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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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. R., Hadsall, K. Z., Euler, D. E., Mehra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapericardial+therapeutics:+a+pharmacodynamic+and+pharmacokinetic+comparison+between+pericardial+and+intravenous+procainamide+delivery.">Intrapericardial therapeutics: a pharmacodynamic and pharmacokinetic comparison between pericardial and intravenous procainamide delivery.</a> J Cardiovasc Electrophysiol. 13 (6), 605-611 (2002).</li></ol>

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 procedu...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SelectSecure&#174; 3830 lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>Pacing Lead&#160;</td>
		</tr>
		<tr>
			<td>C304 Deflectable Catheter&#160;</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>Steerable catheter for placing leads&#160;</td>
		</tr>
		<tr>
			<td>SelectSecure&#174; 3830 lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>active fixation pacing leads&#160;</td>
		</tr>
		<tr>
			<td>Grass S48 Stimulator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Electrical Stimulator&#160;</td>
		</tr>
		<tr>
			<td>Premium 6500 Unipolar Pacing&#160;</td>
			<td></td>
			<td>N/A</td>
			<td>Plunge pacing lead for LAA</td>
		</tr>
		<tr>
			<td>EnSite&#8482; Cardiac Mapping</td>
			<td></td>
			<td>N/A</td>
			<td>Electrophysiology mapping system</td>
		</tr>
		<tr>
			<td>CareLink Programer 2092</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>programmer for pacing leads</td>
		</tr>
		<tr>
			<td>GEM II &#174; pacemaker&#160;</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>pacemaker can&#160;</td>
		</tr>
		<tr>
			<td>DLP &#174; Aortic Root Cannula</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>aortic root cannula for transplant&#160;</td>
		</tr>
		<tr>
			<td>C-Arm Fluoroscopy</td>
			<td>Ziehm&#160;</td>
			<td>N/A</td>
			<td>fluoroscopic imaging&#160;</td>
		</tr>
		<tr>
			<td>Oscilliscope</td>
			<td>Tektronix&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>11F Hemostasis introducer</td>
			<td>SafeSheath</td>
			<td>N/A</td>
			<td>Hemostasis introducers</td>
		</tr>
		<tr>
			<td>Swan-Ganz Catheter 8.0F</td>
			<td>ICU Medical&#160;</td>
			<td>N/A</td>
			<td>thermal dilution catheter&#160;</td>
		</tr>
		<tr>
			<td>Venogram balloon&#160;</td>
			<td>Oscor</td>
			<td>N/A</td>
			<td>pressure monitoring&#160;</td>
		</tr>
		<tr>
			<td>Ultraview SL</td>
			<td>Spacelabs</td>
			<td>N/A</td>
			<td>EKG and blood pressure&#160;</td>
		</tr>
		<tr>
			<td>s/5 Avance</td>
			<td>General Electric</td>
			<td>N/A</td>
			<td>Anesthesia machine</td>
		</tr>
		<tr>
			<td>Atrial 6492 &#8211; Unipolar Temporary Atrial Pacing Lead</td>
			<td>Medtronic</td>
			<td>N/A</td>
			<td>temporary pacing lead&#160;</td>
		</tr>
		<tr>
			<td>VIVID i</td>
			<td>General Electric</td>
			<td></td>
			<td>2D electrocardiography unit&#160;</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 ...

Watch this Scientific Journal Video about Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine at JoVE.com

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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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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

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 ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...