The physical most underlying cardiac fibrillation and defibrillation remain not completely understood. Optogenetics can offer a way to gain more insight into cardiac phenomena in a well-controlled environment. Local light stimulation using micro LED's permits the innovation of specific cardiac tissue areas.
This targeted stimulation is a new tool to develop heart gentle anti arithmetic therapy methods. The shown protocol can be used to study heart behavior during cardiac arrhythmia and especially during termination. In prospective, the developed techniques can be used to study more clinically relevant large animal models.
Before every experiment, clean all tubes with fully de-mineralized water. Aerate thyroid solutions with carbogen for 30 minutes at room temperature and adjust the pH to 7.3 with sodium hydroxide and hydrochloric acid. Heat the perfusion system to 37 degrees Celsius with a water heat pump.
Keep the perfusate temperature constant within the water bath by using an additional heating element such as a waterproof heating cable. Add 500 milliliters of each thyroid solution to the corresponding reservoir and de-aerate the tubes as well as the bubble trap by running thyroid solution through the perfusion system until no more trapped air bubbles are seen in the tubes or in the bubble trap. Continue aerating the thyroid solutions during the whole experiment in the reservoirs with carbogen to ensure that the pH of the perfusate remains stable later during perfusion.
After removing the heart, perform the fine preparation under a stereoscopic microscope. Attach the aorta onto the blunt needle and fix the vessel with suture material. As a control, inject ice cold thyroid solution through the needle into the heart, and check that the heart is tightly mounted.
Transfer the mounted heart to the perfusion system and ensure that the perfusate is flowing to prevent air from entering the heart while connecting the needle with the bubble trap. Check that the heart is covered with thyroid solution in the water bath and that it is beating within a few minutes. Place one of the ECG electrodes as close as possible to the heart surface to ensure good signal quality.
Suspend the second ECG electrode in the thyroid solution, making sure that the ECG is being recorded. Place the micro LED array on the area of interest. Change the perfusion to low potassium thyroid with penecidle and peruse the heart for 15 to 30 minutes.
To induce arrhythmia, illuminate the heart with LED one and LED two with a train of 20 to 50 light pulses with a frequency of 25 to 35 hertz, pulse duration of two to 15 milliseconds, and light intensity of 2.8 milli watts per square millimeters. Repeat the process until arrhythmia is induced. Once a sustained arrhythmia is visually detected, apply a burst of pulses with different widths and frequencies using three, six, or nine micro LEDs of the array at a pulsing current of 15 milliamp piers.
If the arrhythmia keeps going after five micro LED array based defibrillation trials, classify the attempt as unsuccessful and start backup defibrillation, using LED one and LED two with same timing parameters. Perfuse the heart with the blebbistatin solution and wait until mechanical uncoupling occurs, which is accomplished when the heart stops beating, but an ECG signal is still measurable. Give the one milliliter voltage dye, dye 4-A-N-B-D-Q-P-Q as a bolus in the bubble trap of the Langendorff perfusion and wait for five to 10 minutes to allow the dye to peruse the heart uniformly.
Turn on LED three. Focus the camera on the heart's surface and apply 1.27 milli watts per square millimeter optical power. Turn off the laboratory lights and start recording, making sure that an optical signal is being acquired by comparing the frequency of the obtained signal to the frequency of the recorded ECG.
A series of experiments with different frequencies, number of micro LED and pulse durations were tested for 11 mice, demonstrating that pulses of 1 to 20 milliseconds can defibrillate with different success rates. There is a significant increase in success rate of the nine LEDs with pulse durations of one millisecond and 20 milliseconds at a defibrillation frequency of 18 and 20 hertz, which is closer to the analyzed mean arrhythmia frequency of 22.55 of all the arrhythmias with an error interval of plus or minus four point zero three hertz. Two different defibrillation attempts with 14 hertz frequency with their ECG recordings and their respective spectrograms are shown here.
In an example of defibrillation with 30 millisecond pulses, the dominant frequency of the arrhythmia slightly increases until photo stimulation begins. The VF is turned into a VT where the dominant frequency drops to 14 hertz followed by unsuccessful termination and return to arrhythmic behavior with a dominant frequency of 24 hertz. In the second example, segment one shows a VT with dominant frequency of 23 hertz and it's harmonic components until photo stimulation begins, with a pulse width of 20 milliseconds.
Segment three displays a successful termination which leads to a normal sinus rhythm with a fundamental frequency of 3.5 hertz and the resulting harmonics. Optical mapping with high speed cameras showed a change of fluorescence intensity during a single beat of the heart during a normal sinus rhythm. The length of perfusion must maintain all conditions to keep the heart healthy, namely the temperature, the pH value, and the oxidation need to remain constant.
It is particularly important that the tyroid solution do not run empty as air bubbles in the system may damage the heart. The micro reality array offers great flexibility. You can target different locations of the heart or even combine multiple arrays in one experiment in order to properly terminate arrhythmia.
