NOTE: All Procedures were approved by the Lifespan Institutional Animal Care and Use Committee and all animals and procedures according to the National research Council Guide for Care and Use of Laboratory Animals10.
1. Balloon Fabrication and Left Ventricular Pressure Monitoring Circuit
<ol>
	<li>Construct LV balloons according to Miller et al.11 In a beaker with a stir bar, mix 9.5 ml of distilled water, 14.2 ml of light corn syrup and 33.8 g of sucrose, and heat on a hot plate, mix until the sugar is dissolved. Continue heating solution until it reaches approximately 150 &#186;C. 
	NOTE: Numerous methods exist to construct LV balloons including modification of condom tips and cling film construction9,12. We find the above method to be relatively easy to construct leak-free balloons, however an advantage of cling film balloons includes previous characterization for appropriate frequency-response relationships9.</li>
	<li>Break dry spaghetti strands into pieces about 5 cm in length and dip one side of each piece about 1 cm deep into the sugar solution and slowly remove.</li>
	<li>Place the dry end of the pasta strand into a polystyrene foam block and suspend so the sugar mix drips down and forms a teardrop shape mold. Leave O/N in a desiccator so the mold hardens.</li>
	<li>The following day, dip the molds into silicone dispersion gel. Place pasta strands back into polystyrene foam block and place in a a 37 &#186;C oven for 2 hr or until dry. Repeat this step once more so two layers of silicone are applied.</li>
	<li>Once dry, place in water for several hours to assist in removal of balloon from the mold. Store balloons in 0.02% sodium azide solution.</li>
	<li>Use a 23 G blood collection set to produce a customized balloon cannula by cutting a needle to create a blunt tip and placing notches in the needle.</li>
	<li>Connect the tubing to a pressure transducer and flush with water while keeping tubing and cannula completely submerged to prevent air from entering the system. Place a balloon that has been filled with water onto the cannula and use 2-0 silk sutures to tie onto cannula. Test balloon by ability to maintain pressure (~100 mmHg for at least 1 hr)</li>
</ol>
2. Preparation of the Isolated Heart Perfusion System
<ol>
	<li>Initially, wash and warm the system. Turn on a 37 &#186;C warm water bath circulator that has been connected to the Langendorff apparatus, and fill buffer reservoir with distilled water. Connect a new glass fiber filter to tubing flowing into apparatus and turn on pump to flush out the system. During prep time, monitor the temperature of water expelled from the Langendorff block using a temperature probe, to ensure that water bath is set accurately. Also, turn on a refrigerated circulator pump and set to 20 &#186;C that will be used during cardioplegic arrest of the heart.</li>
	<li>In the meantime prepare the following solutions. Prepare 1 L of cardioplegia solution (110 mM NaCl, 16 mM KCl, 16 mM MgCl2, 1.5 mM CaCl2, 10 mM NaHCO3) and filter. Also, prepare 2 L of KHB (118 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.7 mM MgSO4, 2 mM Na-pyruvate, 6 mM dextrose, 24.9 mM NaHCO3 (pre-aerated with CO2),1.4 mM CaCl2 (added last). Once dissolved, remove precipitates by filtering into a flask using a 5 &#181;m glass fritted funnel. Place some into a small dish on ice to be used during surgery and isolation.</li>
	<li>Oxygenate the perfusate (KHB) with 95% O2/5% CO2 for at least 30 min prior to use. Perfuse Langendorff system with KHB to remove any remaining water from system and buffer reservoir. For mice, set the initial pump speed to ~2.0 ml/min. Place 2-0 and 4-0 silk sutures near perfusion cannula. Run the pump until the tubing and apparatus are filled with perfusate and ensure the bubble strup is filled with perfusate. 
	NOTE: For long periods of perfusion and stable function it is highly recommended to install an in-line glass fiber filter (~1 &#181;m) in the perfusion circuit to collect any precipitates that could form clots in the heart</li>
</ol>
3. Mouse Surgery
3.1) Mouse Anesthesia and Handling
<ol>
	<li>Prepare syringe with anesthetic dose of 80 mg/kg ketamine and 5 mg/ml xylazine mixture, and add sterile 0.9% saline to bring up volume to 0.2 ml. Inject Heparin I.P. (50 &#181;l of 1,000 U/ml solution).</li>
	<li>Place mouse back into transport container (~10-20 min) and wait for it to lose consciousness as heparin takes effect. Periodically do a toe pinch to monitor pain reflex.</li>
</ol>
3.2) Remove the Heart
<ol>
	<li>Once mouse is fully unconscious and does not respond to toe pinch, secure it to an appropriate operating table using pins or 25 G syringe tips through the extremities. Perform a thoracotomy to expose the heart. Cut a small hole just below the sternum and extend the cut to the sides of the mouse avoiding the diaphragm.</li>
	<li>Quickly cut the diaphragm and then rapidly cut up the sides of the rib cage. Flip back the rib cage like a clam shell to expose the thoracic cavity. Gently take hold of the heart, place scissors underneath and remove the heart. 
	NOTE: It is essential to quickly remove the heart once it is exposed in the thoracic cavity and the lungs are non-functional.</li>
</ol>
3.3) Clean the Heart
<ol>
	<li>Place the heart into the dish containing ice cold KHB and trim off any large pieces of lung tissue attached. Use forceps to pick up the heart and locate the aorta. Gently squeeze the heart and look for any blood that appears, as this should be the open end of the aorta. Use fine tip tweezers to hold the heart by the open end of the aorta.</li>
</ol>
4. Mounting the Heart, Starting Perfusion, and Placing the Balloon
4.1) Mounting and Cannulating the Heart
<ol>
	<li>Before transporting the heart to the Langendorff apparatus, turn on the pump ensuring that the perfusion pressure is low (~20 mmHg). Hold the heart by the aorta just under the cannula. Using another set of fine tip tweezers, carefully open the aorta and slide heart up onto the cannula. Hold it in place with one set of tweezers and then use a soft edged clip to temporarily secure it to cannula. 
	NOTE: Quickly mounting the heart to the perfusion cannula is critical for a good preparation. With experience, the procedures from opening the thoracic cavity until mounting the heart should take between 1 and 2 min.</li>
	<li>Tie and initiate perfusion as follows, using 4-0 silk suture secure aorta to the cannula directly below the clip. Ensure the tie is also around the metal aortic cannula, so the aorta does not get tied off when tightening the knot. Once one tie is firmly placed, remove the clip.</li>
	<li>Use additional silk sutures to fasten the aorta securely to the cannula making sure to tie off below any vessel branches that may be coming off the aorta. These can often be spotted by perfusate leaking or shooting out from the aorta. 
	NOTE: During the initial stages of perfusion the blood should be washing from the heart and the entire heart should appear a soft pink color. Dark discolorations that do not wash out indicate ischemic areas likely due to air emboli or clots and the heart should not be used.</li>
</ol>
4.2) Establishing Perfusion and LVP Measurement
<ol>
	<li>Slowly increase the perfusion pressure and increase pump speed until the perfusion pressure reaches 70 mmHg. 
	NOTE: Standard perfusion pressures for mouse perfusion can be 70-90 mmHg, but must be kept constant from animal to animal within an experiment.</li>
	<li>Clean the remaining tissue (lung, thyroid etc.) that may still be attached to the heart. Use scissors to cut off the left atria in order to create an opening into the left ventricle.</li>
	<li>Place the balloon onto the holder and deflate. Position the balloon cannula near the aortic cannula, directly over the opening into the left ventricle. Carefully insert balloon downward into the left ventricle while holding the heart in place so the aorta does not tear.</li>
	<li>Once it is in place slowly start to inflate the balloon until the LVEDP reaches ~8 mmHg. Place the temperature probe against the bottom of the heart so it measures the temperature of the effluent.</li>
</ol>
4.3) Basal Measurement
<ol>
	<li>Seal the heart in a water jacketed perfusion chamber. During this time continuously perfuse the heart with KHB, and ensure that the temperature continues to rise. Monitor the temperature and adjust the water bath accordingly until it reaches approximately ~37 &#186;C. 
	NOTE: Monitoring temperature in the initial phases of perfusion is critical as coronary flows and subsequently temperature can vary from heart to heart. Temperature of the effluent perfusate is monitored for desired myocardial temperature via a temperature probe placed at the apex of the heart. In addition, in comparison to other Langendorff perfusion protocols, the heart is not submerged in CP or KHB during perfusion or arrest, this is mainly done to quickly adjust temperature as well as provide efficient CP delivery without diffusion.</li>
	<li>Begin to continuously record functional measurement using a data acquisition system attached to the appropriate sensors, including perfusion pressure, left ventricular pressure (pressure transducer attached to LVP balloon), temperature, and electrophysiological parameters (EKG, MAP if so equipped) (attachment will be according to manufacturers and specific to the individual sensors and data acquisition system). Once the effluent KHB reaches ~37 &#186;C for at least 15 min and cardiac functional parameters are stable, note the time for baseline measurements. 
	NOTE: Inclusion/exclusion criteria are applied to each heart. A LVDP of &#60;60 mmHg at baseline indicates a heart that should be removed from analysis. In addition, coronary flow &#62;4.5 ml/min, or inability to maintain perfusion pressure at baseline likely indicates a leaking or torn aorta. Also any hearts that have clear ischemic areas which can be confirmed visually (i.e.,&#160;clot) or exhibit ischemia-associated reductions in cardiac function (i.e., increasing LVeDP, extremely arrhythmic) are removed from analysis.</li>
	<li>Measure coronary flow via collection of the coronary effluent in a graduated cylinder for 1 min. Close the chamber and allow temperature and function to return to baseline values. Alternatively measure the coronary flow continuously in the perfusion line via a flow probe placed after the pressure relief circuit.</li>
</ol>
5. Initiating Cardioplegic Arrest
<ol>
	<li>Place 100 ml of cold cardioplegia buffer into another reservoir and. transfer the KHB reservoir cannula to the cardioplegia reservoir.</li>
	<li>Detach the water jacketed housing and the perfusion warming circuit from the warm water bath and connect to the refrigerated circulator using tubing quick disconnects. Use separate heating and cooling circulators to allow rapid temperature changes of the heart and perfusate.</li>
	<li>Follow the air bubble that gets introduced into the tubing during the switch, and once it reaches near the isolated heart start the timing for cardioplegia. Deliver cardioplegia for 2 min. After the initial dose stop the pump. Ensure to arrest the heart in diastole and be close to the desired CP temperature. 
	NOTE: A sufficient bubble trap is necessary to avoid air emboli in the heart. Alternatively a delivery system for both reservoirs switched by a stopcock can be easily constructed, however we find following the small air bubble that is introduced when swapping the cannula between reservoirs, an easy method of timing CP delivery given potentially different pump speeds between experiments (i.e., due to mouse/rat, perfusion pressure, filter resistance, etc.)</li>
	<li>Keep the heart in cardioplegia for 2 hr at ~20 &#186;C. Every half hour during CP turn the pump back on for 1 min&#160;so another dose is administered. 
	NOTE: Intermittent doses of CP every 30 min results in functional impairment of the heart without necrosis of the tissue. Longer periods between doses of CP (i.e., &#62;45 min) can result in necrosis and ischemic contracture and would be more appropriate for a model of necrotic injury associated with unprotected ischemia.</li>
</ol>
6. Reperfusion
<ol>
	<li>At the end of CP, transfer the reservoir cannula back to the oxygenated KHB. Connect the temperature control circuits to the heating circulator and turn the pump on to start reperfusion. At this point the cardioplegia washes out, observe the temperature rise and the heart starts beating again after 2-4 min of reperfusion. When the heart starts the heart beat is slow and often arrhythmic. 
	NOTE: There usually is not a large rise in diastolic LVP (i.e., &#8804;10 mmHg rise) which is often characteristic in pure ischemia models. Arrhythmias can persist well into reperfusion and occasionally the heart will need to be defibrillated. This can be achieved with the use of a stimulator set to higher voltages (~10-50 V) and electrodes placed at the base and apex of the heart.</li>
	<li>Allow the heart to reperfuse for 30 min, and during this time period take coronary flow measurements as necessary. Measure coronary flow by collecting the effluent with a graduated cylinder for 30-60 sec.</li>
</ol>
7. Collecting the Tissue
<ol>
	<li>Deflate the balloon and remove it from the left ventricle. Take the heart off the cannula and weigh it. In initial experiments, at least one transverse slice of the heart should be taken and TTC stained to evaluate necrosis to ensure that the heart is free of any necrotic injury.</li>
	<li>Collect heart slices for microscopy studies, wet/dry weights, etc. The remainder or whole heart should be rapidly frozen by placing into liquid nitrogen. Store frozen hearts in a -80 &#186;C freezer. 
	NOTE: For measurement of high energy phosphate compounds (i.e.,&#160;ATP, creatine phosphate), the hearts should be frozen immediately.</li>
</ol>

<ol>
	<li>Kloner, R. a., Jennings, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Brief+Ischemia:+Stunning,+Preconditioning,+and+Their+Clinical+Implications:+Part+1.">Consequences of Brief Ischemia: Stunning, Preconditioning, and Their Clinical Implications: Part 1.</a> Circulation. 104 (24), 2981-2989 (2001).</li><li>Mentzer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+protection+in+heart+surgery.">Myocardial protection in heart surgery.</a> J Cardiovasc Pharmacol Ther. 16 (3-4), 290-297 (2011).</li><li>Chambers, D. J., Fallouh, H. 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M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+the+Langendorff+technique+of+isolated+heart+perfusion).">Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion).</a> J Mol Cell Cardiol. 50 (6), 940-950 (2011).</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Sutherland, F. J., Shattock, M. J., Baker, K. E., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+isolated+perfused+heart+characteristics+and+cautions.">Mouse isolated perfused heart characteristics and cautions.</a> Clin Exp Pharmacol Physiol. 30 (11), 867-878 (2003).</li><li>Miller, A., Wright, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Murine+Ventricular+Balloons+for+the+Langendorff+Heart+Preparation.">Fabrication of Murine Ventricular Balloons for the Langendorff Heart Preparation.</a> J Biotecnol Biomater. 1 (101), 1-4 (2011).</li><li>Curtis, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation,+utilisation+and+clinical+relevance+of+isolated+perfused+heart+models+of+ischaemia-induced+ventricular+fibrillation.">Characterisation, utilisation and clinical relevance of isolated perfused heart models of ischaemia-induced ventricular fibrillation.</a> Cardiovasc Res. 39 (1), 194-215 (1998).</li><li>Clements, R. T., Feng, J., Cordeiro, B., Bianchi, C., Sellke, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p38+MAPK-dependent+small+HSP27+and+&#945;B-crystallin+phosphorylation+in+regulation+of+myocardial+function+following+cardioplegic+arrest.">p38 MAPK-dependent small HSP27 and &#945;B-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest.</a> American journal of physiology. 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The authors have nothing to disclose.

The preceding protocol details methods to evaluate myocardial stunning secondary to global ischemia associated with cardioplegic arrest. In our hands this protocol produces an approximate ~40% reduction in cardiac function (LVDP, +/- dP/dt) with minimal changes in heart rate at the 30 min post-reperfusion time point. As the heart is reperfused and rewarmed all parameters of cardiac function are reduced at initial time points with a greatly reduced heart rate before stabilizing between 20 and 30 min. Coronary flow is gene...

Myocardial stunning is defined as reversible reduced contractile activity despite restoration of adequate blood flow following a brief period of ischemia or prolonged periods of ischemic insults with cardioprotection1,2,3,4,5. The method presented is specifically used to model clinically relevant ischemic insults which can result in reversible impairments in contractile function (i.e., ischemic insults associated with cardiac surgery utilizing cardioplegic arrest, brief periods of ischemia, angina, etc.). In contrast to severe ischemia studies (myocardial infarction, necrosis) this protocol was developed to evaluate myocardial functional recovery and cardioprotection without tissue injury, remodeling, and cell death. The majority of the paper discusses a standard cardioplegic arrest protocol with elements similar to a cardiac surgery using hypothermia and intermittent cardioplegia delivery.
Myocardial protection during the majority of cardiac surgeries relies on cardioplegia and cardiopulmonary bypass. Although cardioplegia (CP) solutions and strategies vary widely (blood, crystalloid, cold, warm etc.) the most common elements are 1) hyperkalemia and/or other strategies to arrest the heart in diastole, thereby limiting energy utilization resulting from myocardial contraction, and 2) hypothermia to slow metabolism and help maintain ATP and other energy reserves while arrested. Current cardioplegia solutions provide protection to the heart against ischemic insults that would otherwise prove lethal. However, cardioprotective strategies during surgical ischemic insults are not perfect, and the resultant mild ischemic injury can result in reversible cardiac contractile dysfunction despite adequate blood flow (myocardial stunning), acidosis, cardiomyocyte damage, and vascular effects including reduced coronary perfusion and vasospasm.
This protocol differs from standard isolated heart ischemia models evaluating myocardial infarction and severe ischemia in that it evaluates milder ischemic insults which can result in impaired cardiac function following brief ischemia or ischemic insults associated with cardioplegic arrest. (for review on Langendorff perfusion techniques and I/R studies see6&#8211;8). For general guidelines and a thorough analysis of experimental parameters associated with mouse isolated perfused hearts see Sutherland et al. 20039 The technique presented here details the necessary equipment, reagents, steps, strategies and tips to reliably induce stunning in mouse hearts. Minor modifications are necessary to apply the technique to rats.
Briefly isolated mouse hearts are Langendorff perfused for approximately 30 min with physiologic Krebs-Henseleit buffer (KHB), followed by cold protected cardiac arrest via delivery of a hyperkalemic hypothermic cardioplegia solution. Following arrest, cardiac functional recovery is monitored during rewarming and reperfusion of the heart with KHB. Changes in the degree of recovery of cardiac contractile function can be evaluated to assess cardioprotective agents and different cardioprotection strategies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Cardioplegia Solution (St Thomas II)</td>
			<td ></td>
			<td ></td>
			<td >Symbol / Concentrations (mM)</td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td ></td>
			<td ></td>
			<td>NaCl; 110</td>
		</tr>
		<tr >
			<td >Potassium chloride</td>
			<td ></td>
			<td ></td>
			<td>KCl; 16</td>
		</tr>
		<tr >
			<td >Calcium Chloride</td>
			<td ></td>
			<td ></td>
			<td>CaCL2; 1.5</td>
		</tr>
		<tr >
			<td >Magnesium Chloride</td>
			<td ></td>
			<td ></td>
			<td>MgCL2; 16</td>
		</tr>
		<tr >
			<td >Sodium Bicarbonate</td>
			<td ></td>
			<td ></td>
			<td>NaHCO3; 10</td>
		</tr>
		<tr >
			<td >Krebs-Heinslet Buffer</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td ></td>
			<td ></td>
			<td>NaCl; 118</td>
		</tr>
		<tr >
			<td >Potassium Chloride</td>
			<td ></td>
			<td ></td>
			<td>KCl; 4.8</td>
		</tr>
		<tr >
			<td >Magnesium Sulfate</td>
			<td ></td>
			<td ></td>
			<td>MgSO4; 1.7</td>
		</tr>
		<tr >
			<td >Sodium Bicarbonate</td>
			<td ></td>
			<td ></td>
			<td>NaHCO3; 24.9</td>
		</tr>
		<tr >
			<td >Potassium Phosphate (monobasic)</td>
			<td ></td>
			<td ></td>
			<td>KH2PO4; 1.2</td>
		</tr>
		<tr >
			<td >Calcium Chloride</td>
			<td ></td>
			<td ></td>
			<td>CaCL2; 1.4</td>
		</tr>
		<tr >
			<td >Sodium Pyruvate</td>
			<td ></td>
			<td ></td>
			<td>Na pyruvate; 2</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td ></td>
			<td ></td>
			<td>C6H12O6; 6</td>
		</tr>
		<tr >
			<td >Balloon reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Corn Syrup</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Spaghetti</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Silicon Dispersion Gel</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >styrofoam block</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >lab oven/incubator ( 50C)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Langendorff Perfusion equipment</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Isolated perfused heart sytem (IH-SR (Hugo-Sachs) or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Data acquisition system (DSI, ADinstruments or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Heated water circulator</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cooling water circulator</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Perfusion pump capable of 2-30 ml/min</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Inline perfusion filters - 1 um glass fiber</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Pressure sensors and amplifiers for LVP and perfusion pressure</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Small graduated cylinder (~10 mL)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Small temperature probe and thermometer (Werner or equivalent)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >perfusion resevoir (1L)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >cardioplegia resevoir (~200 mL)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >gas bubbler</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >95/5 O2/CO2 mix</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical tools and reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Metzenbaum and Potz surgical scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont size 5 forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >ketamine</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >xylazine</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >heparin</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >small clamp with soft sides to hold aorta (i.e. terminal clamp with taped ends)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Silk 2-0 and 4-0 sutures</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine isolated heart model of myocardial stunning associated with cardioplegic arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

Figure 1 presents typical results from one mouse experiment. LVP (red line), dP/dt (green line), and temperature (purple line) were continusouly recorded over ~3 hr. Letters indicate a - baseline measurement, b, c, d, e - delivery of cardioplegia solution, f - start of reperfusion, g, h, i, j - measurement of cornary flow during reperfusion. Note, depressed LVDP and dP/dt upon reperfusion compared to baseline. Figure 1B includes the data from A recorded over ~2 sec. Note, the decrease in...

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Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

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11.6K Views.  Brown University. The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

Video: Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

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.

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. ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.