NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

The overall goal of this procedure is to study metabolism. Using NADH imaging in a working heart preparation, this is accomplished by first isolating the rabbit heart. The second step of the procedure is to successfully cannulate all four chambers of the heart to achieve a working heart model.

The final step is setting up imaging equipment and collecting NADH data at different pacing rates. Ultimately, results can show metabolic changes of the heart through imaging of fluorescent NADH. The main advantage of this technique over existing methods like the lanor retro profusion, is that the heart is operating in its physiologic state with flow in the normal direction and with the heart providing its own pressure for coronary perfusion.

This method can help answer key questions in electrophysiology and the mechanisms of lethal arrhythmias. This is done by providing a physiological model to study the link between coronary flow metabolism and electrical activity. Visual demonstration of this method is critical because vessel and heart chamber cannulation steps are difficult to learn.

Improper technique can damage the heart and jeopardize the rest of the experiment. Prior to running an experiment, rinse out all tubes in chambers of the working heart system with purified water, and then purge the water from the system by running the pumps. Add cellulose membrane filters in line with the LOR perfusion pump, left heart perfusion pump, and right heart perfusion pump.

After adding perfuse eight to the system, perform a two point calibration for each pressure sensor. Turn on the heated circulating water bath that warms the water jacketed tubes and the heat exchangers Turn on the water bath. Used to heat the PERFUSE eight.

Turn on the pumps that circulates the PERFUSE eight in a closed loop, turn on the circulating tank to oxygenate the Perfuse. Eight, make sure the microfiber oxygenator or hemo filters are supplying 95%oxygen to the perfuse eight at 80 kilopascals. Lastly, make sure the heat exchangers are keeping the temperature of the circulating perfuse.

Eight at 37 degrees Celsius, begin the heart excision by first setting the working heart system to operate in constant pressure. LRF mode. Set the pressure of the aortic block between 50 and 60 to after sedating a rabbit.

Inject pentobarbital and heparin into either the marginal ear vein or the lateral saphenous vein on the inside of the hind limb. Once the rabbit lacks a pain reflex, quickly open the thoracic cavity. Slice the pericardium clamp the aorta and excise the heart and lungs.

The lungs are kept attached to make isolating of the pulmonary veins easier. Next, fill a syringe with 60 milliliters per F eight and 200 units of heparin. Attach a five millimeter cannula and cannulate the aorta.

Secure the aorta to the cannula with size zero silk sutures. Then slowly depress the syringe to flush out the blood through the heart. Set up the working heart system by first connecting the heart to the aortic block.

Approach the connector at an oblique angle and allow Perfuse eight to gently drip from the connector into the cannula while it is attached. This prevents air from entering the aorta, thus preventing coronary emboli while the heart is perfused in constant pressure. L endorf mode.

Remove the fat and connective tissue and locate the following vessels. Inferior and superior vena cva, a aose vein, pulmonary artery and pulmonary veins. Next, ligate the superior vena cva.

Cut the pulmonary artery just below where it branches to the right and left pulmonary arteries. The remaining vessels are the pulmonary veins. Group them between the heart and lungs and ligate them all using one suture.

This is critical for two reasons. First, tying the vessels connected to the lungs will ensure no leakage once the cannulation is complete. And second, it's important to isolate both VNA cavas furl later cannulation.

Now remove the lungs. Cut an appropriate size hole in the corner of the left atrial appendage and make sure it is filled with profuse eight. Next, cannulate the appendage while the cannula is completely filled with perfuse eight.

Then while holding the tissue, suture the cannula to the appendage. The tissue of the left atrial appendage is extremely delicate and it can tear easily. So it's important to cannulate the left atrium on the first try, or else it might not be possible.

Turn on the number two pump to provide flow to the left atrium. Set the preload between two and six tor throughout the experiment. The pressure may need to be adjusted.

Keep it within two millimeters of the target pressure. Now switch the heart to working heart mode by turning off pump number one, the langor pump momentarily. Decrease the aortic pressure to tend tor and then slowly increase it to within the range of 80 to 100 tor.

This allows the aortic valve to open and function as it would during normal physiologic conditions. Set the final afterload pressure to a value that is approximately 20. Tore less than peak LV pressure using a cannula directly attached to the system.

Cannulate the right atrium through the inferior vena CVA and suture the cannula to the vein. Avoid introducing any air bubbles. Turn on pump number three, the right side pump to provide flow to the right atrium.

Set the pressure to approximately three tor. Lastly, cannulate the right ventricle without allowing air to enter and suture the cannula to the pulmonary artery ery. This completes the biventricular cannulation.

Now proceed to signal acquisition. Carefully insert the pressure transducer catheter into the aorta via the aorta cannula. Gently navigate it past the aortic valve and into the lv.

The LV pressure signal will appear as expected when the catheter tip is properly positioned. Next, gently press the monophasic action potential electrode against the ventricular epicardium. A slight motion artifact in the electrode signal is normal.

Next place a bipolar stimulus electrode on the right atrium to pace the heart according to the experimental design position. And focus the CCD camera on the region of interest of the heart. There should be an emission filter attached to the camera.

To pass the NADH fluorescence turn off the ambient lights to uniformly illuminate the epicardium with a UV light source at the workstation, which the camera is attached. To set the software to acquire images at two frames per second. Then select live update mode to monitor the mean pixel intensity within the region of interest.

If the profusion was adequate, NADH levels represented by the fluorescence should be low and stable over the epicardial surface. Now, run the experiment when it is complete. Remove the heart.

Drain the PERFUSE eight and the system tubing and chambers with purified water. Periodically rinse the system with mucosal TM solution or a diluted hydrogen peroxide solution using the outlined protocol. Hearts were pasted at cycle lengths between 200 and 400 beats per minute.

Diastolic LVP was usually between zero and 10 tor the minimum diastolic aortic pressure was approximately 60 Tor peak systolic LVP is dependent upon filling pressure and contractility and optimally should be between 60 and 80. Tor the maximum aortic pressure and maximum LVP should closely match monophasic action Potentials typical of a rabbit's heart have a fast depolarization phase and characteristic repolarization phase. Action potentials were easily recorded from the preparations, but usually had a small motion artifact during diastole.

Average fluorescent NADH values from a region of interest in the red box were measured to monitor changes in mitochondrial redox state during three pacing cycles at psycho lengths of 300, 200 and 150 milliseconds when pacing rate was close to sinus rhythm. Baseline NADH level was relatively constant as cycle length was shortened. Below 300 milliseconds baseline NADH levels increased with the largest increase at 150 milliseconds, the signal oscillates with contraction and the frequency of oscillation corresponds to the heart rate.

The amplitude of these oscillations were always less than any longer timescale trends caused by ischemia or hypoxia. High resolution fluorescent NADH imaging of the full anterior surface at 200 beats per minute were constant and spatially homogeneous at 400 beats per minute. NADH 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 Once mastered. This technique can be done in under 45 minutes if it is performed correctly. While attempting to perform this procedure, it's important to remember to prevent any air bubbles from entering the aorta or the left atrium Following this procedure.

Additional methods like loading the heart with a voltage sensitive dye can be used to answer additional questions like linking the electrical and metabolic activity on the surface of the heart during pathological scenarios such as local ischemia After its development. This technique paved the way for researchers in cardiac physiology to study heart metabolism and conduction disturbances during pathologic conditions using a more physiologically relevant model.