This work was supported by a grant from the NIH (R01-HL095828 to M. W. Kay).

1. Setting Up for the Study
<ol>
	<li>Prepare four liters of modified Krebs-Henseleit solution 16 (in mM: 118 NaCl, 3.30 KCl, 2.00 CaCl2, 1.20 MgSO4, 24.0 NaHCO3, 1.20 KH2PO4, 10.0 glucose, 2.00 NaPyruvate, and 20.0 mg/L albumin). The solution should be prepared as close to the start of the experiment as possible. The pH should be adjusted to 7.4 after sterile filtering (pore size: 22 &mu;m, Corning). Solution osmolality should be between 275 and 295 mOsm/kg.</li>
	<li>Rinse all tubes and chambers of the working heart system with purified water. Run pumps until all water has been removed from the system.</li>
	<li>Add cellulose membrane filters (pore size: 5 &mu;m, Advantec) in line with each of the perfusion pumps (Langendorff perfusion pump, left heart perfusion pump, and right heart perfusion pump).</li>
	<li>Perform a two-point calibration (0 and 60 mmHg) for each pressure sensor.</li>
	<li>Turn on the water baths. A heated circulating water bath (Cole Palmer) is used to warm the water-jacketed tubes and heat exchangers. Perfusate is pre-warmed in a separate water bath (Oakton Instruments). Both baths are set to maintain a solution temperature of 37 &deg;C.</li>
	<li>Turn on the pumps to circulate the perfusate in a closed loop. Perfusate passes through microfiber oxygenators (hemofilters) gassed with 95% O2 and 5% CO2 at 80 kPa. Oxygenated perfusate then flows through heat exchangers to maintain it at a temperature of 37 &deg;C before entering the heart cannulae.</li>
</ol>
2. Heart Excision
<ol>
	<li>Begin by setting the working heart system to operate in constant pressure Langendorff mode. Set the pressure of the aortic block within the range of 50 to 60 mmHg.</li>
	<li>Anesthetize the rabbit with an intramuscular injection of ketamine (44 mg/Kg) and xylazine (10 mg/Kg). After the rabbit is sedated, pentobarbital (50 mg/Kg) and heparin (2000 U) is intravenously injected via the marginal ear vein or the lateral saphenous vein on the inside of the hind limb.</li>
	<li>When the rabbit is completely non-responsive, as determined by a lack of pain reflex, the thoracic cavity is quickly opened, the pericardium is sliced, the aorta is clamped, and the heart and lungs are excised. At this point the lungs should be left attached to the heart to help with isolating the pulmonary veins.</li>
	<li>Isolate and cannulate the aorta with a 5 mm diameter cannula that is attached to a syringe filled with 60 mL perfusate and 200 units of heparin. Secure the aorta to the cannula with size zero silk suture and slowly depress the syringe to flush the heart of blood.</li>
</ol>
3. Biventricular Cannulation
<ol>
	<li>Connect the heart to the aortic block of the working heart system. Prevent air from entering the aorta, which can cause coronary emboli. It is best to attach the cannula to the aortic block by approaching the aortic connector at an oblique angle and allowing perfusate to gently drip from the connector into the cannula while it is attached.</li>
	<li>While the heart is perfused in constant pressure Langendorff mode, remove the fat and connective tissue and locate the following vessels: inferior and superior vena cava, azygos vein, pulmonary artery, pulmonary veins.</li>
	<li>Ligate the superior vena cava. Cut the pulmonary artery just below where it branches to the right and left pulmonary arteries.</li>
	<li>Group all the remaining vessels (the pulmonary veins) between the heart and lungs and ligate them all using one suture. Remove the lungs.</li>
	<li>Cut a small hole in the corner of the left atrial appendage. Ensure that the LA is filled with perfusate. Cannulate the LA while ensuring that the cannula is completely filled with perfusate while it is inserted. Suture the cannula to the LA appendage.</li>
	<li>Turn on the left side pump (pump #2) to provide flow to the left atrium. Set preload pressure between 2 - 6 mmHg and adjust &plusmn;2 mmHg, as determined by atrial dilation.</li>
	<li>Switch the heart to the working heart mode by turning off the Langendorff pump (pump #1).</li>
	<li>Momentarily decrease the aortic pressure to 10 mmHg and then slowly increase it to within the range of 80 to 100 mmHg. This will allow the aortic valve to open and function as it would during normal physiologic conditions. The final afterload pressure will depend upon the contractility of the LV. It should be set to a value that is approximately 20 mmHg less than peak LV pressure.</li>
	<li>LV cardiac output can be determined by measuring the flow rate of perfusate exiting the aortic block (mL/min). Normal cardiac output is between 14.77 and 16.43 mL/min per 100 g of body weight 17 and averages 340 mL/min for a 2.2 kg rabbit. Aortic pressure should resemble the pressure signal shown in Figure 1.</li>
	<li>Cannulate the RA through the inferior vena cava. Ensure that both the RA and the cannula are completely filled with perfusate and insert the cannula while preventing the formation of air bubbles. Suture the cannula to the vein.</li>
	<li>Turn on the right side pump (pump #3) to provide flow to the right atrium. Set the pressure to approximately 3 mmHg.</li>
	<li>Ensure that the RV is filled with perfusate and cannulate the pulmonary artery. Ensure that the cannula is completely filled with perfusate while it is inserted to prevent air bubbles. Suture the cannula to the pulmonary artery.</li>
</ol>
4. Signal Acquisition: Pressures, Monophasic Action Potentials, and fNADH
<ol>
	<li>Once biventricular cannulation is complete, carefully insert the pressure transducer catheter (Millar) into the aorta via the aorta cannula. Gently navigate it past the aortic valve and into the LV. Monitor the LV pressure signal to ensure proper positioning of the catheter tip. An example of LV pressure is shown in Figure 1.</li>
	<li>Gently press the monophasic action potential electrode against ventricular epicardium. Monitor the signal to achieve appropriate action potential measurements. Slight motion artifact in the signal is normal.</li>
	<li>Place a bipolar stimulus electrode on the right atrium to pace the heart. In our protocol, hearts were paced at cycle lengths between 300 and 150 msec, corresponding to 200 and 400 bpm, respectively.</li>
	<li>Measure the temperature of the LV epicardial surface. If the study requires that the temperature be maintained at 37 &deg;C then position the heart inside a water-jacketed heart chamber or submerge the heart in a warmed superfusate bath to maintain a constant temperature throughout the heart.</li>
	<li>Position the CCD camera (Andor iXon DV860, 128x128 pixels) and focus the lens such that an appropriate field of view is observed. The camera is connected to a workstation and images are acquired at 2 fps using Andor SOLIS software.</li>
	<li>Turn on the mercury lamp light prior to the start of imaging. Light is directed through an excitation filter (350&plusmn;25 nm, Chroma Technology) and into a fiber optic light guide (Horiba Jobin Yvon model 1950-1M) to illuminate the surface of the heart. The attenuation of UV light through the light guide is small. UV illumination could also be provided using a high power LED system consisting of LED spotlights (Mightex PLS-0365-030-S) and a control unit (Mightex SLC-SA04-US).</li>
	<li>Turn off the room light and minimize any ambient lighting. Aim the ferrules of the light guide (or LED spotlights) at the heart to achieve uniform epicardial illumination. Emitted NADH fluorescence (fNADH) passes through an emission filter (460&plusmn;20 nm Chroma Technology) and is imaged by the CCD camera.</li>
	<li>Monitor fNADH changes over time by selecting a region of interest using the imaging software. Select live-update mode to monitor the mean pixel intensity within the region of interest.</li>
	<li>The heart should be functioning in biventricular working mode to generate appropriate pressures. fNADH levels should be low and stable over the epicardial surface to confirm adequate coronary perfusion. At this point in the study a specific experimental protocol should be implemented to test a hypothesis.</li>
	<li>When the study is completed, remove the heart from the system and drain all perfusate. Rinse the system tubing and chambers with purified water. For routine maintenance, the system should be periodically rinsed with Mucasol solution or a diluted hydrogen peroxide solution, as needed.</li>
</ol>
5. Off-line Processing of fNADH Images
<ol>
	<li>One way to compare NADH datasets (fNADH(i,j,t)) between experiments is to normalize each fluorescence image using a reference image (fNADH(i,j,t0)) from the dataset9, as shown in the equation below. Another way to normalize NADH fluorescence is to place a small piece of uranyl glass in the field of view before the experiment9, 18, 19. Uranyl glass will fluorescence (450 - 550 nm) when illuminated with UV light to provide a signal that can be used as a stable reference.</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/4115/4115eq1.jpg" />
6. Representative Results
Anterior and basal views of a biventricular working rabbit heart preparation are shown in Figure 1. Left ventricular pressure was measured by navigating a pressure transducer catheter (Millar SPR-407) past the aortic valve and into the left ventricle. Aortic, pulmonary artery, and left ventricular pressures (LVP) are shown in Figure 1C. Diastolic LVP is usually between 0 and 10 mmHg. The minimum diastolic aortic pressure is approximately 60 mmHg. Peak systolic LVP is dependent upon filling pressure (the preload or LA pressure) and contractility and, optimally, should be between 80 and 100 mmHg. The maximum aortic pressure and maximum LVP should closely match, as shown in Figure 1C.
Monophasic action potentials (MAPs) with a fast depolarization phase and a repolarization phase that are typical for rabbit hearts are shown in Figure 1D. MAPs can be recorded relatively easily from a contracting heart but will usually have small motion artifact during diastole, as shown in Figure 1D. MAPs are useful for confirming successful entrainment of the heart (capture) during pacing and can also be used to measure local electrophysiological changes due to ischemia or other acute perturbations. An ECG could also be measured by submerging the heart in a bath of warm superfusate and placing an electrode in the bath on the left and right sides of the heart. A third indifferent electrode is either placed in the bath, away from the heart, or is attached to the aorta. An ECG will provide information regarding the global excitation and repolarization process, which is useful for evaluating overall electrical function and for revealing the presence of ischemia.
fNADH imaging reveals changes in the mitochondrial redox state of the heart, which can be used to measure the spatiotemporal progression of ischemic or hypoxic regions. For this study, epicardial fNADH was measured to monitor changes in redox state during three pacing rates at cycle lengths (CLs) of 300, 200, and 150 msec. Average fNADH values from a region of interest (red box, Figure 2) show that baseline fNADH levels increase as the cycle length is shortened. When pacing rate is close to sinus rhythm (CL=300 msec) baseline fNADH level is relatively constant. As cycle length is shortened below 300 msec, baseline fNADH levels increase, with the largest increase at the shortest CL (150 msec). High resolution fNADH imaging of the full anterior surface at 200 and 400 bpm is shown in Figure 3. fNADH levels at 200 bpm were constant and spatially homogeneous. At 400 bpm, fNADH levels increased substantially throughout the epicardium. Significant spatial heterogeneity was observed with the largest increases occurring within the septal regions of the RV and LV.
The fNADH signal oscillates with contraction (motion artifact) and the frequency of oscillation corresponds to heart rate (Figure 2). In biventricular cannulation, the base of the heart is held by 4 cannulae, which helps to prevent the heart from swinging during contraction. Therefore, oscillation amplitude is always less than any longer time scale (5-10 sec) trends in fNADH that are caused by ischemia or hypoxia.
<img alt="Figure 1" src="/files/ftp_upload/4115/4115fig1.jpg" /> 
Figure 1. Typical pressures and monophasic action potentials from an isolated biventricular working rabbit heart. A. Basal view of the heart showing the four cannulae: 1, aortic; 2, pulmonary artery; 3, left atrial; and 4, right atrial. B. Anterior view of the heart showing the left ventricle (LV) and the right ventricle (RV). C. Representative pressures. Top: left ventricular pressure (solid line) and the aortic pressure (dotted line). Bottom: pulmonary pressure. D. Representative monophasic action potentials. The signal is aligned with the pressures shown in panel C. <a href="https://www.jove.com/files/ftp_upload/4115/4115fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4115/4115fig2.jpg" /> 
Figure 2. fNADH imaging of an isolated biventricular working rabbit heart. Top: A cartoon of the field of view (left) and three fNADH images are shown. The corresponding pacing cycle length (CL) is indicated on each image. The region of interest for the fNADH signal in the bottom panel is indicated by the red box. The tip of the monophasic action potential electrode is seen to the right of the region of interest. The epicardium was illuminated using the mercury lamp and light guide, as shown in Figure 5. Only the epicardial surface surrounding the region of interest was illuminated. Bottom: Average fNADH for the region of interest indicated by the red box in the top panel. Average fNADH increases with reduced cycle length.
<img alt="Figure 3" src="/files/ftp_upload/4115/4115fig3.jpg" /> 
Figure 3. fNADH images of the full anterior surface of an isolated biventricular working rabbit heart. The heart was paced from the RA at 200 bpm and 400 bpm. fNADH was imaged (2 fps, 128x128 pixels at a resolution of 0.4 mm) while illuminating the entire anterior epicardium using two high power LEDs (Mightex PLS-0365-030-S, 365 nm, 4% intensity, 50 mW max).

<ol>
	<li>Langendorff, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+am+uberlebenden+saugethierherzen+[investigations+on+the+surviving+mammalian+heart].">Untersuchungen am uberlebenden saugethierherzen [investigations on the surviving mammalian heart].</a> Arch. Gesante Physiol. 61, 291-332 .</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to langendorff---still viable in the new millennium.</a> J. Pharmacol. Toxicol. Methods. 55, 113-126 (2007).</li><li>Neely, J. R., Liebermeister, H., Battersby, E. J., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pressure+development+on+oxygen+consumption+by+isolated+rat+heart.">Effect of pressure development on oxygen consumption by isolated rat heart.</a> Am. J. Physiol. 212, 804-814 (1967).</li><li>Feng, H. Z., Jin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+cardiac+troponin+T+variants+reduces+heart+efficiency.">Coexistence of cardiac troponin T variants reduces heart efficiency.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H97-H105 (2010).</li><li>Clemens, M. G., Forrester, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+adenosine+triphosphate+in+the+coronary+sinus+effluent+from+isolated+working+rat+heart+in+response+to+hypoxia.">Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working rat heart in response to hypoxia.</a> J. Physiol. 312, 143-158 (1981).</li><li>Cole, M. A., Murray, A. J., Cochlin, L. E., Heather, L. C., McAleese, S., Knight, N. S., Sutton, E., Jamil, A. A., Parassol, N., Clarke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+fat+diet+increases+mitochondrial+fatty+acid+oxidation+and+uncoupling+to+decrease+efficiency+in+rat+heart.+Basic+Res.">A high fat diet increases mitochondrial fatty acid oxidation and uncoupling to decrease efficiency in rat heart. Basic Res.</a> Basic Res. Cardiol. 106, 447-457 (2011).</li><li>Demmy, T. L., Curtis, J. J., Kao, R., Schmaltz, R. A., Walls, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load-insensitive+measurements+from+an+isolated+perfused+biventricular+working+rat+heart.">Load-insensitive measurements from an isolated perfused biventricular working rat heart.</a> J. Biomed. Sci. 4, 111-119 (1997).</li><li>Demmy, T. L., Magovern, G. J., Kao, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+biventricular+working+rat+heart+preparation.">Isolated biventricular working rat heart preparation.</a> Ann. Thorac. Surg. 54, 915-920 (1992).</li><li>Kay, M., Swift, L., Martell, B., Arutunyan, A., Sarvazyan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locations+of+ectopic+beats+coincide+with+spatial+gradients+of+NADH+in+a+regional+model+of+low-flow+reperfusion.">Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2400-2405 (2008).</li><li>Swift, L., Martell, B., Khatri, V., Arutunyan, A., Sarvazyan, N., Kay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+regional+hypoperfusion+in+langendorff+heart+preparations.">Controlled regional hypoperfusion in langendorff heart preparations.</a> Physiol. Meas. 29, 269-279 (2008).</li><li>Kay, M. W., Swift, L. M., Sangave, A., Zderic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+contrast+ultrasound+and+NADH+fluorescence+imaging+of+myocardial+perfusion+in+excised+rat+hearts.">High resolution contrast ultrasound and NADH fluorescence imaging of myocardial perfusion in excised rat hearts.</a> , 1-4 (2008).</li><li>Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyridine+nucleotide+as+an+indicator+of+the+oxygen+requirements+for+energy-linked+functions+of+mitochondria.">Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of mitochondria.</a> Circ. Res. 38, I31-I38 (1976).</li><li>Mayevsky, A., Rogatsky, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+in+vivo+evaluated+by+NADH+fluorescence:+From+animal+models+to+human+studies.">Mitochondrial function in vivo evaluated by NADH fluorescence: From animal models to human studies.</a> Am. J. Physiol. Cell Physiol. 292, C615-C640 (2007).</li><li>Barlow, C. H., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+areas+in+perfused+rat+hearts:+Measurement+by+NADH+fluorescence+photography.">Ischemic areas in perfused rat hearts: Measurement by NADH fluorescence photography.</a> Science. 193, 909-910 (1976).</li><li>Mayevsky, A., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+states+of+NADH+in+vivo:+From+animals+to+clinical+use.">Oxidation-reduction states of NADH in vivo: From animals to clinical use.</a> Mitochondrion. 7, 330-339 (2007).</li><li>Gillis, A. M., Kulisz, E., Mathison, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+electrophysiological+variables+in+blood-perfused+and+buffer-perfused,+isolated,+working+rabbit+heart.">Cardiac electrophysiological variables in blood-perfused and buffer-perfused, isolated, working rabbit heart.</a> Am. J. Physiol. 271, H784-H789 (1996).</li><li>Ôta, K., Peaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactation+in+the+rabbit:+Mammary+blood+flow+and+cardiac+output.">Lactation in the rabbit: Mammary blood flow and cardiac output.</a> Experimental Physiology. 64, 225-238 (1979).</li><li>Ashruf, J. F., Ince, C., Bruining, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+ischemia+in+hypertrophic+langendorff-perfused+rat+hearts.">Regional ischemia in hypertrophic langendorff-perfused rat hearts.</a> Am. J. Physiol. 277, H1532-H1539 (1999).</li><li>Ashruf, J. F., Coremans, J. M., Bruining, H. A., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+of+cardiac+work+is+associated+with+decrease+of+mitochondrial+NADH.">Increase of cardiac work is associated with decrease of mitochondrial NADH.</a> Am. J. Physiol. 269, 856-862 (1995).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ. Res. 95, 21-33 (2004).</li><li>Rogers, J. M., Walcott, G. P., Gladden, J. D., Melnick, S. B., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Panoramic+optical+mapping+reveals+continuous+epicardial+reentry+during+ventricular+fibrillation+in+the+isolated+swine+heart.">Panoramic optical mapping reveals continuous epicardial reentry during ventricular fibrillation in the isolated swine heart.</a> Biophys. J. 92, 1090-1095 (2007).</li><li>Qu, F., Ripplinger, C. M., Nikolski, V. P., Grimm, C., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+panoramic+imaging+of+cardiac+arrhythmias+in+rabbit+heart.">Three-dimensional panoramic imaging of cardiac arrhythmias in rabbit heart.</a> J. Biomed. Opt. 12, 044019 (2007).</li><li>Chinchoy, E., Soule, C. L., Houlton, A. J., Gallagher, W. J., Hjelle, M. A., Laske, T. G., Morissette, J., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+four-chamber+working+swine+heart+model.">Isolated four-chamber working swine heart model.</a> Ann. Thorac. Surg. 70, 1607-1614 (2000).</li><li>Hill, A. J., Laske, T. G., Coles, J. A., Sigg, D. C., Skadsberg, N. D., Vincent, S. A., Soule, C. L., Gallagher, W. J., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+human+hearts.">In vitro studies of human hearts.</a> Ann. Thorac. Surg. 79, 168-177 (2005).</li><li>Schenkman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+performance+as+a+function+of+intracellular+oxygen+tension+in+buffer-perfused+hearts.">Cardiac performance as a function of intracellular oxygen tension in buffer-perfused hearts.</a> Am. J. Physiol. Heart Circ. Physiol. 281, H2463-H2472 (2001).</li><li>Pijl, A. J., Pfaffendorf, M., Mathy, M., Van Zwieten, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+by+nifedipine+in+isolated+working+hearts:+A+comparative+study+on+three+different+types+of+experimental+ischemia.">Cardioprotection by nifedipine in isolated working hearts: A comparative study on three different types of experimental ischemia.</a> J. Cardiovasc. Pharmacol. 21, 70-76 (1993).</li><li>Khatib, S. Y., Boyett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+glyburide+(glibenclamide)+on+myocardial+function+in+langendorff+perfused+rabbit+heart+and+on+myocardial+contractility+and+slow+calcium+current+in+guinea-pig+single+myocytes.">Effects of glyburide (glibenclamide) on myocardial function in langendorff perfused rabbit heart and on myocardial contractility and slow calcium current in guinea-pig single myocytes.</a> Mol. Cell Biochem. 242, 81-87 (2003).</li><li>Kates, R. E., Yee, Y. G., Hill, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+albumin+on+the+electrophysiologic+stability+of+isolated+perfused+rabbit+hearts.">Effect of albumin on the electrophysiologic stability of isolated perfused rabbit hearts.</a> J. Cardiovasc. Pharmacol. 13, 168-172 (1989).</li><li>Combs, C. A., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+imaging+of+dehydrogenase+activity+within+living+cells+using+enzyme-dependent+fluorescence+recovery+after+photobleaching+(ED-FRAP).">Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP).</a> Biophys. J. 80, 2018-2028 (2001).</li><li>Coremans, J. M., Ince, C., Bruining, H. A., Puppels, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(Semi-)quantitative+analysis+of+reduced+nicotinamide+adenine+dinucleotide+fluorescence+images+of+blood-perfused+rat+heart.">(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.</a> Biophys J. 72, 1849-1860 (1997).</li><li>Scholz, T. D., Laughlin, M. R., Balaban, R. S., Kupriyanov, V. V., Heineman, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+substrate+on+mitochondrial+NADH,+cytosolic+redox+state,+and+phosphorylated+compounds+in+isolated+hearts.">Effect of substrate on mitochondrial NADH, cytosolic redox state, and phosphorylated compounds in isolated hearts.</a> Am. J. Physiol. 268, 82-91 (1995).</li><li>Holcomb, M. R., Woods, M. C., Uzelac, I., Wikswo, J. P., Gilligan, J. M., Sidorov, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+dual+camera+systems+for+multimodal+imaging+of+cardiac+electrophysiology+and+metabolism.">The potential of dual camera systems for multimodal imaging of cardiac electrophysiology and metabolism.</a> Exp. Biol. Med. (Maywood). 234, 1355-1373 (2009).</li></ol>

No conflicts of interest declared.

The isolated Langendorff perfused heart remains a prominent tool for studying cardiac physiology2. It is especially useful in studies of cardiac arrhythmias, particularly those that use fluorescence imaging of transmembrane potential20. An advantage is that the entire epicardium of the isolated heart can be observed21, 22. Another advantage is that, in contrast to blood, perfusion with a clear crystalloid buffer solution does not interfere with fluorescence signals. A limitation is that the Langendorff technique is not well-suited for studies of cardiac metabolism, which often require the heart to perform work within the context of physiologic preload and afterload pressures.
To elevate the relevance of isolated heart preparations for metabolic studies, 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. The LV working heart model is superior to the Langendorff model for functional evaluations, yet it does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model (LV &amp; RV) 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 improves septal function by diminishing abnormal pressure gradients across the septum. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, mean pulmonary pressure, heart rate and myocardial ATP, and creatine phosphate levels for up to 3 hours8. Biventricular working heart studies typically use hearts from small animals, such as rats and rabbits, because the cardiac output and the required volume of perfusate are much less than that for hearts of larger animals. However, biventricular working heart studies have been conducted using hearts from swine, canines, and even humans23, 24.
The metabolic demand of isolated hearts in biventricular working mode is considerably higher than that of Langendorff perfusion. It is important that the perfusate solution provide enough oxygen and metabolic substrate to support biventricular heart function. Standard crystalloid buffer solutions, such as Krebs-Henseleit16, 17, 25 or Tyrodes26, 27, have oxygen solubilities as high as 5.6 mg/L. When these solutions are gassed with carbogen (a gas blend of 95% O2 and 5% CO2) and contain suitable metabolic substrate (glucose, dextrose, and/or sodium pyruvate), they are appropriate for biventricular working hearts beating at normal sinus rates (approximately 180 bpm for a rabbit).
Metabolic demand increases for fast rhythms and the amount of oxygen dissolved in standard perfusates might not be enough to fully support a biventricular working heart that is contracting at high rates. Crystalloid buffer solutions containing erythrocytes or mixed with whole blood have been used in working heart preparations to ensure adequate oxygen availability. Previous studies have shown that adding erythrocytes to a Krebs-Henseleit solution improved working heart function during rigorous pacing protocols and also reduced the incidence of ventricular fibrillation16. A limitation of using erythrocytes or mixtures of whole blood is that hemoglobin interferes with light wavelengths that are used for fluorescence imaging13. Other substrates, such as albumin, may also be added to perfusate solutions to prolong heart viability and reduce edema28.
During fluorescence imaging the intensity of excitation light should be high and the light distribution should be uniform. Achieving uniform illumination is not always easy due to the curvature of the epicardial surface. In our studies, we image fNADH by filtering light (350&plusmn;25 nm) from a mercury lamp. A bifurcated fiber optic light guide is used to direct the UV light onto the epicardial surface. Uniform lighting can be achieved by appropriate positioning the two output ferrules. UV LED light sources could also be used, as we have demonstrated in Figure 3. LED sources are relatively inexpensive so multiple sources could be incorporated into an imaging system. LEDs can also be cycled on and off at high rates to synchronize excitation light with image acquisition.
Photobleaching of NADH should be minimized29 by reducing the time of tissue illumination. This can be done by cycling the illumination on and off using an electronic shutter and a lamp or with an LED lighting system and a controller. If illumination is synchronized with the cardiac cycle, then fNADH image acquisition could be confined to diastole, which would reduce motion artifact in the fluorescence signals. Trigging illumination and image acquisition using a pressure signal, such as LV pressure, would be one way to do this.
In our studies we have observed that changes in fNADH per unit time can be more than 5X higher at 400 bpm than at 200 bpm. This indicates that fast rhythms elevate the redox state of the heart. Whether or not this is caused by hypoxia or the inability of myocytes to oxidize NADH to NAD+ quickly enough to avoid the accumulation of NADH is still an unanswered question.
The performance of a biventricular working heart preparation is contingent upon multiple factors. One of the most important is to set appropriate preload and afterload pressures to mimic the physiological conditions that are under investigation. In particular, the LV afterload (aortic pressure) must be adjusted to represent systemic pressure. If it is too high, the LV will not be able to overcome the pressure, resulting in regurgitation. Pressure that is too low will adversely affect coronary perfusion. The LV preload pressure (left atrial pressure) should also be adjusted to provide an end diastolic volume that is appropriate for the experimental protocol.
fNADH imaging of living tissue is an established mode of fluorescence imaging13. Its application to cardiac tissue was illustrated by Barlow and Chance when they reported striking elevations of fNADH within regionally ischemic tissue after ligation of a coronary vessel14. Their fNADH images were recorded on film using a Fairchild oscilloscope camera and UV flash photography. Coremans et al. expanded upon this concept using the NADH fluorescence/UV reflectance ratio to measure the metabolic state of the epicardium of Langendorff blood-perfused rat hearts30. A videofluorimeter was used for imaging and data was recorded using a video recorder. Later, Scholz et al. used a spectrograph and photodiode array to measure average fNADH from a large area of the LV. This approach reduced the effects of epicardial fluorescence heterogeneities and local variations in circulation while revealing macroscopic work-related variations of fNADH31. This approach is similar to computing average fNADH levels for a region of interest across all frames of an fNADH imaging dataset, as illustrated in Figure 2. As we have presented in this article, today's technology provides high-speed CCD cameras and digitally controlled high-power UV spotlights. These technologies enable the spatiotemporal dynamics of fNADH and cardiac metabolism to be studied from many new perspectives. The relatively low-cost of the optics and light source makes fNADH imaging a useful accessory for conventional cardiac optical mapping systems.9, 32

<table border="1">
 <tbody>
 <tr>
 <td>Chemical</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>S-3014</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>P3911-500G</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>Fisher Scientific, Fair Lawn, NJ </td>
 <td>C77-500</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>M-7506</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Fisher Scientific, Fair Lawn, NJ </td>
 <td>S-233</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Fisher Scientific, Fair Lawn, NJ </td>
 <td>423-316</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>158968-500G</td>
 </tr>
 <tr>
 <td>NaPyruvate</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>P2256-25G</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich, St. Louis, MO </td>
 <td>A9418-100G</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts at JoVE.com

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

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 ...

nadh-fluorescence-imaging-isolated-biventricular-working-rabbit

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17.8K vues.  The George Washington University. L'objectif est de surveiller l'état redox mitochondrial de coeurs isolés dans le contexte de la précharge physiologique et les pressions postcharge. Un modèle de travail biventriculaire coeur de lapin est présenté. Haute résolution spatio-temporelle imagerie par fluorescence de NADH est utilisée pour surveiller l'état mitochondriale redox de tissu épicardique.

Vidéo: NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

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.

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 ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

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 ...

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) ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

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.

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.

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