Hi, I am Yon Ziv from the laboratory of Dr.Cohen in the Department of Anesthesiology at Children's Hospital and Howard Medical School. Today we'll show a procedure of optical mapping in Lal perfused red hearts. We use this procedure in our laboratory to study the spread of electrical activity on the cardiac surface.
So let's get started. On the morning of the experiment, mix up four liters of Krebs Hensel light solution. After mixing up the solution, remove undissolved particles with a 200 micron filter using vacuum filtration.
First, use one liter of the prepared Krebs Henzel lite solution to dissolve 11 millimoles of BDM. Second, take another 100 milliliters of Krebs Henzel lite and mix in 10 millimolar D eight NFS in DMSO to a final concentration of five Micromolar D eight annex in solution. The water jacketed, glass reservoirs, the bubbler and the glassware used here are all from red.Naughty.
Transfer the two solutions to the reservoirs pre-war to 41 degrees Celsius. Oxygenate the solutions with 0.2 micron filtered gas using a submerged bubbler pump the solutions from the three stock reservoirs through the wall-mounted Ling Endorf system with peristaltic pumps and low absorption silicone tubing to harvest the rat heart. First intraperitoneal inject a 200 to 250 gram Lewis rat with ketamine and xylazine to induce deep general anesthesia and with heparin to prevent blood coagulation and myocardial ischemia during explanation.
Once the rat is completely anesthetized and does not respond to a toe pinch, proceed with removing the heart. Begin the surgery by first removing the anterior chest wall to easily access the heart and great vessels. Then carefully dissect the surrounding tissue and open the pericardial sac.
After identifying the inferior vena cava, ligate this vessel with five O Proline sutures and explan the entire heart lung block. Immediately place the tissue in ice cold Krebs Henzel light solution in a 50 milliliter beaker on ice, quickly identify the ascending aorta and dissect it from the surrounding tissue. Insert an appropriately sized cannula into the aorta.
Taking care to avoid inserting the cannula too far into the aortic root and interrupting obligatory perfusion of the coronary arteries. Secure the cannula to the ascending aorta. Then place the rat heart on the Ling Endorf apparatus without introducing air bubbles into the cannula.
Retrograde coronary vascular perfusion is now established with the warm oxygenated Krebs Hensel lite solution. From the pressure head of the Ling Endorf apparatus, now remove extra tissue including the lungs after cleaning, perfuse the heart for 20 minutes to permit recovery of function and stabilize the rhythm as the heart is perfusing. Introduce a very thin thermocouple temperature probe into the left ventricular cavity.
Suture the probe in place with five oh prolene sutures and adjust the settings of the water circulatory pumps to maintain the temperature of the heart. At 37 degrees Celsius, minimize motion from the Perfuse eight dripping from the cardiac apex. By placing a piece of gauze in the effluent receptacle, the heart is now ready to be loaded with potentia metric dye to acquire electrographic and optical signals to load the potential metric dye dye eight NEPs into the heart.
Switch over to the profusion line that contains Krebs, hence light mixed with fluorescent dye. Because the atria are not sufficiently perfused with dye introduced through the coronary arteries, an 18 gauge cannula is placed in the left and or right atrium. And an additional 50 milliliters of dye solution is slowly administered into each of these chambers.
During the loading procedure, gently place three ECG leads on the surface of the heart. That is not facing the optics used for mapping. The first electrode is positioned on the posterior apical portion of the left ventricle.
The second is placed on the left atrium and the third acts as a reference electrode on the aortic root. The atrial and ventricular electrographic signals are subsequently amplified, digitized, and displayed optical signals using the software. An oscilloscope is also used to visualize the surface ECGs in real time and to assure adequate pacing.
Set the ECG amplifier at 0.1 hertz for the high pass filter and 150 hertz for the low pass filter position the CMOs camera using the X, Y, Z adjustments. A piece of glass that is positioned in the focal plane of the camera is placed on the surface of the heart so that the area of interest is in focus and centered in the acquisition frame. The camera and optics are mounted on a vibration isolation table to minimize resonant frequencies.
At the same time. On the right atrium place a coaxial pacing electrode controlled with an isolated S 48 electrical stimulation unit. This will pace the heart at 300 beats per minute, set the electrical stimulation as follows.
The rate should be five pulses per second, a delay of 0.2 milliseconds, a two millisecond stimulation duration with a strength of six to 12 volts and mode set to repeat and single pulse. For optical recordings that lack motion artifacts from contraction, the heart needs to be electro mechanically uncoupled. Do this by again, switching perfusion lines to Krebs Hensel solution containing 11 millimolar BDM.
Between acquisitions. Perfuse the heart with unadulterated Krebs HENSEL light to help preserve the viability of the preparation. Configure the software recording parameters to 2000 hertz with a 1 28 by 1 28 pixel array.
The frame interval should be set to 0.5 milliseconds and gains should be set to five x for the camera amplifier and eight x for the on-chip gain. The on-chip gain should also be set to 12. Set the software's camera control for a shutter delay of 500 milliseconds and record 4, 000 frames at a duration of 2000 milliseconds.
Turn off or shield all room and equipment lights to eliminate background noise during the recording, the LED light illuminates the heart only during the optical recording. To reduce photobleaching and dye toxicity, control the light source shutter with a five volt pulse delivered through the control panel by way of a D two A board in the computer following acquisition. Process the data using different filter settings.
The default settings are generally used except when adjusting the band stop pass filter, which is set with the left boundary at 44.0 and the right boundary at 98.0. Afterwards, the recorded information is processed and used to generate a movie. Data from one acquisition corresponds to the local electrical activation at 16, 384 sites on the heart surface over a period of two seconds.
The software allows these local signals to be directly compared with one another and with the atrial and ventricular electrographic recordings data by mapping local electrical activation to color and render this information as an animation showing the spatiotemporal electrical activation on the cardiac surface. To create such an animation, first, use the software to temporally and or spatially filter the data. Next, select a start and end time for the animation.
Third, map optical signals to color based on resting light intensity of each pixel. Fourth, overlay the resulting color data with a picture of the heart, and finally generate the animation if the perfused heart preparation was motionless During recording, the optical signals show one distinct peak for every pixel involved in a change of the emission intensity of D eight anep. These real time imaging movies demonstrate an excitation wavefront propagating across the epicardial surface of the heart, as well as the simultaneously acquired electrographic recordings.
We've just demonstrated a high resolution technique to optically image action potential movement on the surface of Alanor Perfuse Red Heart. When doing this procedure, it is important to quickly remove the heart from the anesthetized red. To avoid myocardial ischemia, the myocardial cells need to be adequately perfused and the heart must be completely loaded with water sensitive dye.
So that's it. Thank you for watching and good luck with your experiments.
