Name: testing the efficacy of pharmacological agents in a pericardial target delivery model in the swine
Uploaded: 2016-07-07T09:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine at JoVE.com

We would like to give a big thanks to the Visible Heart Laboratory staff and students that have helped with this project: Nate Menninga, Lars Mattison and Megan Schmidt.

This protocol was approved by the University of Minnesota Institutional Animal Care and Use Committee.
<img alt="Figure 1" src="/files/ftp_upload/52600/52600fig1.jpg" /> 
Figure 1. Diagram of Study Paradigm <a href="https://www.jove.com/files/ftp_upload/52600/52600fig1large.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>
1. Surgical Preparation of the Swine
<ol>
	<li>Administer 5-7mg/kg of tiletamine/zilazipam to the swine (70-80 kg) via intramuscular injection. Place an ear vein catheter and administer 5-7 mg/kg methohexital via intravenous access. Intubate the animal with endotracheal tube and ventilate.</li>
	<li>Deliver isoflurane &#62;1.2% MAC in a mixture of house air and oxygen to maintain a surgical plane of anesthesia. Ensure the animal is in a deep plane of anesthesia prior to any manipulations by checking for the absence of jaw tone and keeping the anesthesia above the minimum alveolar concentration for swine.&#160;Initial anesthetic depth check is conducted by assuring the absence of jaw tone and the isoflurane levels are monitored continuously throughout the procedure (&#62;1.5 MAC) to ensure a deep plane of anesthesia.</li>
	<li>Carefully access the right external jugular and carotid artery with cautery and blunt dissection. Place an 8.5F Swan-Ganz catheter in the external jugular and inflate the balloon to 1.5 cc. Then traverse through the right atrium, into the ventricle and through the pulmonic valve down the pulmonary artery until a wedge pressure is felt.</li>
	<li>Deflate the balloon and leave the catheter in this position. Record the right atrial pressure and pulmonary artery pressure using the balloon. Additionally, record the wedge pressure or pressure in the lung (or if the lungs are considered normal, then also record the left atrial pressure). &#160; 
	NOTE: This catheter also allows for estimates of the cardiac output.</li>
	<li>Place an additional 5.0F balloon pressure catheter in the external jugular and into the right ventricle to continuously record pressure.</li>
	<li>Place a 5.0F balloon pressure catheter in the carotid artery, prolapse through the aortic valve and maintain in the left ventricle. Flush all pressure catheters with 20 units/ml&#160;heparanized saline connected to a pressure transducer, and record all pressure data from the left ventricle, right ventricle (and estimated cardiac output), pulmonary artery pressure and right atrial pressure.</li>
	<li>For peripheral monitoring, access a branch of the femoral artery by placing an 18F catheter for real-time blood pressure or blood gas monitoring. Record heart rate and temperature during the procedure.</li>
	<li>Carefully access the left external jugular with cautery and blunt dissection. Place two 11.0F hemostasis introducers in the jugular and secure with 0 silk sutures. Place steerable catheters in the introducers.</li>
	<li>Under fluoroscopic guidance, place active fixation leads in the right atrial appendage and the apex of the right ventricle. 
	NOTE: A fixation lead is a surgically implanted wire that connects the heart to an implanted pacemaker.</li>
	<li>Connectanalyzer cables to the leads and test for capture utilizing a programmer. Set the parameters to 8V, 0.25 msec duration, and set the pacing rate at a minimum of 10 bpm higher than the intrinsic rate at that time. Record the relative impedance for each lead.</li>
	<li>Make a medial surgical incision from the xiphoid process to near the insertion of the sternocleidomastoid muscles. Use a sternal saw to remove the anterior protrusion from the sternum (keel bone) to enable the partial medial dissection of the sternum. Use a shear cut through the remaining portions of sternal bone structure. Dissect the sternal-pericardial ligament and retract the sternum.</li>
	<li>Use blunt dissection to separate the pericardium from the pleural linings. Make a medial sagittal incision (3-5 cm) in the pericardium and create a pericardial cradle with four square knot sutures at each corner (right, left, superior and inferior sites).</li>
	<li>Place a temporary bipolar lead in the apical region of the left ventricle and place a unipolar plunge temporary pacing lead into the left atrial appendage.</li>
	<li>Euthanasia is accomplished by removal the heart under deep anesthesia.</li>
</ol>
2. Preparation for Electrophysiological Monitoring
<ol>
	<li>Cardiac Mapping System.
	<ol>
		<li>Select an AF study in a cardiac mapping system Connect the ventricular epicardial bipolar pacing leads and the epicardial leads in the atrial appendage with alligator clips to the appropriate settings in the breakout box. Connect an EKG and ground pads to the system.</li>
	</ol>
	</li>
	<li>Oscilloscope and Grass Stimulator.
	<ol>
		<li>Connect the bipolar left ventricular pacing lead and unipolar atrial lead in the breakout box to a Grass stimulator which is connected to an oscilloscope for electrical verification.</li>
	</ol>
	</li>
	<li>Programmed Electrophysiological Stimulation.
	<ol>
		<li>Secure the right atrial leads and right ventricle leads in a pacemaker Place the programmer telemetry head over the pacemaker. Select the pacing electrode stimulation (PES) study and set the parameters to 8V; remove all patient parameters. Set the pacing duration set to 8 pulses at 400 msec (or 300 msec).</li>
	</ol>
	</li>
</ol>
3. In Situ Study
<ol>
	<li>Refractory Period: Study Paradigm.
	<ol>
		<li>Determine the atrial effective refractory period (AERP), atrial ventricular node refractory period (AVNERP) and the ventricular effective refractory period (VERP) at baseline, and at 30 and 60 min&#160;post pericardial delivery of compound/drug/control treatment; additional time points may be added as desired.</li>
	</ol>
	</li>
	<li>Refractory Period: Programmer Settings.
	<ol>
		<li>Burst induce the PES study parameter to 8 paces (400 msec or 300 msec) followed by a minimum of 300 msec pacing. Reduce the pacing until the ventricles/atrium (respectively) fail to contract. This is noted from the sensing of the pacing leads; thus determining the relative refractory period.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52600/52600fig2.jpg" /> 
Figure 2. EnSiteSystem: AERP, AVNERP and VERP Determination <a href="https://www.jove.com/files/ftp_upload/52600/52600fig2large.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Determination of Relative AF Burden: Parameters.
	<ol>
		<li>Sustain the animal&#8217;s heart in AF, after 1 min, allow it to remain in AF for up to 10 min: i.e., before shocking the atria with direct paddles at 5 joules to terminate AF. Elicit AF induction up to 10 times at each time point. Note, in this study. AF is defined as an erratic rhythm with an absence of the P wave.</li>
	</ol>
	</li>
	<li>AF Burden: Grass Stimulator and Oscilloscope Settings.
	<ol>
		<li>Optimize the parameters for the Grass stimulator to deliver 4 Hz&#160;with a 2 sec&#160;pulse duration to the left atrial appendage (LAA) via the plunge pacing lead; verify this by a standard oscilloscope. Deliver a single pulse to the LAA to induce AF.</li>
	</ol>
	</li>
</ol>
4. Heart Explantation and Reanimation (Transplant Model)
<ol>
	<li>Aortic Root Cannula.
	<ol>
		<li>Carefully dissect the pericardial tissue around the ascending aorta and remove the pericardium.Suture two points (2-3 cm apart) of 2.0 Ethibond suture in the ascending aorta, and administer 30,000 units of Heparin intravenously.</li>
		<li>Place the aortic root cannula between the suture points and secure the aorta. With the delivery system pressurized, remove the stylet-bevel and place a clamp on the cannula not allowing for flow.</li>
	</ol>
	</li>
	<li>Cardioplegia Preparation.
	<ol>
		<li>Administer cold St. Thomas cardioplegia solution into the ascending aorta under a pressure of 150 mm/hg via a pressurized bag. Secure an irrigation catheter that is attached to the pressurized cardioplegia with a 3-way stop cock on the distal end. Secure the stop cock to the aortic root cannula, release a steady flush of cardioplegia through the catheter and secure to the aortic root cannula.</li>
	</ol>
	</li>
	<li>Cross-Clamp.
	<ol>
		<li>Clamp the inferior vena cava with an atraumaticclamp 1-2 cm just superior to the diaphragm. Clamp the ascending aorta with atraumatic angled camp 1-2 cm on the ascending aorta superior to the aortic root cannula.</li>
	</ol>
	</li>
	<li>Cross-Clamp/Cardioplegia.
	<ol>
		<li>Remove the clamp from the aortic root cannula and flush the cardioplegia at 150 mm/hg toward the aortic valve, closing the aortic valve and perfusing the coronaries.</li>
		<li>Clamp the superior vena cava and make a small incision in the pulmonary artery, so as not to over pressurize the heart. Once the heart is stopped (asystolic), excise and remove heart from the chest and place in a bath of cold Krebs-Henseleit buffer (~3-8 &#176;C).</li>
	</ol>
	</li>
	<li>Reanimation.
	<ol>
		<li>Cannulate the great vessels and prepare the heart utilizing Visible Heart&#160;methodologies (3). Once the perfused heart reaches 37&#177;5 &#176;C, restore a native sinus rhythm by delivering 34 J shocks to the ventricles via epicardial patch electrodes.</li>
	</ol>
	</li>
</ol>
5. In Vitro Study Paradigm
<ol>
	<li>Continuously monitor cardiac function for 60 min&#160;post reanimation. Continuously monitor echocardiography in 2D mode with a short-axis view: an LV view just below the mitral valve.</li>
	<li>Calculate ventricular wall thickness and ejection fractions every 10 min&#160;post reanimation (see below). Take samples from the coronary sinus (1 cc) at -5 min&#160;and every 5 min&#160;during the evaluation period.</li>
	<li>Monitoring.
	<ol>
		<li>Monitor the hemodynamics for both the left and right ventricles, including pressures, flows and volume changes.</li>
	</ol>
	</li>
	<li>Echocardiography: Ejection Fraction.
	<ol>
		<li>Place a transthoracic echo probe directly on the heart, capturing a mid-papillary short-axis view. Calculate the ejection fraction (EF %) in 4-chamber working mode every 10 min&#160;using the equation: LV End Diastolic Volume &#8722; LV End Systolic Volume X 100/LV End Diastolic volume</li>
	</ol>
	</li>
	<li>Echocardiography: Left Ventricular Wall Thickness.
	<ol>
		<li>Measure ventricular wall thickness (cm) from a mid-papillary short-axis window at 10 min&#160;intervals for an hour post reanimation. Measure four corners of the left ventricle using a screen calipers and take the average.</li>
	</ol>
	</li>
	<li>Myocardial Metabolism.
	<ol>
		<li>Place a steerable catheter in the coronary sinus (CS). Sample 1 cc from the CS 5 min&#160;before reanimation and every 5 min&#160;post reanimation for 1 hr. Test the samples for lactate and glucose levels; other blood gases can also be assessed.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Richardson, E. S., Rolfes, C., Woo, S. O., Elmquist, W. F., Benditt, D. G., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Responses+to+the+Intrapericardial+Delivery+of+Metoprolol:+Target+Delivery+Compared+to+Intravenous+Administration.">Cardiac Responses to the Intrapericardial Delivery of Metoprolol: Target Delivery Compared to Intravenous Administration.</a> J. Cardiovasc. Translational Research. 5 (4), 535-540 (2012).</li><li>Xiao, Y. F., Sigg, D. C., Ujhelyi, M. R., Wilhelm, J. J., Richardson, E. S., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardial+delivery+of+omega-3+fatty+acid:+a+novel+approach+to+reducing+myocardial+infarct+sizes+and+arrhythmias.">Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias.</a> Am J Physiol Heart Circ Physiol. 294 (5), 2212-2218 (2008).</li><li>Chinchoy, E., 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=Isolated+four-chamber+working+swine+heart+model.">Isolated four-chamber working swine heart model.</a> Ann Thorac Surg. 70 (5), 1607-1614 (2000).</li><li>Moreno, R., Waxman, S., Rowe, K., Verrier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapericardial+beta-adrenergic+blockade+with+esmolol+exerts+a+potent+antitachycardic+effect+without+depressing+contractility.">Intrapericardial beta-adrenergic blockade with esmolol exerts a potent antitachycardic effect without depressing contractility.</a> J Cardiovasc Pharmacol. 36 (6), 722-727 (2000).</li><li>Jansen, B. J., 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=Intrapericardial+delivery+enhances+cardiac+effects+of+sotalol+and+atenolol.">Intrapericardial delivery enhances cardiac effects of sotalol and atenolol.</a> J Cardiovasc Pharmacol. 44 (1), 50-56 (2004).</li><li>Vereckei, A., Gorski, J. C., Ujhelyi, M., Mehra, R., Zipes, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapericardial+ibutilide+administration+fails+to+terminate+pacing-induced+sustained+atrial+fibrillation+in+dogs.">Intrapericardial ibutilide administration fails to terminate pacing-induced sustained atrial fibrillation in dogs.</a> Cardiovas Drugs Ther. 18 (4), 269-277 (2004).</li><li>Kumar, K., Nguyen, K., Waxman, S., Nearing, B. D., Wellenius, G. A., Zhao, S. X., Verrier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+antifibrillatory+effects+of+intrapericardial+nitroglycerin+in+the+ischemic+porcine+heart.">Potent antifibrillatory effects of intrapericardial nitroglycerin in the ischemic porcine heart.</a> J Am Coll Cardiol. 41 (10), 1831-1837 (2003).</li><li>Fei, L., Baron, A. D., Henry, D. P., Zipes, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapericardial+delivery+of+L-arginine+reduces+the+increased+severity+of+ventricular+arrhythmias+during+sympathetic+stimulation+in+dogs+with+acute+coronary+occlusion:+nitric+oxide+modulates+sympathetic+effects+on+ventricular+electrophysiological+properties.">Intrapericardial delivery of L-arginine reduces the increased severity of ventricular arrhythmias during sympathetic stimulation in dogs with acute coronary occlusion: nitric oxide modulates sympathetic effects on ventricular electrophysiological properties.</a> Circulation. 96 (11), 4044-4049 (1997).</li><li>Ayers, G. M., Rho, T. H., Ben-David, J., Besch, H. R., Zipes, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amiodarone+instilled+into+the+canine+pericardial+sac+migrates+transmurally+to+produce+electrophysiologic+effects+and+suppress+atrial+fibrillation.">Amiodarone instilled into the canine pericardial sac migrates transmurally to produce electrophysiologic effects and suppress atrial fibrillation.</a> J Cardiovasc Electrophysiol. 7 (8), 713-721 (1996).</li><li>Darsinos, J. T., Karli, J. N., Samouilidou, E. C., Krumbholz, B., Pistevos, A. C., Levis, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+amiodarone+in+heart+tissues+following+intrapericardial+administration.">Distribution of amiodarone in heart tissues following intrapericardial administration.</a> Int. J. Clin. Pharacol Ther. 37 (6), 301-306 (1999).</li><li>Ujhelyi, M. 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The authors have nothing to disclose

Here, we have demonstrated a unique approach for testing the potential efficacy of target delivered compounds into the pericardial space. This study paradigm can be utilized to test current market released pharmaceutical products or experimental compounds, thus providing direct translational applications to various clinical settings. Within this treatment strategy there are two major clinical applications for delivering pharmaceuticals to the pericardial space: 1) open-heart or minimally invasive cardiac surgical procedures; and 2) heart transplantation and organ preservation. In addition, this study paradigm has multiple parameters that can be analyzed to better understand the predictive efficacy of the pharmaceutical agents themselves within these described treatment strategies. In situ parameters include: 1) the relative refractory periods of the AVN, atria and/or ventricles; 2) the determination of relative AF susceptibility/burden; and 3) hemodynamic and heart rate responses. In vitro parameters include: 1) relative ejection fractions; 2) LV wall thickness responses; 3) cardiac metabolic alterations; and 4) hemodynamics and heart rate changes. The historical data we present here demonstrate a selection of these described parameters. The most notable translational aspects of these studies are for open- or minimally invasive heart procedures, as well as organ procurement prior to transplantation.
More specifically, a formed pericardial cradle placed during an open-heart surgery, either minimally invasive or following a full-sternotomy, provides an important opportunity to administer localized compounds: i.e, at higher concentrations than may be administered intravenously and/or without systemic side effects. For even less invasive cardiac procedures (e.g., subxyphoid access), a small incision is made in the pericardium that could also be used as a conduit for target drug delivery. Myocardial irritability/insult is greatly increased while the heart is manipulated during various cardiac procedures. Treating for arrhythmias and/or functional deficits directly may be a beneficial and easy means for improving patient outcomes.
Similar applications could be easily performed during procedures for organ recovery, for example, preconditioning the heart prior to procurement. It is crucial that the myocardium is optimally conditioned for a complicated procedure that includes defibrillating the heart back into a normal sinus rhythm after transplantation. In other words, there are increased risks of atrial and ventricular tachycardias and/or fibrillation during heart procurement and after transplantation. Also, it is important to preserve the other organs in the donor during the procurement process, where peripherally administered drugs can compromise these organs. For example, intravenously administered metoprolol can result in acute kidney dysfunction when given to treat arrhythmias during surgical procedures/procurements 1. Currently, the heart must be transplanted to the recipient within approximately 4-6 hr. This time constraint remains as one of the limiting factors in performing heart transplantations today. Thus, employing the experimental paradigm we describe here could be an important means for evaluating compounds that may be beneficial in prolonging the acceptable range of ischemic time after organ recovery.
Previously collected data from our laboratory utilizing this study paradigm have demonstrated that it can be highly useful for obtaining multiple parameters that can be analyzed to predict the efficacy of the pharmaceutical agents themselves within the described treatment strategy. More specifically, studying the effective refractory periods following pericardial delivered DHA, DHA infusion, metoprolol and other drugs in situ has been important so to understand their antiarrhythmic potential for this target delivery method. Prolonging the effective refractory periods and/or reducing the conductance velocities within the AVN can often terminate certain types of arrhythmias 4. Here we have demonstrated notable increases in these effective refractory periods following a DHA infusion, as well as after delivering metoprolol as compared to control (previously published, 1. In addition, it is of interest to determine the relative AF burden because of its relationship to the probability for arrhythmic potentiation in a given patient; here we also noted alterations in this response following pharmacological preconditioning.
The investigation of clinically relevant clinical parameters after the reanimation of a preconditioned heart also gives translational insights as to the potential benefits of this pharmacological administration on a transplant recipient. To date using this overall target delivery/preconditioning research approach, our laboratory has investigated a variety of clinically administered agents as well as novel pharmacological compounds that may minimize arrhythmias or ischemic damage to the heart (metoprolol, amiodarone, lidocaine, delta opioids, omega-3 fatty acids, ursodyoxycholic acid, lipovenous, docosahexaenoic acid and/or combinations of these). It should also be noted that these agents could also be administered as post-conditioning agents in the in vitro aspect of the protocols described within. Observing the in vitro hemodynamics, the representative data for the LV pressures are notably higher in hearts that have been treated with DHA or a DHA infusion compared to a vehicle control. In addition, our laboratory has observed changes in ejection fraction and LV wall thickness in treated hearts compared to controls. The clinical parameter/factors that can be studied are important so to evaluate effective function and to observe the onset of edema. Further, samples from the coronary sinus can also be obtained for analysis of various metabolic factors: e.g., lactate and glucose levels. These parameters are imperative to assess the relative cardiac metabolism and/or overall heart function. For example, increased lactate levels are often indicative of acidosis, in turn resulting in poor cardiac function.
The data we have obtained from such studies have demonstrated a viable experimental model (with noted limitations because of the acute nature of the study) to determine the practicality of utilizing either clinically available drugs or experimental agents for a pericardial target delivery strategy. We consider that the investigative approach we describe here is highly reproducible and provides novel insights relative to the target and systemic benefits of the delivery of various pharmacological agents within the pericardial space. The results one may obtain in such designed protocols could have important translational implications for both cardiac surgery and heart transplantation.

Currently in open-heart procedures, clinicians utilize antiarrhythmic and other treatment agents systemically. Yet, this can be problematic for many patients, especially those who are already clinically compromised. For example, intravenous treatments can result in systemic drops in blood pressure or renal dysfunction; further, they may create anesthesia management issues and/ or other long-term side effects.
Here we have created a model for testing the efficacy of administering pharmacological agents into the pericardial space. For example, this approach can be utilized for testing antiarrhythmic drugs, studying compounds that could increase cardiac function and/or promoting recovery of the myocardium after surgical procedures. There have been observed benefits to the target delivery of treatments into the pericardial space versus intravenous administration: e.g., our laboratory demonstrated that localized delivery of antiarrhythmic drugs, such as metoprolol, is protective against the incidence of arrhythmias while minimizing reductions in blood pressure 1. This target delivery strategy also provides the opportunity for administering higher focal concentrations while minimizing systemic levels. For example, high levels of intravenously delivered concentrations of fatty acids may result in hemolysis, but pericardial delivery minimizes this concern 2.
This study paradigm consists of three major objectives to determine the efficacy of pericardial delivered compounds: 1) in situ determination of refractory periods of the atrial ventricular node, the atria and ventricles, before treatment and 30 and 60 min&#160;post treatment; 2) the relative in situ AF burden before treatment and 30 and 60 min&#160;post treatment (additional timepoints were often added) 3) functional analysis of the heart after it has been reanimated 3 including hemodynamic monitoring, heart rate, heart metabolism (lactate and glucose) sampled from the coronary sinus, ejection fraction (EF%) and ventricular wall thickness (cm) monitored every 10 min&#160;post reanimation This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery or transplantation.

<table><tbody><tr><td>SelectSecure 3830 lead</td><td>Medtronic</td><td></td><td>Pacing Lead&amp;nbsp;</td></tr><tr><td>C304 Deflectable Catheter&amp;nbsp;</td><td>Medtronic</td><td></td><td>Steerable catheter for placing leads&amp;nbsp;</td></tr><tr><td>SelectSecure 3830 lead</td><td>Medtronic</td><td></td><td>active fixation pacing leads&amp;nbsp;</td></tr><tr><td>Grass S48 Stimulator</td><td></td><td></td><td>Electrical Stimulator&amp;nbsp;</td></tr><tr><td>Premium 6500 Unipolar Pacing&amp;nbsp;</td><td></td><td></td><td>Plunge pacing lead for LAA</td></tr><tr><td>EnSite Cardiac Mapping</td><td></td><td></td><td>Electrophysiology mapping system</td></tr><tr><td>CareLink Programer 2092</td><td>Medtronic</td><td></td><td>programmer for pacing leads</td></tr><tr><td>GEM II pacemaker&amp;nbsp;</td><td>Medtronic</td><td></td><td>pacemaker can&amp;nbsp;</td></tr><tr><td>DLP Aortic Root Cannula</td><td>Medtronic</td><td></td><td>aortic root cannula for transplant&amp;nbsp;</td></tr><tr><td>C-Arm Fluoroscopy</td><td>Ziehm&amp;nbsp;</td><td></td><td>fluoroscopic imaging&amp;nbsp;</td></tr><tr><td>Oscilliscope</td><td>Tektronix&amp;nbsp;</td><td></td><td></td></tr><tr><td>11F Hemostasis introducer</td><td>SafeSheath</td><td></td><td>Hemostasis introducers</td></tr><tr><td>Swan-Ganz Catheter 8.0F</td><td>ICU Medical&amp;nbsp;</td><td></td><td>thermal dilution catheter&amp;nbsp;</td></tr><tr><td>Venogram balloon&amp;nbsp;</td><td>Oscor</td><td></td><td>pressure monitoring&amp;nbsp;</td></tr><tr><td>Ultraview SL</td><td>Spacelabs</td><td></td><td>EKG and blood pressure&amp;nbsp;</td></tr><tr><td>s/5 Avance</td><td>General Electric</td><td></td><td>Anesthesia machine</td></tr><tr><td>Atrial 6492 &amp;ndash; Unipolar Temporary Atrial Pacing Lead</td><td>Medtronic</td><td></td><td>temporary pacing lead&amp;nbsp;</td></tr><tr><td>VIVID i</td><td>General Electric</td><td></td><td>2D electrocardiography unit&amp;nbsp;</td></tr></tbody></table>

testing the efficacy of pharmacological agents in a pericardial target delivery model in the swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

These results are characteristic of the data that can be collected utilizing this target delivery model of pharmacological agents in the swine. This data demonstrated notable increases in these ventricle effective refractory periods (VERP) following a DHA infusion in situ. In addition, the data establishes an increase in left ventricular pressure of DHA compared to control in vitro. The LV pressure in the DHA treated hearts were significantly higher compared to control at several time-points. This data validates a pharmacological window for testing treatment strategies in situ and in vitro.
<img alt="Figure 3" src="/files/ftp_upload/52600/52600fig3.jpg" /> 
Figure 3. Change in the Ventricle Effective Refractory Period (VERP). The Ventricle Effective Refractory Period (VERP) was determined 5 min&#160;before pericardial delivery of control agent (saline) or docosahexaenoic acid (DHA), or DHA infusion, in addition to 30 and 60 min&#160;post-pericardial delivery of either the treatments or control. The VERP of the DHA group trended toward increasing compared to controls. <a href="https://www.jove.com/files/ftp_upload/52600/52600fig3large.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>

<img alt="Figure 4" src="/files/ftp_upload/52600/52600fig4.jpg" /> 
Figure 4.&#160;Pressure of Left Ventricle in 4-Chamber Working Model&#160;The maximum pressure (mmHg) was determined every 5 min&#160;for 60 min&#160;after the heart was reanimated using the visible heart methodologies. DHA and DHA infusion (treated for over 10 min) treated hearts trended to elicit initial higher pressure than control for the majority of the testing period. An un-paired T-test was completed for each time-point. (*, P=0.065, +, P=0.056, &#8224;, P=0.059 &#8225;.P=0.058) <a href="https://www.jove.com/files/ftp_upload/52600/52600fig4large.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>
The Ventricle Effective Refractory Period (VERP) was determined 5 min&#160;before pericardial delivery of control agent (saline) or docosahexaenoic acid (DHA), or DHA infusion (over 10 min), in addition to 30 and 60 min&#160;post-pericardial delivery of either the treatments or control. The VERP of the DHA group trended toward increasing compared to controls.
The maximum pressure (mmHg) was determined every 5 min&#160;for 60 min&#160;after the heart was reanimated using the visible heart methodologies. DHA and DHA infusion treated hearts trended to elicit initial higher pressure than control for the majority of the testing period. An un-paired T-test was completed for each time-point. (*, P=0.065, +, P=0.056, &#8224;, P=0.059 &#8225;.P=0.058)

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Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

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Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

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8.2K Views.  University of Minnesota. We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated. 

Video: Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Measuring Cardiac Output in a Swine Model

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume and cardiac output are essential in this type of research. Contrast ventriculography, thermodilution, and pressure-volume loop (PV-loop) catheters can be used to accurately obtain cardiac performance data depending on which resources and expertise are available. For this study,five Yorkshire swine were anesthetized and intubated. Central venous and arterial access was obtained to place the necessary measurement instruments.A temperature probe was placed in the aortic root. A cold saline bolus was delivered to the right atrium and temperature deflection curve was recorded. Integration of the area under the curve allowed for the calculation of the current cardiac output.A pigtail catheter was percutaneously placed in the left ventricle and 30 mL of iodinated contrast was power injected over 2 seconds. Digital subtraction angiography images were uploaded to volumetric analysis software to calculate the stroke volume and cardiac output. A pressure volume-loop catheter was placed into the left ventricle (LV) and provided continuous pressure and volume data of the LV, which allowed the calculation of both stroke volume and cardiac output.All three methods demonstrated good correlation with each other. The PV-loop catheter and thermodilution exhibited the best correlation with a 3% error and a Pearson coefficient of 0.99, with 95% CI=0.97 to 1.1, (p=0.002). The PV-loop catheter against ventriculography also showed good correlation with a 6% error and a Pearson coefficient of 0.95, 95% CI=0.96 to 1.1 (p=0.01). Finally, thermodilution against ventriculography had a 2% error with r=0.95, 95% CI=0.93 to 1.11, (p=0.01). In conclusion, we state that the PV-loop catheter, contrast ventriculography, and thermodilution each offer certain advantages depending on the researcher's requirements. Each method is reliable and accurate for measuring various cardiac parameters in swine such as the stroke volume and cardiac output.

Thermodilution, pressure-volume loop catheters, and contrast ventriculography are reliable and accurate methods for determining cardiac physiology such as stroke volume and cardiac output in a laboratory setting in swine.

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume ...

Sterile Pericarditis in Aachener Minipigs As a Model for Atrial Myopathy and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only target the electrical abnormalities and not the underlying atrial myopathy. For the development of novel therapies, a reproducible large animal model of atrial myopathy is necessary. This paper presents a model of sterile pericarditis-induced atrial myopathy in Aachener minipigs. Sterile pericarditis was induced by spraying sterile talcum and leaving a layer of sterile gauze over the atrial epicardial surface. This led to inflammation and fibrosis, two crucial components of the pathophysiology of atrial myopathy, making the atria susceptible to the induction of AF. Two pacemaker electrodes were positioned epicardially on each atrium and connected to two pacemakers from different manufacturers. This strategy allowed for repeated non-invasive atrial programmed stimulation to determine the inducibility of AF at specified time points after surgery. Different protocols to test AF inducibility were used. The advantages of this model are its clinical relevance, with AF inducibility and the rapid induction of inflammation and fibrosis-both present in atrial myopathy-and its reproducibility. The model will be useful in the development of novel therapies targeting atrial myopathy and AF.

We describe a sterile pericarditis model in minipigs to study atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only ...

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 ...

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.