The overall goal of this procedure is to simultaneously optically map transmembrane potential and intracellular calcium transient in intact isolated LOR perfused mouse hearts. This is accomplished by first excising the intact mouse heart, then cannulating the aorta and beginning LOR perfusion. The next step is to immobilize the heart by loading an excitation contraction on coupler and then pinning the heart to the perfusion chamber.
The final steps are to load the optical dyes and make preparations to reduce motion artifacts during the experiment. Optical images are obtained by collecting the light emitted from the heart with my cam Ultima LC Moss cameras. Hey, my name's di.
I'm a graduate student in Washington University. The main advantage of this technique over existing method, like micro electrode, is that optical mapping allows for greater spatial resolution over the entire surface of the heart. Hi, I'm Matt Sulkin.
I'm a graduate student at Washington University. This method, if done correctly, can help answer many questions in cardiac electrophysiology such as the basis of arrhythmogenesis. Begin by configuring one peristaltic pump to circulate the solutions at 40 milliliters per minute.
Then set a second peristaltic pump to 80 milliliters per minute. This pump returns the Perfuse eight back to the holding and oxygenating reservoir. Turn on the pumps and wash the system with one liter, 70%ethanol for 30 minutes with recirculation.
Follow this wash with two liters of deionized water without recirculation. Once the water is evacuated, circulate two liters of Tyro solution and pass it through a five micron filter. Warm the perfuse eight to 37 degrees Celsius with a water jacket and circulator bubble oxygenated gas into the solution.
Adjust the rate of gas bubbling to maintain the pH at 7.35 plus or minus 0.05. Monitor both the pH and temperature during experiment. The dual optical mapping apparatus consists of two high speed C moss cameras that can capture a 100 by 100 pixel image at 3000 frames per second.
Attach a band pass filter to the calcium imaging camera and attach a long pass Filter to the voltage imaging camera on a dual camera holder. Attach a diic mirror and lens so that emission light coming from the heart is focused onto the mirror by the lens. Also, attach two cameras to the holder and arrange them so they are perpendicular to one another.
Next, set up the excitation light. The light is generated by a halogen lamp and directed through a flexible light guide. Pass the light through a heat filter, a shutter to limit exposure, and an excitation band pass filter.
Finally, prepare silver and silver chloride electrodes by installing them in the chamber. After double checking and adjusting the levels on the amplifiers and filters, the setup is ready for a heart. Use a cocktail of ketamine and xylazine with 100 units of heparin to anesthetize a mouse before proceeding.
Assure that the animal has no pain reflex using a toe pinch. After making a mid sternal incision, quickly remove the heart and the surrounding tissues. Then using dissecting microscope rapidly, identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery.
Now attach a short section of aorta to a custom made 21 gauge cannula using four oh black braided silk sutures After cannulation retro greatly perfu and super fuse the heart. With Tyro solution, adjust the flow rate to keep the aortic pressure between 60 and 80 millimeters. Mercury as measured by a pressure transducer.
Now dissect and remove the surrounding lung thymus and fat tissue. Now insert a silicone tube into the left ventricle through the pulmonary veins. Suture the tube to nearby connective tissue.
This very important step prevents congestion and acidification of Perfuse eight trapped in the left ventricle, especially after the suppression of ventricular contractions. To prevent Perfuse eight induced heart movements, pin the heart to the bottom of the sill guard coated perfusion chamber at its apex. Furthermore, stretch and pin down the right and left atrial appendages to increase the surface area of the atria in the upright configuration.
This will maximize recording surface area. To conduct the pacing stimulations, attach a custom made electrode to the surface of the heart. To reduce motion artifacts, fix a small cover glass over the heart on the solution surface.
Do this by wedging the glass between several pins. As final preparations, focus the excitation light on the heart and position the cameras to get an optical image then commence with loading the dye while loading dies. Continuously monitor ECG recordings to assure normal electrical function of the heart.
Begin with warming up BLEs statin to room temperature. Then mix one half milliliter of BLEs statin into the profuse eight in the holding reservoir, and dilute the remaining one 10th milliliter of BLEs statin into one milliliter of Tyros solution. Over the next 20 minutes, slowly inject the BLEs statin in tyros through a drug port near the cannula.
The BLEs statin will gradually reduce the motion artifact. Next, dilute 30 microliters of voltage sensitive dye into one milliliter tyro solution, and slowly inject this mixture over five to seven minutes into the same drug port. Then make the same mixture again.
This time, adding an additional 30 microliters of calcium indicator. Slowly apply this mixture over five to 10 minutes through the same port. After the calcium indicator is injected, wait an additional five to 10 minutes for the BLEs statin to fully suppress motion, and for the dyes to enter the cells before proceeding with the experiment.
This heart preparation was made. According to the protocol, ECG signals were recorded during ventricular pacing from S one S one stimulation. Action potentials and calcium transient signals were recorded from the atria on the left and ventricles on the right.
The arrow shows where signal scattering from the ventricles occurred by binning the recordings from the camera in units of three pixels by three pixels, an array of calcium transients and action potentials can be displayed from the data from the array data. A figure showing action potential conduction can be constructed. Data from three consecutive points on the transverse and longitudinal axis show the amplitude change with the propagation of the action potential across a ventricular surface.
Likewise, a figure showing action potential and calcium transient durations can be constructed. This shows a control condition on the left on the right. An injection of isoproterenol was given to the heart, which clearly shortens the duration of both calcium transients and action potentials.
Once mastered, this technique can be completed in two to three hours Following this procedure. Other methods like pacing protocol can be performed in order to answer additional questions such as those regarding refractory period of AV node.
