All animal experiments in this protocol were approved by the Institutional Animal Care and Use Committee at the University of Michigan, Ann Arbor. All mice were&#160;housed at a 12:12 light cycle in pathogen-free rooms. See the Table of Materials for details related to all materials, animals, and equipment used in this protocol.
1. Construction of the silicone balloon catheter
NOTE: The silicone balloon is made as described previously13.
<ol>
	<li>Add 9.5 mL of distilled water, 14.2 mL of light corn syrup, and 33.8 g of sucrose to a 100 mL beaker. Heat and stir the solution until the sugar is completely dissolved.</li>
	<li>Prepare the dough by mixing 10 g of wheat flour and 5 g of water until an even consistency is achieved and allow it to rest for 10 min.</li>
	<li>Shape a small piece of dough into an oval-the &#34;head&#34;-and attach it to the end of a dry spaghetti strand. Then, dip this head into the sugar solution and slowly remove from the solution, as the head is now completely coated. 
	NOTE: The dough should be smooth and of even texture. The sizes of dough can be varied to generate different sizes of balloons, from 5 mm (short diameter) to 7 mm (long diameter). Try to cover them with a thin film of sugar solution.</li>
	<li>Suspend the spaghetti strand on a polystyrene foam block, or other holders, to form a glossy cover evenly over the head and dry overnight.</li>
	<li>Dip the mold into silicone dispersion (silicone elastomer dispersed in xylene). Place the spaghetti strand back into the polystyrene foam block at 37 &#176;C for 2 h or until dry. Repeat this step once. 
	NOTE: It is essential to prevent the silicone dispersion gel from oxidizing due through air exposure, as this will generate an uneven balloon thickness.</li>
	<li>Place the mold into the water to separate and collect the balloon. Store the balloon in 0.02% sodium azide.</li>
	<li>Cut a two-blunt-end tip from a 22 G needle; mount one blunt end to the silicone balloon and another blunt end to PE tubing. Use 4-0 silk to tie the balloon in place on the needle. 
	&#8203;NOTE: Test the balloon integrity by injecting water into it. Once the balloon is filled, press the balloon softly to test if the balloon maintains tension inside. Use a new balloon if it is leaking. The mounted balloons can be stored for future use.</li>
</ol>
2. Preparation of the heart perfusion system
<ol>
	<li>Make 1 L of Krebs-Henseleit (KH) perfusion buffer and transfer it to the water reservoir Langendorff system.</li>
	<li>Connect the air tube to the water reservoir and turn on the airflow to balance the KH buffer with 5% CO2 and 95% O2 for at least 30 min.</li>
	<li>Set the water bath at 41.5 &#176;C and circulate the water in the outer layer of the Langendorff system to warm up the system and KH buffer. 
	&#8203;NOTE: The water bath temperature requires optimization for each system. For this system, the water bath temperature will maintain the KH at 37-37.5 &#176;C when perfusing into the heart.</li>
</ol>
3. Isolation, mounting, and cannulating of the mouse heart
<ol>
	<li>For anticoagulation, adminster 200 units of heparin in Saline by intraperitoneal (i.p.)&#160;injection into the right-hand quadrant of C57/B6 mouse abdomen.&#160;Aspirate the syringe before injection to confirm the bevel of the needles&#160;is not in the bladder or lumen of the GI tract.&#160;Use at least four mice in each experimental condition (but also consider the size of the treatment effect).
	<ol>
		<li>After 30 min, administer 80 mg/kg ketamine and 10 mg/kg of xylazine i.p. to anesthetize the mouse. Check if the anesthetized mouse is unconscious by performing a toe pinch and ensuring no response is observed.&#160;Acepromazine 2mg/kg can be added&#160;to the ket/xyl cocktail if the used strain&#160;of mice does&#160;not reach&#160;an adequate level of anesthesia with ket/xyl only.</li>
		<li>Make an incision right below the sternum. Use scissors to open the chest by cutting the diaphragm and the ribs. Fold the anterior chest wall to fully expose the chest. Cut at the descending aorta (closed to the aorta arch). Transfer the heart, lungs, and thymus of the mouse to cold histidine-tryptophan-ketoglutarate (HTK) buffer. Isolate the organs under ice-cold HTK buffer. Expose the aorta by removing any connective tissues. 
		NOTE: Maximize the aorta length by including both the ascending aorta and aortic arch region in the excision to allow enough space to connect to a needle.</li>
	</ol>
	</li>
	<li>Connect the end of the aorta to a 22 G needle, and tie with a 6-0 silk suture. Ensure the cannula is above the aortic root so as not to interfere with the aortic valve. Perfuse the aorta with 10 mL of cold (4 &#176;C) HTK buffer over approximately 10 min. 
	NOTE: It takes less than 15 min from heart removal to cannulation of the aorta; However, it is important to keep the perfusion speed at the appropriate level. Injections that are too fast and vigorous can generate high pressures and cause vascular/cardiac damage.</li>
	<li>Store the heart in a 50 mL tube with ice-cold HTK for 8 h or immediately perform the perfusion (do not store the control) and avoid direct contact with ice. 
	NOTE: Direct contact of the heart tissue with ice may lead to cold injury.</li>
	<li>Connect the needle-mounted heart to the cannula in the Langendorff apparatus and tie it with a silk suture. 
	NOTE: To standardize the procedure, wait for a total of 3 min for the cannulation process before perfusion.</li>
	<li>Start the perfusion with a constant flow mode at 3 mL/min; then, change to constant pressure mode at 70-80 mmHg and adjust the heart to ~6 mL/min. 
	NOTE: Palpating the heart softly can help accelerate cardiac reanimation. If the perfusion flow rate is much higher than 6 mL/min at constant pressure mode, there could be a leak in the cannulation or the aorta valve may not be working properly. Adjust the connections to fix the leak. The constant flow mode overrides the vascular tone self-regulation of the heart. The constant pressure mode allows the heart to regulate its coronary perfusion flow. Therefore, the constant pressure mode will precisely measure the cardiac function and preservation quality of the heart.</li>
	<li>Connect a deflated, water-filled balloon to a pressure transducer and a water-filled syringe with a three-way tap. After a 15-20 min equilibration period, cut the right atrium (RA) and insert the balloon to the RV through the RA. Use tape to hold the balloon inside the RV. Minimize the open area of the RA to help constrain the balloon in the ventricle (see Figure 1 for the setup). 
	NOTE: A period of equilibration is necessary, as the cardiac contraction and relaxation are not stable at the beginning, and the measure is less accurate and representative. If the AV node is damaged during the opening of the RA, the heart will display frequent arrhythmias.</li>
	<li>After 20 min of RV functional data collection, cut the left atrium (LA) and insert a deflated water-filled balloon to the LV through the LA. Use tape to hold the balloon inside the LV. 
	&#8203;NOTE: The heart should maintain stable hemodynamics for more than 1.5 h.</li>
</ol>
4. Functional data recording
<ol>
	<li>Calibration of the pressure transducer
	<ol>
		<li>Fill a 10 mL syringe with warm saline and connect the syringe to the dome through a three-way tap. Open the tap and slowly fill the dome with saline, and then close all taps and remove the syringe. Attach the filled dome to the transducer; connect the pressure gauge to the third end of the three-way tap.</li>
		<li>In the recording software, select the Bridge Amp from the dropdown menu of the channel connecting to the transducer. Rename the channel as Perfused pressure. Click Zero to zero the transducer.</li>
		<li>Start the recording by clicking Start so that the transducer is now reading 0 mmHg. After several seconds of recording, slowly push the syringe and increase the pressure to 100. Click stop to stop recording.</li>
		<li>In the Units conversion dialog, select an area of recording for 0 mmHg and click the arrow to Point 1 and type 0 mmHg. Select area of recording for 100 mmHg, click the arrow to Point 2, and type 100 mmHg. Click OK to calibrate the transducer.</li>
	</ol>
	</li>
	<li>Rename the channel corresponding to the pressure transducer with the balloon as Ventricle pressure. Start the recording when the heart is connected to the system. After inserting the balloon into the ventricle, adjust the water volume in the balloon using a micrometer syringe through the three-way tap to maintain the end-diastolic pressure at 5-10 mmHg. 
	NOTE: The end-diastolic measure could decrease during the measurement, preferably starting at close to 10 mmHg.</li>
	<li>Rename an empty channel as dP/dt. From the dropdown menu, select Derivative | source channel as Ventricle Pressure. The channel will record the ratio of pressure change in the ventricular cavity during the contraction period.</li>
	<li>Select a stable period of measure, then click Setting on the Blood pressure module.
	<ol>
		<li>Select the Ventricle pressure as the input channel and click selection for a period of calculation | OK.</li>
		<li>Click Classifier view to remove the outlier cardiac cycle (e.g., abnormal cycle time or pressure).</li>
		<li>Click table view to generate the tables of the average of max dP/dt (contraction) and min dP/dt (relaxation) for the selected period. 
		NOTE: Store the recording file for each sample and save the table of average heart function for statistical analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kim, I. C., Youn, J. C., Kobashigawa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+of+heart+transplantation.">The past, present and future of heart transplantation.</a> Korean Circulation Journal. 48 (7), 565-590 (2018).</li><li>Gaffey, A. C., 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=Transplantation+of+&amp;#34;high-risk&amp;#34;+donor+hearts:+Implications+for+infection.">Transplantation of &amp;#34;high-risk&amp;#34; donor hearts: Implications for infection.</a> The Journal of Thoracic and Cardiovascular Surgery. 152 (1), 213-220 (2016).</li><li>Hsich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matching+the+market+for+heart+transplantation.">Matching the market for heart transplantation.</a> Circulation: Heart Failure. 9 (4), 002679 (2016).</li><li>Piperata, A., 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+transplantation+in+the+new+era+of+extended+donor+criteria.">Heart transplantation in the new era of extended donor criteria.</a> Journal of Cardiac Surgery. 36 (12), 4828-4829 (2021).</li><li>Huckaby, L. V., Hickey, G., Sultan, I., Kilic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+utilization+of+marginal+donors+for+orthotopic+heart+transplantation.">Trends in the utilization of marginal donors for orthotopic heart transplantation.</a> Journal of Cardiac Surgery. 36 (4), 1270-1276 (2021).</li><li>Singh, S. S. A., Dalzell, J. R., Berry, C., Al-Attar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+graft+dysfunction+after+heart+transplantation:+a+thorn+amongst+the+roses.">Primary graft dysfunction after heart transplantation: a thorn amongst the roses.</a> Heart Failure Reviews. 24 (5), 805-820 (2019).</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 (6), 940-950 (2011).</li><li>Rossello, X., Hall, A. R., Bell, R. 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=Characterization+of+the+Langendorff+perfused+isolated+mouse+heart+model+of+global+ischemia-reperfusion+injury:+impact+of+ischemia+and+reperfusion+length+on+infarct+size+and+LDH+release.">Characterization of the Langendorff perfused isolated mouse heart model of global ischemia-reperfusion injury: impact of ischemia and reperfusion length on infarct size and LDH release.</a> Journal of Cardiovascular Pharmacology and Therapeutics. 21 (3), 286-295 (2016).</li><li>Matsuura, H., 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=Positive+inotropic+effects+of+ATP+released+via+the+maxi-anion+channel+in+Langendorff-perfused+mouse+hearts+subjected+to+ischemia-reperfusion.">Positive inotropic effects of ATP released via the maxi-anion channel in Langendorff-perfused mouse hearts subjected to ischemia-reperfusion.</a> Frontiers in Cell Development Biology. 9, 597997 (2021).</li><li>Tse, G., Hothi, S. S., Grace, A. A., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+arrhythmogenesis+following+slowed+conduction+in+heptanol-treated,+Langendorff-perfused+mouse+hearts.">Ventricular arrhythmogenesis following slowed conduction in heptanol-treated, Langendorff-perfused mouse hearts.</a> The Journal of Physiological Sciences. 62 (2), 79-92 (2012).</li><li>Kobashigawa, 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=Report+from+a+consensus+conference+on+primary+graft+dysfunction+after+cardiac+transplantation.">Report from a consensus conference on primary graft dysfunction after cardiac transplantation.</a> The Journal of Heart and Lung Transplantation. 33 (4), 327-340 (2014).</li><li>Lei, I., 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=Differential+inflammatory+responses+of+the+native+left+and+right+ventricle+associated+with+donor+heart+preservation.">Differential inflammatory responses of the native left and right ventricle associated with donor heart preservation.</a> Physiological Reports. 9 (17), 15004 (2021).</li><li>Miller, A., Wright, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+murine+ventricular+balloons+for+the+Langendorff+heart+preparation.">Fabrication of murine ventricular balloons for the Langendorff heart preparation.</a> Journal of Biotechnology &amp; Biomaterials. 1 (101), (2011).</li><li>Madsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+immunology+of+heart+transplantation.">Advances in the immunology of heart transplantation.</a> The Journal of Heart and Lung Transplantation. 36 (12), 1299-1305 (2017).</li><li>Voelkel, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+function+and+failure:+report+of+a+National+Heart,+Lung,+and+Blood+Institute+working+group+on+cellular+and+molecular+mechanisms+of+right+heart+failure.">Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure.</a> Circulation. 114 (17), 1883-1891 (2006).</li></ol>

The authors do not have any conflicts of interest to disclose.

This protocol describes the retrograde perfusion Langendorff method via aortic cannulation. This technique can be used to evaluate the LV and RV function of murine hearts after cold storage. The results show that the prolonged cold storage of donor hearts leads to reduced cardiac function in both the LV and RV using this protocol. 
The studies of acute and chronic rejection after heart transplantation widely focus on immunobiology14. The effects of native cells on PGD during cold storage are less well examined. PGD occurs in ~10%-20% of heart transplants and accounts for 66% of early death within 30 days following transplantation. In particular, the incidence of PGD affecting the LV versus the RV differ after transplantation11. Without the contribution of recipient cellular responses, this ex vivo method focuses on the contributions of native cardiac cells to PGD after cold preservation of donor hearts. Further studies may incorporate recipient responses in a murine heart transplant model. 
In this protocol, the Langendorff perfusion of cold preserved donor hearts focused on the native cardiac responses to warm crystalloid perfusion without infiltrating cellular immunity. To achieve reproducible results, several critical steps were standardized. The mouse hearts were arrested using HTK solution and stored in ice-cold HTK, similar to clinical practice. The perfusion volume and infusion time of the HTK solution for every heart was closely monitored with a timer. The donor heart was kept in prechilled tubes on ice containing HTK in a 4 &#176;C room. The cannulation time waas standardized to ~3 min prior to perfusion. All these steps ensured that cold preservation duration was the major variable in the study. 
A period of irregular cardiac contractility for ~20 min was commonly seen at the beginning of perfusion. This equilibration and recovery period was facilitated by gradual warming and oxygenation of cardiac tissues. A relatively stable period was expected after the initial 20 min. The balloon was inserted into the ventricle cavity at ~18 min after the initial equilibration period. We started recording hemodynamics after the heart was stable for ~25 min, once the balloon was inserted. Perfusion with KH buffer maintained stable cardiac performance for ~1.5-2 h. We therefore elected to record hemodynamics for 20 min in each of the left and right ventricles.
There are several limitations of retrograde perfusion for studying the PGD of hearts after cold storage. First, due to the balloon size and a lack of space in each ventricular cavity (in particular, the RV), the simultaneous insertion of two balloons into both the LV and RV is very challenging. Thus, we measure the function of RV and LV sequentially. It is important to note that the interventricular septum contributes significantly to both left and right ventricular function. The septum contributes to ~50% of right ventricular function, so there is interventricular dependence15. It is also important to note that, while the procedures for reperfusion of the murine heart in the Langendorff device take ~3 min, surgical implantation of the human heart in the relatively warm surgical field takes ~45 min. In comparison, the murine heart in this Langendorff system incurs less ischemic time. This should be taken into account when considering clinical translation.
Since we used KH buffer to perfuse the heart without blood, this may also have less efficiency in oxygen delivery. However, the heart function is relatively stable through the initial 1.5-2 h of perfusion, thus allowing reliable hemodynamic measurements. Unfortunately, there are currently no viable working heart perfusion models for these smaller murine hearts, and the effect of ventricular loading cannot be evaluated in this system. Despite this, the perfusion system is highly reproducible and less labor-intensive and time-consuming than transplant models. It is also less costly than transplant studies, which may make it more suitable for screening different therapeutic options and various molecular pathways. With modifications to preservation solutions by adding candidate drugs, this platform can be used to evaluate the effects of pharmacological agents on reducing PGD in both the LV and RV.

Heart transplantation improves survival and the quality of life in patients with end-stage heart failure1. Unfortunately, the shortage of heart donors limits the number of patients who could benefit from this therapy and limits the ability of clinicians to optimally match donors with recipients2,3,4. Furthermore, the new allocation system has contributed to longer ischemic times and significantly increased the use of marginal donors since 20185. Consequently, the mean age of heart donors and the ischemic time is increasing over time, leading to a higher rate of primary graft dysfunction (PGD) despite significant improvements in the strategies for heart preservation 6.
PGD can affect the left, the right, or both ventricles, and remains a life-threatening complication that represents the leading cause of early deaths after heart transplantation. Investigating the mechanisms of PDG and the development of strategies for better heart preservation are important considerations, given the potential life-saving impact on heart recipients. Therefore, experimental models that allow a robust and reliable assessment of donor heart function after a prolonged storage time are essential to increase our understanding of PGD and facilitate the development of novel therapies. The ability to accurately assess cardiac function in the mouse heart allows access to a vast repertoire of transgenic murine models that can accurately identify PGD mechanisms.
In physiologic and pharmacologic studies, the Langendorff retrograde perfusion model is widely used to assess heart function7. Specifically, cardiac performance is detected by a silicone balloon connected to a pressure transducer within the left ventricular (LV) cavity. A key feature of PGD is the inadequate contraction and relaxation of the ventricular muscle. Prior Langendorff studies have focused on using an LV balloon to produce reliable and reproducible results in LV functional assessment8,9,10. However, the use of an intracavitary balloon to assess right ventricular (RV) function using the balloon system is less well recognized.
Given a significant PGD rate involving the RV after transplantation11, experimental methods to study both LV and RV function would help determine the molecular and physiological mechanisms that contribute to RV PGD. This protocol shows that intracavitary silicone balloons can provide reliable assessments of LV and RV function in the same murine heart12. To evaluate the potential use of the Langendorff system in the PGD study, we examined the heart functions with different periods of storage and found decreased cardiac function in contraction and relaxation with the prolonged cold storage of murine hearts. Interestingly, the LV has a higher functional reduction than the RV. In summary, the protocol described here can be used for assessing the effect of a candidate drug and molecular pathways on both LV and RV function. The ability to use this method on murine hearts will facilitate the performance of detailed mechanistic studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-0 silk suture</td>
			<td>Braintree Scientific</td>
			<td>SUTS108</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Braintree Scientific</td>
			<td>SUTS104</td>
			<td></td>
		</tr>
		<tr>
			<td>All purpose flour</td>
			<td>Kroger</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD General Use and PrecisionGlide Hypodermic Needles 22 G</td>
			<td>Fisher scientific</td>
			<td>14-826-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Syringe with Luer-Lok Tips (Without Needle)</td>
			<td>Fisher scientific</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn Syrup</td>
			<td>Kroger</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol HTK Solution</td>
			<td>Essential Pharmaceuticals LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors&#160;</td>
			<td>World Precision Instruments</td>
			<td>14393/14394</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical tubes</td>
			<td>Fisher scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa&#160;&#160;</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs Henseleit buffer</td>
			<td>Sigma</td>
			<td>K3753</td>
			<td></td>
		</tr>
		<tr>
			<td>Nusil silicone dispersions</td>
			<td>Avantor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion system</td>
			<td>Radnoti</td>
			<td>130101BEZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab</td>
			<td>ADInstruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma</td>
			<td>X1126</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of ex vivo murine biventricular function in a langendorff model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Adult C57Bl/6 mouse hearts, 3 months of age, were harvested and mounted to the Langendorff system. The donor heart was stored in HTK for 0 and 8 h, and then perfused with oxygenated KH buffer. A silicone balloon connecting to a pressure transducer was used to measure the contraction and relaxation of LV and RV function.
The aortic pressures were maintained in the 70-80 mmHg range. The heart rate was comparable in mouse hearts with 0 and 8 h of storage. The LV and RV function were examined by measuring systolic and diastolic pressure. dP/dt, a derivative to calculate the ratio of pressure change, was calculated to determine the pressure dynamics. The absolute number of max dP/dt and min dP/dt could represent the level of muscular contraction and relaxation. At 0 h of storage, the LV had higher systolic pressure compared to the RV (Figure 2C and Figure 3A). The LV showed more muscular contraction and relaxation than the RV after perfusion of 0 h storage (Figure 2C and Figure 3B,C). However, after 8 h of cold storage, both the LV and RV showed a significant functional reduction compared to a 0 h baseline (Figure 2A-D and Figure 3B,C). The decreases in cardiac contraction were more severe in the LV. After 8 h of storage, the contraction and relaxation of the LV was 25.1% and 30.7% of the 0 h baseline, while the RV had 32.5% and 29.1% of function compared to the 0 h baseline (Figure 3B,C). These results showed that the PGD of the LV after prolonged storage had a more significant cardiac contraction reduction than the RV.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64384/64384fig01.jpg" /> 
Figure 1: Mounting and cannulation of the mouse heart. (A) Overall setup of the perfusion setup. 1. Perfusion reservoir. 2. Oxygenation chamber. 3. Air trap chamber. 4. Heart chamber. 5. Value switch for constant flow and pressure. 6 and 7. Oxygen inflow. (B) Cannulated hearts with the RV in the front. (C) Position of the RV to cut for opening its cavity. (D) Tap the balloon tube with the cannula. Abbreviation: RV = right ventricle. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64384/64384fig02.jpg" /> 
Figure 2: Comparing the function of the LV versus RV. (A) Tracing record of max and min dP/dt in the RV and LV in the donor heart with 0 h of storage. (B) The record of max and min dP/dt in the RV and LV in the donor heart with 8 h of storage. (C,D) Details of dP/dt, LV pressure, heart rate, and perfusion pressure in the LV and RV at 0 h and 8 h. Abbreviations: RV = right ventricle; LV = left ventricle; dP/dt = pressure-time relationship. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64384/64384fig03.jpg" /> 
Figure 3: Comparing the function of the LV versus RV after storage and perfusion. (A) Systolic and diastolic pressure of the LV and RV after 0 h and 8 h of storage. (B) Max dP/dt and (C) Min dP/dt of the LV and RV after perfusion with 0 h and 8 h of storage. This figure is from Lei et al.12. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model at JoVE.com

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

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1.9K Views.  University of Michigan, Ann Arbor. Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system. 

Video: Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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 ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...