Cardiac tissue endothelin-1 levels under basal, stimulated, and ischemic conditions.</a> J Cardiovasc Pharmacol. 26, S44-S46 (1995).</li><li>de Lannoy, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renin-angiotensin+system+components+in+the+interstitial+fluid+of+the+isolated+perfused+rat+heart.+Local+production+of+angiotensin+I.">Renin-angiotensin system components in the interstitial fluid of the isolated perfused rat heart. Local production of angiotensin I.</a> Hypertension. 29 (6), 1240-1251 (1997).</li><li>Strupp, M., Kammermeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+Lactate+And+Glucose-Concentrations+Of+the+Isolated-Perfused+Rat-Heart+before,+during+And+after+Anoxia.">Interstitial Lactate And Glucose-Concentrations Of the Isolated-Perfused Rat-Heart before, during And after Anoxia.</a> Pflugers Arch. 423 (3-4), 232-237 (1993).</li><li>Wienen, W., Jungling, E., Kammermeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-Release+into+the+Interstitial+Space+of+the+Isolated+Rat-Heart+Induced+by+Changes+in+Contractile+Performance.">Enzyme-Release into the Interstitial Space of the Isolated Rat-Heart Induced by Changes in Contractile Performance.</a> Cardiovasc Res. 28 (8), 1292-1298 (1994).</li><li>De Deckere, E. A., Ten Hoor, ., P, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+Langendorff+technique+for+metabolic+investigations.">A modified Langendorff technique for metabolic investigations.</a> Pflugers Arch. 370 (1), 103-105 (1977).</li><li>Tschubar, F., Rose, H., Kammermeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+transfer+across+the+myocardial+capillary+wall.">Fatty acid transfer across the myocardial capillary wall.</a> J Mol Cell Cardiol. 25 (4), 355-366 (1993).</li><li>Wienen, W., Kammermeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+extracellular+markers+in+interstitial+transudate+of+perfused+rat+hearts.">Intra- and extracellular markers in interstitial transudate of perfused rat hearts.</a> Am J Physiol. 254 (4 Pt 2), H785-H794 (1988).</li><li>Sasse, A., Ding, Z. P., Wallich, M., Godecke, A., Schrader, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+transfer+of+adenovirus+is+augmented+by+nitric+oxide+in+the+rat+heart.">Vascular transfer of adenovirus is augmented by nitric oxide in the rat heart.</a> Am J Physiol Heart Circ Physiol. 287 (3), H1362-H1368 (2004).</li><li>Gnecchi, M., Zhang, Z., Ni, A., Dzau, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+mechanisms+in+adult+stem+cell+signaling+and+therapy.">Paracrine mechanisms in adult stem cell signaling and therapy.</a> Circ Res. 103 (11), 1204-1219 (2008).</li><li>Herr, D. J., Aune, S. E., Menick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+Assessment+of+Ischemia-reperfusion+Injury+in+Langendorff-perfused+Rat+Hearts.">Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts.</a> J Vis Exp. (101), e52908 (2015).</li><li>Ding, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-Derived+Cells+Formed+After+Myocardial+Injury+Display+Phagocytic+Activity+Permitting+In+Vivo+Labeling+and+Tracking.">Epicardium-Derived Cells Formed After Myocardial Injury Display Phagocytic Activity Permitting In Vivo Labeling and Tracking.</a> Stem Cells Transl Med. 5 (5), 639-650 (2016).</li><li>Hartwig, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+profiling+of+primary+human+skeletal+muscle+cells.">Secretome profiling of primary human skeletal muscle cells.</a> Biochim Biophys Acta. 1844 (5), 1011-1017 (2014).</li><li>Smolenski, R. T., Lachno, D. R., Ledingham, S. J. M., Yacoub, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J Chromatogr. 527 (2), 414-420 (1990).</li><li>Decking, U. K., Juengling, E., Kammermeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+transudate+concentration+of+adenosine+and+inosine+in+rat+and+guinea+pig+hearts.">Interstitial transudate concentration of adenosine and inosine in rat and guinea pig hearts.</a> Am J Physiol. 254 (6 Pt 2), H1125-H1132 (1988).</li><li>Heller, L. J., Mohrman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+interstitial+adenosine+from+surface+exudates+of+isolated+rat+hearts.">Estimates of interstitial adenosine from surface exudates of isolated rat hearts.</a> J Mol Cell Cardiol. 20 (6), 509-523 (1988).</li></ol>

The reversed-heart model is based on the well-established Langendorff heart perfusion technique12 and is performed by simply inverting the heart into an upside-down position and holding this position using a rigid intra-ventricular balloon catheter. In such a way, cardiac interstitial transudate can be physically separated from coronary venous effluent perfusate, dripping by gravity from the base of the heart9. The CT can be continuously collected by means of a thin and fle...

Heart failure (HF) is the leading cause of death in humans worldwide1. HF often occurs because of myocarditis, ischemic insults to the myocardium, and left ventricular remodeling, leading to the progressive deterioration of cardiac contractile function and patients&#39; quality of life. Although advances in cardiology and cardiac surgery have remarkably lowered HF mortality, they merely serve as transient &#34;delayers&#34; of an inevitably progressive disease process that carries signi&#64257;cant morbidity. Therefore, the current lack of effective treatment underscores the need to identify novel molecular targets that can prevent or even reverse HF. This includes changes in the extracellular matrix, uncontrolled cardiac immune response, and interactions between cardiac and non-cardiac cells2.
It is important to recognize that the microenvironment that cardiac cells are exposed to directly shapes the immune and regenerative response of the injured heart. In the isolated, saline-perfused heart, CT is generated on the heart surface in the form of small droplets that are derived from the interstitial fluid space (i.e., microenvironment), both under physiological and pathophysiological conditions3,4,5. Therefore, analysis of the CT (i.e., interstitial fluid) may help to identify factors that regulate cardiac metabolism and contractile function6 or influence immune cell functions after migration into the injured heart. Potentially, this may lead to the development of novel therapeutic strategies for the treatment of HF.
The collection of CT from murine hearts is technically challenging. In regular Langendorff-perfused hearts, the exclusive collection of CT is difficult because the mixture of the CT with coronary venous effluent perfusate unpredictably dilutes any concentration of metabolites/enzymes released from the interstitial space. One possible strategy to overcome this limitation is to exclude the venous effluent by cannulating the pulmonary and simultaneously ligating the pulmonary vein7. However, this method faces difficulties associated with the cannulation and ligation of the pulmonary artery and vein, causing potential leakage of venous effluent into the cardiac transudate. The concept of using a reverse heart model was first introduced by the group of Kammermeier, who inverted the isolated, perfused heart into an upside-down position and placed a thin latex cap on the epicardial surface to continuously sample CT without the contamination of venous effluent8,9. Using this procedure, CT was shown to provide a very sensitive measure of the metabolites released from the heart9, the capillary transfer of fatty acids8, and viral particles10.
More recently, paracrine factors that may regulate the local immune response and augment cardiac angiogenesis11 have been implicated in the beneficial effects of stem cell-based therapy for heart disease. The analysis of CT in the reversed heart may help to chemically identify these individual paracrine factors. In addition, CT may help to identify the factors involved in the in vivo activation of immune cells in the heart.
The detailed description of CT collection from the heart surface, provided here, is experimentally useful for researchers studying the interplay of immune cells, fibroblasts, endothelial cells, and cardiomyocytes in relation to overall cardiac function. As mentioned above, the interstitial fluid carries the information for cell-to-cell communication within the heart, which can conveniently be assessed by the collection of CT. The detailed technical description, including a video protocol of how to collect CT from the reversed heart, should facilitate the future application of this unique technique.

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			<td height="32" style="height:32px;width:301px;">Latex Solution</td>
			<td style="width:129px;">ProChemie</td>
			<td style="width:184px;">Z-Latex LA-TZ</td>
			<td style="width:201px;">&#160;http://kautschukgesellschaft.de/%E2%80%A8z-latexla-tz%E2%80%A8</td>
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			<td height="32" style="height:32px;width:301px;">Aluminum Mold</td>
			<td style="width:129px;">Home made</td>
			<td style="width:184px;">-</td>
			<td>Reverse heart model</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Universal Ovens</td>
			<td style="width:129px;">Memmert</td>
			<td style="width:184px;">UNB 400</td>
			<td>Reverse heart model</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Latex Balloon</td>
			<td style="width:129px;">Hugo Sachs</td>
			<td style="width:184px;">Size 4</td>
			<td>Reverse heart model</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Milling Machine</td>
			<td style="width:129px;">Proxxon</td>
			<td style="width:184px;">MF70</td>
			<td>Reverse heart model</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Sodium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:184px;">SZBD0810V</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Sodium Hydrogen Carbonate</td>
			<td style="width:129px;">Roth</td>
			<td style="width:184px;">68852</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Potassium Chloride</td>
			<td style="width:129px;">Merck</td>
			<td style="width:184px;">49361</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Magnesium Sulphate Heptahydrate</td>
			<td style="width:129px;">Merck</td>
			<td style="width:184px;">58861</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Potassium Dihydrogen Phosphate</td>
			<td style="width:129px;">Merck</td>
			<td style="width:184px;">48731</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">D(+)-Glucose Anhydrous</td>
			<td style="width:129px;">Merck</td>
			<td style="width:184px;">83371</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Calcium Chloride Dihydrate</td>
			<td style="width:129px;">Fluka</td>
			<td style="width:184px;">21097</td>
			<td>Chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Balance</td>
			<td style="width:129px;">VWR</td>
			<td>SE 1202&#160;</td>
			<td>Weighing chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Double Distilled Water</td>
			<td style="width:129px;">Millpore</td>
			<td style="width:184px;">-</td>
			<td>Disolving chemicals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Medical Pressure Transducer</td>
			<td style="width:129px;">Gold</td>
			<td style="width:184px;">-</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Medical Flow Probe</td>
			<td style="width:129px;">Transonic</td>
			<td style="width:184px;">3PXN</td>
			<td>Langendorff apparatus</td>
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		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Heating Circulating Bath</td>
			<td style="width:129px;">Haake&#160;</td>
			<td style="width:184px;">B3 ; DC1</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Laboratory and Vaccum Tubing</td>
			<td style="width:129px;">Tygon</td>
			<td style="width:184px;">R-3603</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Animal Research Flowmeters</td>
			<td style="width:129px;">Transonic</td>
			<td style="width:184px;">T206</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">PowerLab Data Acquisition Device</td>
			<td style="width:129px;">AD Instruments</td>
			<td style="width:184px;">Chart 7.1</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">LabChart Data Acquisition Software</td>
			<td style="width:129px;">AD Instruments</td>
			<td style="width:184px;">Chart 7.1</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Peristaltic Pump</td>
			<td style="width:129px;">Glison</td>
			<td style="width:184px;">MINIPULS 3</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Glass Water Column</td>
			<td style="width:129px;">home made</td>
			<td style="width:184px;">-</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Water Bath Protective Agent</td>
			<td style="width:129px;">VWR</td>
			<td style="width:184px;">462-7000</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:301px;">Sterile Disposable&#160; Filters (0.2&#181;m)</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:184px;">595-4520</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Blood gas analyzers</td>
			<td>Radiometer</td>
			<td style="width:184px;">ABL90 FLEX PLUS</td>
			<td>Gas analyzer</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">70% ethanol</td>
			<td style="width:129px;">VWR</td>
			<td style="width:184px;">UN1170</td>
			<td>Cleaning&#160; tubings</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">100% ethanol</td>
			<td style="width:129px;">Merck</td>
			<td style="width:184px;">64-17-5</td>
			<td>Cleaning tubings</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Wistar Rats</td>
			<td style="width:129px;">Janvier</td>
			<td style="width:184px;">-</td>
			<td>Animals</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Stainless Scissors</td>
			<td style="width:129px;">AESCULAP</td>
			<td style="width:184px;">BC702R</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Stainless Scissors</td>
			<td style="width:129px;">AESCULAP</td>
			<td style="width:184px;">BC257R</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">Big Forceps&#160;</td>
			<td style="width:129px;">AESCULAP</td>
			<td style="width:184px;">-</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">8m/m Stainless Forceps</td>
			<td style="width:129px;">F.S.T</td>
			<td style="width:184px;">11052-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:301px;">superfine (10/0) emery paper</td>
			<td style="width:129px;">3M</td>
			<td style="width:184px;">051111-11694</td>
			<td>Reverse heart model</td>
		</tr>
	</tbody>
</table>

the inverted heart model for interstitial transudate collection from the isolated rat heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

The reversed-heart model enables the collection of cardiac interstitial transudate in an isolated, retro-perfused rat heart (Figure 1A-C). When perfused at a constant pressure of 100 cmH2O, the rate of interstitial fluid formation ranged between 17 and 147 &#181;L/min, amounting to 0.1-1% of the coronary venous effluent in the isolated heart.
The protein content of the CT...

Watch this Scientific Journal Video about The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart at JoVE.com

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

The overall goal of this inverted heart model is to collect cardiac interstitial transudate from an isolate rat heart. Within the beating heart, the individual sense of the heart release paracrine factors and they release those into the surrounding interstitial fluid space. This is important, because the interstitial fluid space, it carries information for the communication between the cardiac cells.And that is particularly important under conditions of inflammation and regeneration of the infarcted myocardium. In this video, my collaborators, Dr.Ding and Tan, will show you the techniques by which we here, in this uh-love collect interstitial fluid from the surface of an isolated perfused rat heart. To make the latex heart cover, or cap, have an aluminum mold that matches the average size of a rat's heart and has been polished with superfine emery paper.Vertically fix the neck of the mold to the milling machine and start to slowly rotate the mold. Then, pour 20 milliliters of liquid latex into a 50 milliliter glass beaker, and lower the rotating mold into the latex until it is immersed. Then, slowly lift it out and keep rotating the mold for an additional 15 minutes until the latex has solidified.The rotation is vital to make this mousse and evenly distribute layer of the liquid latex on the mold's surface. So, make certain that the mold is rotating as it is lifted from the latex. Once the latex is dry, coat the latex with talcum powder to prevent damage to the latex.Stop the rotation, then gently detach the mold from the machine. Carefully remove the latex from the mold. Now, attach a 25 centimeter length of silicon tubing to the outlet on the latex cap.This tube is used to collect the interstitial cardiac transudate, or CT.Next, firmly attach the rat-sized ventricular balloon onto an L-shaped metal cannula, connected to a one milliliter water-filled syringe. And fill the rat-sized ventricular balloon with water. Make sure that the balloon is airtight by inflating and deflating it several times using the syringe.Then, connect the cannula to a pressure transducer, via a three way stop. Lastly, make a mark on the syringe where the balloon will fill the ventricular cavity for the measurement of intraventricular pressure. Before proceeding with preparation of the heart, ready the Langendorff perfusion system according to the text protocol.Now, after fully anesthetizing a 300 to 350 gram male Wistar rat, transfer it to a room temperature operating table. With experience, the following steps leading to the cannulation of the heart should only take about three minutes. Lift the skin and muscle just below the sternum, using forceps, and cut around the lower margin of the ribs using heavy scissors.Next, with fine scissors, make a small cut into the diaphragm at the rib margin. Next, cut the rib's caudally to make a flap of the entire ventral chest wall. Now, gently take hold of the heart, and slowly life it until the cardiac vessels start to stretch.Then, excise the heart until the aorta is fully exposed and transfer the heart to a 100 milliliter beaker containing 50 milliliters of ice cold KHB. Now, immediately take the heart to the profusion apparatus, and mount the aorta onto a dripping cannula. Secure it tightly with a 4-0 suture, so that no air bubbles can enter the heart.Then, apply either a constant profusion pressure or a profusion flow. To establish the reversed heart set up, first gently rotate the aortic cannula until the posterior wall of the hart is en face. Then, snip away the connective tissue to expose the opening to the left atrium.Next, gently grasp the heart and turn it upside-down. Mount the heart onto the prepared ventricular balloon, with the balloon deflated, and feed it into the left ventricle. The heart is now mechanically stabilized in an upright position, using the balloon catheter assembly as a support.Inflate the balloon until it fills the entire ventricular cavity, by advancing the plunger to the fill mark. Secure the heart so there is minimal stress on the aortic root. This part of the procedure takes practice.Now, observe the gauge on the balloon, and adjust the diastolic pressure to three to five millimeters of mercury. Small droplets should form on the epicardium if the pressure is set correctly. Next, gently pull the latex cap over the heart.Even the most ventricular surface should be covered. Before proceeding, check for air bubbles inside the cap. To remove any air bubbles, use gentle suction from the syringe.Now, position the end of the letting tube 10 centimeters below the heart. Collect the dripping CT into a 1.5 milliliter tube and keep the tube below four degrees Celsius by embedding it in a 1:1 mixture of ice and sodium chloride. Collect between 0.15 and 1.5 milliliters of CT, then weight the sample and snap freeze it in liquid nitrogen.Store it at minus 80 degrees Celsius until it can be processed. When retroperfusing the reversed heart model at a constant pressure of 100 centimeters of water, between 1/10 to 1%of the coronary venous effluent could be collected. The protein content of the CT was measured with the BCA assay, and averaged 1.1 milligram per milliliter.Simple gel electrophoresis revealed a wide spectrum of proteins. Normal samples were compared to samples from hearts subjected to 50 minutes of ischemia reperfusion using 2D DIGE. Several protein were found to be upregulated in the CT of the ischemic hearts.Proteomic analysis was performed on the upregulated proteins. Purines were also of great interest. Purines are pivotal signaling molecules that regulate the cardiac immune response, vasomotor tone, and cardiac function, particularly after ischemic injury.HPLC analysis of the CT showed elevated concentrations of many purines in the injured hearts. While attempting this procedure, it's important to remember that a cardiac trans-in-date being formed in an isolated heart, will not entirely mimic the true composition of the in-vee-vuh conditions.

the-inverted-heart-model-for-interstitial-transudate-collection-from

Research

JoVE Journal

Medicine

8.3K ビュー.  Second Military Medical University. The overall goal of this inverted heart model is to collect cardiac interstitial transudate from an isolate rat heart. Within the beating heart, the individual sense of the heart release paracrine factors and they release those into the surrounding interstitial fluid space. This is important, because the interstitial fluid space, it carries information for the communication between the cardiac cells.And that is particularly important under conditions of inflammation and regeneration of...