Name: an isolated working heart system for large animal models
Uploaded: 2014-06-11T14:30:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about An Isolated Working Heart System for Large Animal Models at JoVE.com

There are no acknowledgments.

1. Building the Langendorff Apparatus (See Figure 1)
<ol>
	<li>Using 3/8&#8221; tubing, connect the heart reservoir to the blood reservoir.
	<ol>
		<li>Ensure that this tubing goes through a roller pump. NOTE: This may require using two 3/8&#8221; to 1/4&#34; tubing connectors to create a piece of 1/4&#34; tubing to go through the roller pump.</li>
		<li>Connect the blood reservoir to heater/oxygenator with 3/8&#8221; tubing.</li>
		<li>Use 3/8&#8221; tubing to connect the heater/oxygenator to a Y-connector.</li>
		<li>Connect one arm of Y-connector to the centrifugal pump, then connect the centrifugal pump to a second Y-connector (all with 3/8&#8221; tubing).</li>
		<li>Attach a piece 3/8&#8221; tubing securing a hemostasis valve to the upward-facing arm, which will serve as both a bubble trap and means of inserting the pressure transducer.</li>
		<li>Attach a piece of 3/8&#8221; tubing to the downward arm. This portion will attach to the aortic cannula (i.e. the afterload line).</li>
		<li>Connect the other arm of the Y-connector to the inflow of the pre-load chamber using 3/8&#8221; tubing. Ensure this tubing goes through a second roller pump.&#160;</li>
		<li>Connect excess 3/8&#8221; tubing to the outflow of this chamber. This portion will attach to the left atrium (i.e. the preload line).</li>
	</ol>
	</li>
	<li>Connect the oxygen tank and heating apparatus to the heater/oxygenator.</li>
	<li>Clamp the line going from the Y-connector to the pre-load chamber, as this line will not be used until the heart is put into working mode.</li>
</ol>
2. Pressure-Volume Catheter Preparation
<ol>
	<li>In a 37 &#176;C water bath, warm a bottle of saline solution.</li>
	<li>Soak the PV conductance catheter and pressure transducer in the warm saline for at least 30 min.</li>
	<li>Turn on the data acquisition systems, allowing both to warm-up for at least 30 min.</li>
</ol>
3. Preparing the Langendorff Apparatus
<ol>
	<li>Turn on the oxygen tank, heating apparatus, roller pump connecting the two reservoirs, and centrifugal pump.&#160;The heating apparatus should be set to the animal&#8217;s body temperature (~36 &#176;C).</li>
	<li>Wash the blood according to manufacturer&#8217;s instructions. Slower wash speeds are recommended for more complete removal of waste products from the blood (e.g., excess electrolytes, lysed cellular material).</li>
	<li>Once the blood is washed, check hematocrit level prior to hemodilution.</li>
	<li>Reconstitute the washed red blood cells with normal saline for desired hematocrit concentration (recommended: 20-25%) and add to the Langendorff apparatus.</li>
	<li>Adjust the speeds of the two pumps to begin blood flow through the system (excluding the preload chamber).</li>
	<li>Check the pH and electrolytes of the blood mixture and adjust until physiologic for the species used. NOTE: To prevent deleterious influx of calcium upon reperfusion, the calcium levels on the Langendorff apparatus should initially be kept low (0.3-0.5 mmol/L).
	<ol>
		<li>If there is a decrease in the hematocrit with concurrent increase in potassium, check lactate dehydrogenase and plasma free hemoglobin to rule out hemolysis.</li>
		<li>In case hemolysis does occur, ensure all connections are tight and there are no areas of obvious sheering.</li>
	</ol>
	</li>
	<li>Attach the Millar catheter into the Secondary Pressure slot of the PowerLab system.</li>
	<li>Calibrate the pressure transducer according to manufacturer&#8217;s instructions.</li>
</ol>
4. Preparing the Heart for Attachment to the Langendorff Apparatus
NOTE: A properly arrested heart should be used for any large animal experiments involving an isolated heart system.&#160;Lack of cardioplegic arrest can damage the heart such that it will not produce measureable work.&#160;Celsior, or low-potassium University of Wisconsin (UW) solution is recommended, as not only are these solutions similar to those used clinically, but the low potassium of the solution helps prevents hyperkalemia while on the circuit.&#160;Volume of cardioplegic solution will depend on heart size, with 1&#160;liter sufficient for porcine hearts.
<ol>
	<li>Quickly remove the heart from the storage container, pour out any storage solution in the ventricles, blot dry and weigh.</li>
	<li>To help maintain a cold myocardial temperature until the heart is ready for the Langendorff, return the heart to storage container and orient it so that aorta is facing upwards.</li>
	<li>Insert an&#160;3/8&#8221; cannula into the aorta and secure with a zip-tie.</li>
</ol>
5. Attaching the Heart to the Langendorff
<ol>
	<li>Decrease the centrifugal pump to a slow trickle.</li>
	<li>Trickle the blood into the aorta until it is filled with blood and completely de-aired.</li>
	<li>Carefully attach the aortic cannula to the aortic tubing on the Langendorff. Make note of attachment time.</li>
	<li>Insert the calibrated pressure transducer through the hemostasis valve[DS1]&#160;into the native aorta.</li>
	<li>Begin pressure measurements and adjust centrifugal pump speed until desired reperfusion pressure is achieved. NOTE: Pressure may change as coronary resistance changes.&#160;Therefore, monitor aortic pressure closely, especially during initial reperfusion.</li>
	<li>Increase temperature on warming unit intramyocardial temperature is measured at 37 &#176;C. NOTE: There will be a delay between adjustments made to warming unit and changes in intramyocardial temperatures.&#160;Therefore, temperature changes should be made incrementally.</li>
	<li>Obtain a baseline (T = 0) sample from the venous blood reservoir to measure pH, electrolytes, and other biochemical measurements.</li>
	<li>Insert temperature probe into septum and monitor myocardial temperature. Decrease temperature of warming unit if myocardial temperature rises above 39 &#176;C.</li>
	<li>Take blood samples every 15 min, adjusting the physiologic parameters as desired for the experiment.
	<ol>
		<li>Add approximately 1 mmol of calcium to the blood solution every 5 min, ensuring that&#160;ionic calcium is &#62; 0.8 mmol/L prior to the initiation of working mode.</li>
	</ol>
	</li>
</ol>
6. Putting the Heart into Working Mode
<ol>
	<li>Insert an appropriately sized cannula into the left atrium/pulmonary vein. This can be done with either a purse-string suture or zip-tie as appropriate.</li>
	<li>Close any holes in the left atrium that may leak, such as other pulmonary vein origins with suture or staples as needed.</li>
	<li>Adjust the height of the preload chamber such that the column height gives the desired preload pressure. NOTE:&#160;Assuming the density of blood/crystalloid mixture is equal to the density of water, 1 mmHg = 1.36 cm of the distance from the aortic valve to the top of the blood level in the preload reservoir (e.g., 15 mmHg = 20.4 cm).</li>
	<li>Unclamp the tubing going to the preload chamber and slowly start the preload roller pump, allowing the preload chamber and preload tubing to fill completely with blood.</li>
	<li>Once the preload tubing is completely de-aired, slowly fill the left atrium and cannula with blood.</li>
	<li>Without allowing any air to enter the system, connect the preload tubing to the left atrial cannula.</li>
</ol>
7. Obtaining Ventricular Pressure-Volume (PV) Recordings
<ol>
	<li>Follow the manufacturer&#8217;s instructions for pressure and Rho cuvette calibration for the data acquisition systems.</li>
	<li>Place a purse-string suture using a 3-0 polypropylene suture at the left ventricular (LV) apex.</li>
	<li>Using a 16 G&#160;needle, make a stab incision within the purse-string.</li>
	<li>Insert the PV conductance catheter into the apical incision. NOTE: Ideal catheter placement will depend on having all sensing electrodes within the LV and two excitation electrodes outside the LV. Ensure that a properly sized animal and catheter have been selected (see Discussion).</li>
	<li>Press the &#8220;Start&#8221; button in the top right corner to begin recording data and determine how many volume segments are active.
	<ol>
		<li>If all segments are not active, adjust the catheter position until all segments are active. NOTE:&#160;Slight twisting of the catheter may be necessary to optimize loop morphology</li>
		<li>If unable to obtain signals in all segments, adjust the location of excitation electrodes and sensing electrodes per manufacturer instructions.</li>
	</ol>
	</li>
	<li>Once the desired configuration is obtained, follow the manufacturer&#8217;s instructions for volume and alpha calibration.</li>
	<li>Using a properly calibrated catheter, obtain at least 30 sec&#160;of baseline pressure-volume data. NOTE: These pressure-volume loops will provide volume dependent measurements of cardiac function (e.g., cardiac output, stroke volume).
	<ol>
		<li>Once sufficient loops are obtained, continue to the next step without stopping the data recording, so as to obtain occlusion pressure-volume data.</li>
	</ol>
	</li>
	<li>Occlude the preload tube slowly using a tubing clamp. NOTE: The pressure-volume loops should begin to become smaller and shift down and to the left.&#160;This is called the &#8220;walk down&#8221;.
	<ol>
		<li>Obtain 10-15 sec&#160;of the walk down, then release the tubing clamp to allow for preload to reenter the left atrium. NOTE: These pressure-volume loops will provide volume independent measurements of cardiac function (e.g., preload recruitable stroke work, end systolic pressure-volume relationship).</li>
		<li>Stop recording data by hitting the &#8220;Stop&#8221; button at the top right corner of the screen.</li>
		<li>Wait at least 5 min&#160;before repeating the occlusion.</li>
	</ol>
	</li>
	<li>Repeat steps 7.7 and 7.8 to obtain replicate measurements.</li>
</ol>

<ol>
	<li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> Journal of Pharmacological and Toxicological Methods. 55, 113-126 (2007).</li><li>Cheung, P. 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L., 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=Sensitivity+and+specificity+of+isolated+perfused+guinea+pig+heart+to+test+for+drug-induced+lengthening+of+QTc.">Sensitivity and specificity of isolated perfused guinea pig heart to test for drug-induced lengthening of QTc.</a> Journal of Pharmacological and Toxicological Methods. 49, 15-23 (2004).</li><li>Lee, M. S., Lill, M., Makkar, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+low+oxygen-carrying+capacity+of+Krebs+buffer+causes+a+doubling+in+ventricular+wall+thickness+in+the+isolated+heart.">The low oxygen-carrying capacity of Krebs buffer causes a doubling in ventricular wall thickness in the isolated heart.</a> Canadian Journal of Physiology and Pharmacology. 83, 174-182 (2005).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+the+Langendorff+technique+of+isolated+heart+perfusion.">Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.</a> Journal of Molecular and Cellular Cardiology. 50, 940-950 (2011).</li><li>Hearse, D. J., Sutherland, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+the+study+of+cardiovascular+function+and+disease.">Experimental models for the study of cardiovascular function and disease.</a> Pharmacological Research: the Official Journal of the Italian Pharmacological Society. 41, 597-603 (2000).</li><li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+blood+and+perfusion+fluid+perfused+heart.">The isolated blood and perfusion fluid perfused heart.</a> Pharmacological Research: the Official Journal of the Italian Pharmacological Society. 41, 613-627 (2000).</li><li>Hill, A. 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=In+vitro+studies+of+human+hearts.">In vitro studies of human hearts.</a> Ann Thorac Surg. 79, 168-177 (2005).</li><li>Colah, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+perfusion+of+the+swine+heart+as+a+method+for+pre-transplant+assessment.">Ex vivo perfusion of the swine heart as a method for pre-transplant assessment.</a> Perfusion. 27, 408-413 (2012).</li><li>Ozeki, T., 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=Heart+preservation+using+continuous+ex+vivo+perfusion+improves+viability+and+functional+recovery.">Heart preservation using continuous ex vivo perfusion improves viability and functional recovery.</a> Circ J. 71, 153-159 (2007).</li><li>Garbade, 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=Functional,+metabolic,+and+morphological+aspects+of+continuous,+normothermic+heart+preservation:+effects+of+different+preparation+and+perfusion+techniques.">Functional, metabolic, and morphological aspects of continuous, normothermic heart preservation: effects of different preparation and perfusion techniques.</a> Tissue engineering. 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The authors have nothing to disclose.

The Langendorff isolated heart perfusion apparatus and working heart model have led to some of the most fundamental discoveries in cardiac physiology, pathology, and pharmacology. This model&#8217;s versatility allows for its use with a variety of species under a variety of normal and pathological conditions1-18. However, the isolated heart model is not commonly used for large mammals, especially human hearts, in part due to the increased complexity of both apparatus design and data collection. Therefore, the protocol presented herein demonstrates an attempt to improve these complexities that results in a relatively reproducible means of studying isolated porcine hearts.
A crucial component of our setup is the replacement of arterial compliance/afterload chamber with a centrifugal pump. This exchange allows for enhanced control of the coronary perfusion pressure and afterload in Langendorff and working heart modes, respectively, allows this set-up to be easily adapted to hearts of different sizes and species. For example, in this design, porcine hearts are reperfused at 40-45 mmHg, while human hearts are reperfused at 60-65 mmHg. This change in pressure is achieved simply by adjusting the settings of the centrifugal pump; no component of the system needs to be physically adjusted. Furthermore, placing a pressure transducer within the aortic root to monitor root pressures enables easy transition between constant flow and constant pressure during Langendorff mode. Although this change removes the classic compliance chamber, the centrifugal pump, by allowing bidirectional flow occurs based upon the pressure gradient, may serve as a compliance chamber. With systole and ejected stroke volume, retrograde flow across the pump serves to diminish afterload pressure, replicating aortic elasticity.
The open design of this apparatus is also important. Having the heart hanging in an open area, instead of a semi-enclosed chamber or funnel, allows for easier instrumentation for pressure-volume measurements. The open design enables use of a transapical incision for LV catheter placement, avoiding of the transvalvular approach. The transvalvular approach is more technically difficult, and usually requires fluoroscopy for proper placement. Furthermore, this approach can also induce valvular insufficiency. By using the transapical approach, we safely and easily place the catheter within the left ventricle while eliminating the extra cost and inconvenience of fluoroscopy. The open design also affords easy access for echocardiography and effluent collection, further expanding the functional and biochemical parameters that can be assessed while on this system.
The open design, while facilitating data collection, does make myocardial temperature regulation more difficult. Maintaining physiologic temperature is one of the known issues with a Langendorff or working heart system1,3,11,13. The Langendorff system typically contains a thermal chamber that helps maintain a proper temperature, but this chamber also makes insertion of a ventricular pressure-volume catheter more difficult. To resolve the inferior temperature regulation of the open design, an oxygenator/heat exchanger was placed after the reservoir. The minimal space between the heat exchanger and the aortic cannula reduces heat loss, and the myocardial temperature probe ensures normothermia. The use of jacketed tubing or external heating sources can also be used to help with temperature control.
Another unique element of this protocol is washing the autologous blood of the pig under study and reconstituting it with normal saline. Although, the use of either whole blood perfusates or red blood cells augmented with crystalloid buffers is not uncommon, it does present with issues. The former usually requires a donor animal, which adds substantial costs to the experiment, while the latter can have immunogenicity issues, since it usually is derived from bovine blood1,11-13. By washing the original pig&#8217;s own blood, the protocol only requires a single animal and immunogenicity issues are ablated. Also, the washing process removes most of the electrolytes, meaning they can be easily manipulated per the experimental parameters. Finally, using a blood conservation unit removes most of the proteins within the blood, which is both an advantage and disadvantage of this process. The advantage is that any coagulation and immunologic/infectious proteins are removed, decreasing the likelihood of clots or contamination. The disadvantage is that this mixture has a low oncotic pressure, which can to lead to myocardial edema and possibly loss of cardiac function over time. This issue can be addressed, however, through the addition of albumin or another colloid.
Ensuring that a properly sized animal and catheter have been selected is as important as using the proper working heart apparatus. Ideally, the catheter will be placed with all sensing electrodes inside the ventricular space, with two excitation electrodes (i.e. the most proximal electrodes) outside of the ventricular space. If the animal&#8217;s ventricular cavity is too small, or the spacing between the electrodes is too large, then all segments will not fit within the LV space. While the location of the excitation electrodes can be adjusted, a small LV cavity can also cause the catheter to bend or curve, making data collection difficult. Therefore, for functional analysis of large animal hearts, an animal size of at least 60 kg is recommended. With an animal of this size, electrode spacing of 7 mm usually allows for complete insertion of the catheter.
In conclusion, this manuscript describes an isolated working heart system that simplifies perfusion pressure regulation, data collection, and overall design, while making temperature control only slightly more difficult. These modifications to the isolated working heart will hopefully allow for its increased usage with large mammalian hearts, including humans, furthering our understanding of cardiac pathology and enabling more clinically-relevant treatment options to be discovered.

Much of our understanding of basic cardiac biology and physiology has come from experiments that utilized the isolated, retrograde-perfused Langendorff heart and the isolated working heart systems. These experimental systems are still widely used today to extend our cardiovascular knowledge of important topics, including ischemia-reperfusion injury2, preconditioning4, cell based therapy for damaged myocardium5,7, the cardiac effects of drugs6,9, and cardiac allograft preservation techniques8,15-18.
While both isolated heart systems can be used for any mammalian species, they are primarily used on small mammals, such as guinea pig, rat, or rabbit3,12,13. Larger animal models, such as pigs and humans, provide more clinically-relevant data, but are less frequently used due to higher cost, greater biological variability, larger volumes of blood perfusion solutions, and bigger pieces of equipment1,12-15. Furthermore, data collection is more difficult, especially for isolated working hearts1,3,12-15. As a result of these complexities, clinically-relevant isolated heart models are rarely used, severely hampering the progress of cardiovascular translational research.
In an attempt to resolve these complexities, the isolated working heart preparation was modified to create a system that can be easily adapted to hearts of different species, including human, under either constant pressure or constant flow Langendorff conditions. The afterload compliance chamber was replaced with a centrifugal pump to simplify the process of adjusting perfusion pressure in Langendorff mode and afterload in working mode. Instead of an enclosed, jacketed reservoir to contain the heart, this system uses an open chamber to make data collection easier, by enabling use of the transapical approach for conductance catheterization. Moreover, this open design allows access for echocardiographic assessment of the heart, further broadening the physiologic parameters that can be measured during these experiments. These improvements will hopefully encourage others to use this system for large animal translational research.

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerLab 16/35 with LabChart Pro</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">PL3516/P</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MPVS Ultra Pressure-Volume Unit</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">880-0168</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ventri-Cath Catheter (5F, 12E, 7mm, DField, Straight, 122cm)</td>
			<td style="width:147px;">Millar</td>
			<td style="width:128px;">VENTRI-CATH-507s</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pressure Catheter (3.5F, Single, Straight, 100cm, Ny, Non Repairable)</td>
			<td style="width:147px;">Millar</td>
			<td style="width:128px;">SPR-524</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PV Extension Cable (10ft)</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">CEC-10PV</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catheter Interface Cable (10ft)</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">PEC-10D</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rho Calibration Cuvette</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">910-1060</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MPVS Ultra BNC Cable Pack</td>
			<td style="width:147px;">ADInstruments</td>
			<td style="width:128px;">880-0172</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autotransfusion system</td>
			<td style="width:147px;">Sorin</td>
			<td style="width:128px;">7320000</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bowl Set with Low Volume (135 ml) Centrifuge Bowl</td>
			<td style="width:147px;">Sorin</td>
			<td style="width:128px;">7135100</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygenator/Heat Exchanger</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:128px;">3CXSX18RX</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Perivascular flow probe</td>
			<td style="width:147px;">Transonic Systems</td>
			<td style="width:128px;">PAU Series</td>
			<td style="width:319px;">Size of flow probe will depend on animal size; for 60 kg pig, recommend 20 or 24 mm probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Perivascular flowmeter module</td>
			<td style="width:147px;">Transonic Systems</td>
			<td style="width:128px;">TS420</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Myocardial temerpature sensor</td>
			<td style="width:147px;">Smiths Medical</td>
			<td style="width:128px;">MTS-40015</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16 G 1&#34; Regular needle</td>
			<td style="width:147px;">BD Inc.</td>
			<td style="width:128px;">305197</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-0 polypropylene suture (double-arm)</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:128px;">8526H</td>
			<td style="width:319px;">For purse-string stitches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-0 polypropylene suture (single-arm)</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:128px;">8833H</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable ties</td>
			<td style="width:147px;">ULINE</td>
			<td style="width:128px;">S-1021</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable tie gun</td>
			<td style="width:147px;">ULINE</td>
			<td style="width:128px;">H-241</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Clear, Flexible PVC Tubing</td>
			<td style="width:147px;">VWR International</td>
			<td style="width:128px;">89068</td>
			<td style="width:319px;">Inner diameter depends on cannulas, pumps and other equipment used; most commonly use 1/4&#34;, 3/8&#34; tubing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight Tubing Connectors</td>
			<td style="width:147px;">VWR International</td>
			<td>46600</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Y-Shaped Tubing Connectors</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td style="width:128px;">6152</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jacketed Bubble Trap</td>
			<td style="width:147px;">Radnoti</td>
			<td style="width:128px;">14040</td>
			<td style="width:319px;">For preload chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal pump</td>
			<td style="width:147px;">Maquet</td>
			<td style="width:128px;">70105</td>
			<td style="width:319px;">The centrifugal pump and roller pumps were obtained used from perfusion department after clinical use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roller pumps</td>
			<td style="width:147px;">Maquet</td>
			<td style="width:128px;">HL-20</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemostasis Valve</td>
			<td style="width:147px;">Merit Medical</td>
			<td style="width:128px;">MAP150</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blood gas analyzer</td>
			<td style="width:147px;">Instrumentation Laboratory</td>
			<td style="width:128px;">570001000</td>
			<td style="width:319px;"></td>
		</tr>
	</tbody>
</table>

an isolated working heart system for large animal models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Figure 1 is a schematic drawing of the circuit, including suggested catheter placement. The important elements of this apparatus include the following: use of a centrifugal pump to control afterload; placement of a pressure catheter (dark blue line) in the aortic root to monitor perfusion pressure; and placement of the pressure-volume (PV) catheter (light blue line) transapically. Although the connections in the figure appear to be straight connections, &#8220;Y&#8221; connectors are recommended, especially for the preload line.
Figure 2 shows the data obtained from the pressure transducer that is placed in the aortic root of a porcine heart during reperfusion on the circuit, which is consistently between 40-42 mmHg for over 20 min. Changes in the coronary resistance can cause fluctuations in the perfusion pressure (Figure 3). These variations can be minor and gradual, correcting themselves over time (Figure 3a). However, in some cases these variations can be abrupt and require adjustment of the flow through the centrifugal pump to maintain the desired reperfusion pressure (Figure 3b). Because changes can occur, monitoring of the aortic root pressure during reperfusion is required.
By utilizing the transapical stab incision, pressure-volume data can be easily obtained on the isolated heart system. In this experiment, a porcine heart that had been stored in cold (4 &#176;C) preservation solution for 2&#160;hr&#160;was used. Upon initial introduction of the PV catheter, the loops were of poor quality (Figure 4a), with multiple areas of crossover and no discernible cardiac cycle components. However, with minimal manipulation of the catheter within the ventricle, the loop morphology improved dramatically (Figure 4b), allowing for measurements to be obtained.
Despite optimization of catheter position, the loops acquired on the ex&#160;vivo circuit (Figure 5, top row) may have a different morphology than the in&#160;vivo loops (Figure 5, bottom row). These changes to loop morphology are likely due to the different orientation of the heart on the circuit compared to in a supine animal, as well as the lack of the anatomical attachments found within a live animal (such as pericardium). Furthermore, the use of pacing wires to help regulate heart rate (recommended attachment site: interventricular septum) introduces an external electrical current, leading to the spikes seen in the bottom right portion of the ex&#160;vivo loops. However, as long as these loops still feature the cardiac cycle components, they can still yield interpretable data. Table 1 lists the multiple functional parameters obtained from these pressure-volume loops using the PV catheter. The cold static storage likely caused some intrinsic damage to the heart, which helps explain some of the changes in the values obtained on the circuit compared to the in-vivo measurements. Some of the variation within the load dependent variables is also due to the likely differences in the preload between the circuit and the live animal.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig1highres.jpg" width="500" /> 
Figure 1. Diagram of apparatus.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig2highres.jpg" width="500" /> 
Figure 2. Representative aortic root pressure measurements during reperfusion.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig3highres.jpg" width="500" /> 
Figure 3. Examples of changes to aortic root pressure that may occur during reperfusion. These changes can be gradual and self-correcting (A), or abrupt and require changes to the settings on the centrifugal pump (B).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig4highres.jpg" width="500" /> 
Figure 4. Pressure-volume loops obtained upon initial insertion of the catheter transapically (A) and after minor catheter manipulation (B). Note the improvement of the loop morphology, whereby the loop crossover is eliminated and the elements of the cardiac cycle are recognizable. The spikes in the bottom right portion of both sets of loops are due to the use of a pacer, which introduces an extrinsic electric signal.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51671/51671fig5highres.jpg" width="500" /> 
Figure 5. Representative pressure-volume measurements taken on the ex&#160;vivo circuit (top row), with in&#160;vivo measurements (bottom row) for comparison. Again, pacer spikes can be seen in the bottom right of both sets of ex&#160;vivo loops.
<img alt="Table 1" fo:content-width="5in" src="/files/ftp_upload/51671/51671table1.jpg" width="500" /> 
Table 1. Functional parameters obtained for a porcine heart in&#160;vivo (left column) and on the working heart apparatus after 2&#160;hr&#160;of cold storage (right column). CO: cardiac output; Ea: arterial elastance; EDPVR: End diastolic pressure-volume relationship; EDV: end diastolic volume; ESPVR: End systolic pressure-volume relationship; PRSW: Preload-recruitable stroke work; PVA: pressure-volume area; SV: stroke volume; SW: stroke work.

Watch this Scientific Journal Video about An Isolated Working Heart System for Large Animal Models at JoVE.com

An Isolated Working Heart System for Large Animal Models

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

an-isolated-working-heart-system-for-large-animal-models

Research

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

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

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

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

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

A 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 ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...