Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

The overall goal of this procedure is to study the cardiac electrophysiological properties such as conduction, repolarization, arrhythmogenesis, and calcium handling by using optical mapping techniques in the langor perfused rabbit heart. This is accomplished by first isolating and retrograde perfusing an intact rabbit heart via the aorta. The next step of the procedure is exposing the surface of the heart, abolishing the contractions of the heart, using excitation contraction on Coler BLEs statin, and properly staining the myocardium using voltage sensitive and calcium fluorescent indicators.

Then recordings of optical action potentials from much of the ventricular epicardium are made using the panoramic optical mapping system. The optical signals are then mapped onto a reconstructed 3D geometry of the heart epicardial surface. During this step, the optical action potential and calcium transient can be recorded simultaneously using the non panoramic dual imaging system.

This procedure provides quantitative data of the spatial and temporal myocardium, excitation and repolarization, as well as congruent characteristics of calcium handling, which can be analyzed during regular rhythm as well as atrial and ventricular arrhythmia. Hi, my name is Dr.Iger Eov. I'm a professor of biomedical engineering at Washington University in St.Louis.

The optical technique which we present today is a unique technology which has a number of advantages over traditional micro and micro electrograms because it is capable to record tens, tens of thousands of action potentials and calcium transients simultaneously. Moreover, this technology is immune to artifacts presented by electrical stimulation and defibrillation, which strongly distort action potentials recorded by any electrograms. Moreover, calcium transients could be only recorded by this technique.

There is just no alternative. This method can help answer the key questions in the field of cardiac electrophysiology, such as the mechanisms of induction, maintenance, and termination of life-threatening atrial and ventricular arrhythmias, as well as the function modeling in the conduction ization and excitation and construction coupling in diseases such as congenital heart disease, metabolic disease, and heart failure. Once prepared for surgery, euthanize a rabbit with a cocktail of sodium pentobarbital and heparin.

As soon as the rabbit fails to respond to a pain reflex, open its chest and excise the heart and lungs. Make a cut at the upper end of ascending aorta before all the branches of aortic arch turn on the pump to start the flow of Tyro solution in the non recirculating LRF perfusion system. Quickly mount the heart to the 16 gauge aorta cannula connected with the LOR perfusion system.

Flush the air out of the aorta and tie off the aorta cannula once fully perfused. Waste no time in cutting open the pericardium. Then remove the lung trachea fat surrounding the heart and connective tissues.

It is very important to prevent fluid buildup in the left ventricle, which would produce ischemia. So insert a silicone drain tube through a pulmonary vein and mitral valve and into the left ventricle. Now move the heart with the cannula to the recirculating lang Andorf perfusion system equipped with optical mapping system.

Place the heart in a custom made hexagon chamber and connect the cannula to the perfusion system. Monitor the aortic pressure using a pressure transducer and maintain it at around 60 plus or minus five millimeters of mercury by adjusting the flow rate of the perfusion pump. Maintain the pH around 7.35 plus or minus 0.05.

Turn off the room light and slowly inject the light sensitive BLEs statin stock solution through the injection port in the air bubble trapper located above the cannula. To reach a concentration of 10 micromolar BLEs statin slowly inject 0.1 milliliters BLEs statin. For every bolus injection, wait for the pressure to stabilize before the next injection.

Position the photo diode arrays or PDAs at three evenly spaced angles around the heart. Then on each PDA, focus the image of the heart on the frosted glass of the image plane. Adjust the position of the cannula in the hexagon chamber and each PDA until the heart fits in the field of view of all three PDAs.

Then take a picture of each focused image on the frosted glass. Slowly inject 10 to 20 microliters of D four Anep stock solution into the perfusion solution through the injection port in the air bubble trap. And wait three minutes.

Next, gently place the pacing electrode onto the desired location. Here it is placed at the center of the anterior view of the heart. Begin making simultaneous optical recording from the PDAs.

Make sure that there are no motion artifacts in the optical signal and that the signal to noise ratio is sufficient for subsequent data analysis. When finished, turn on the room lights and make a photographic record of the heart by taking one photograph for every 10 degrees of rotation from a camera positioned at the location of one of the PDAs. Then proceed with cleaning up the perfusion system.

Place the cannulated heart in a rad naughty glass chamber and connect the cannula to the perfusion system. Pin down the heart to the silicon bottom of the chamber at the ventricular apex and atria. To immobilize the heart, turn off the room light and slowly inject BLEs statin through the injection port to reduce the motion of the solution surface.

Place a glass window cover above the epicardial surface. This part of the procedure is normally done in the dark, but is being done in the light. To improve the video image, move the dual imaging system on top of the heart.

Now focus the two CMOs cameras on the same field of view. Next position the light guides for two halogen lamps with excitation filter. Stain the heart by adding 10 to 20 microliters of voltage sensitive dye RH 2 37 through the injection port.

Then add 200 microliters of road 2:00 AM with onic F1 27 in a one-to-one mixture through the bubble traps port. Wait approximately 20 minutes to allow the DS ification of the road 2:00 AM before mapping starts. Now make a test recording.

Turn off the superfusion pump to avoid motion at the surface of the solution. Turn on the halogen lamps and take an optical recording from both cameras. Then turn off the excitation light.

Turn on the superfusion pump and check the quality of the optical signals. If the signal is weak, restain the heart with additional RH 2 37 and road 2:00 AM.If there is motion artifact, add additional BLEs statin to remove the motion with everything in order. Proceed with the planned experiment and clean up the system.

When finished, the panoramic optical mapping system was used to show this anterior view of a Lang Endorf perfused rabbit heart. By comparison, a reconstruction of the heart geometry shows the same anterior view. The unwrapped epicardial surface shown here is color coded by phase.

The heart was undergoing an episode of tacky arrhythmia from five locations around the rotor. Optical action potentials were presented. Here in red is an activation wavefront propagating during a cycle of stable reran arrhythmia.

The wavefront rotates clockwise around a phase singularity at the anterior surface of the heart. Repolarization is in transparent blue to allow visualization of wave fronts on the posterior surface. The heart was analyzed using the dual mapping system to show simultaneous mapping of action potentials and calcium transients.

A a closeup view of recordings from one site shows action potentials are in blue and calcium transient in red. An array of sample traces are shown from an array of evenly spaced sites as depicted by the dots on this anterior view. These data were collected at optical resolution of 200 microns by 200 microns per pixel.

The implications of the therapy extend defibrillation therapy for atrial and ventricular 10 K arrhythmias because we can directly visualize the effect of strong electric stimuli during the electrotherapy. No alternative method is capable of doing that. Using this method, we develop and validated novel low voltage pain-free deflation therapy for atrial fibrillation and ventricular tachycardia.