This protocol utilizes high spatial and temporal resolution optical mapping to understand the mechanisms underlying stretch induced atrial fibrillation. This is accomplished by first explaning, a sheep heart, and connecting it through the aorta to a LAN endorf profusion system with circulating oxygenated tyro solution. An atrial transseptal puncture is used to seal off all the vein orifices except for the inferior vena cava, which is used to control the intra atrial pressure.
Then under the continuous atrial stretch, a persistent atrial fibrillation is applied using burst pacing from an electrode located on top of the left atrial appendage. The final step is to optically and electrically map the epicardial and endocardial surfaces of the left atrial appendage and posterior left atrium using laser illumination CCD cameras and bipolar electrodes. The results show dominant frequency gradients from the posterior left atrium to the left atrial appendage and to the right atrium.
Additionally, occasional long lasting rotors with centers of rotation in the highest frequency domain are identified. The main advantage of this technique over existing methods like a conventional electrical mapping that is traditionally used in human electrophysiological studies is that optical epicardial and endocardial mapping of the left atrium provides high temporal and spatial resolution over a wide area, which allows the tracking of atrial fibrillation waves in various regions, including regions with the high rotor frequency that maintain atrial fibrillation. This method can help answer key questions in the cardiac electrophysiology and complex cardiac arrhythmias field, such as what are the special temporal patterns of activation underlying the maintenance of the most common sustained arrhythmia in the clinical practice.
The implications of using this technique extend toward therapeutic possibilities either to prevent or terminate atrial fibrillation based on a better understanding of its mechanisms. Generally, individuals new to this technique will struggle to perform seamlessly all the steps in reasonable period of in time. It should be noticed that there is only a limited time around four hours for which they isolate.
Heart reminds in conditions suitable for the optical mapping of arrhythmias. This demand certain surgical skills to ly set up the atrial stretch model combined with technical skills to set up the electrical and optical mapping system. After administering heparin and anesthetizing a 35 to 40 kilogram sheep with propofol and Pentobarbital, use a thoracotomy to remove its heart.
Connect the dissected heart to a LAN perfusion system with circulating oxygenated tyro solution at a pH of 7.4, set the constant flow rate to between 240 and 270 milliliters per minute and maintain the temperature between 35.5 and 37.5 degrees Celsius. Begin by equalizing the intracavitary pressure in both atria of the perfused heart. Using an atrial transseptal puncture, cannulate the inferior vena cavo with an outflow tube.
Then adjust the intra atrial pressure by changing the height of the open-ended outflow cannula above the atria. Now seal off all the vein orifices, including coronary sinus, superior vena kava, and the four pulmonary veins. When sealing off the pulmonary veins, place catheters in each one of them to record bipolar signals from the two distal electrodes through a differential amplifier.
Next, to reduce the tissue motion caused by the contractility of the cardiac myocytes, add 10 micromolar of BLEs statin to the perfusion. Monitor the pressure using a digital sensor at the base of the cannula. Continually adjust the height of the outflow to maintain a stable pressure of 12 centimeters of water throughout the experiment.
Lastly, bipolar electrodes are placed on the roof of the left atrial appendage and on the top of right atrial appendage. Now prepare for optical mapping. Begin by focusing a high resolution high speed CCD camera on about 14 square centimeters of the left atrium free wall surface.
Then position a laser light source to illuminate the region. Now introduce a 10 millimeter diameter dual channel rigid borescope across the mitral valve to get a direct field of view through the anterior wall of the left ventricle. Focus the borescope on the endocardial surface of the posterior left atrium to visualize the four pulmonary veins and the atrial septal pulmonary bundle.
Now sea mount the borescope to a second CCD camera through an eyepiece adapter using a liquid light guide with a 0.2 inch core diameter. Set up a laser on the excitation channel of the borescope. Synchronize the recording from both CCD cameras and configure them to provide a square TTL pulse that will further trigger simultaneous bipolar recordings.
To prepare the system for optical recording, inject five to 10 milliliters of dif four andep into the heart and wait for 10 minutes before beginning the optical mapping recordings. The D four ANEP PS emits voltage sensitive fluorescence upon 532 nanometer laser excitation. So after its addition, proceed immediately with atrial fibrillation recordings to induce atrial fibrillation under the continuous atrial stretch, set the electrode on top, the LAA for burst pacing during atrial fibrillation.
Record five second movies at two minute interval simultaneously from both cameras. Continue recording of atrial fibrillation over a 50 minute period with the raw data collected, apply a high pass filter to the bipolar signals at three hertz and a low pass filter at 35 hertz. Now to analyze both the electrical and optical signals, transform them both using an FFT algorithm.
Finally, to identify regions with high activation rates and to produce frequency gradients generate dominant frequency maps from each optical movie using phase movies generated With the hilbert transformation, three classes of activation patterns can be identified. A rotor pattern is identified by the presence of phases converging toward a singularity point that lasts for more than one rotation. A breakthrough pattern is defined as a wavefront appearing in the field of view and propagating outwards in a target like pattern.
Spatiotemporally organized periodic waves are defined as a minimum of four sequential periodic waves emerging from the same location with similar direction and inter wave interval. During this representative atrial fibrillation episode, the highest dominant frequency was localized on the right inferior pulmonary vein of the posterior left atrium. The presence of high frequency sources in the PLA is consistent with the left to right dominant frequency gradients observed during ablative procedures in per proximal human atrial fibrillation.
Further quantification allows for spatial correlation of the highest dominant frequencies with the most common pattern of activation. Occasionally, it is possible to identify long lasting rotors localized within the highest frequency domain. In some cases, a scroll wave, the 3D equivalent of a rotor is identified because the scroll wave stable center of rotation remains perpendicular to the surface of the mapping area.
This rotor was recorded from the endocardium of the PLA with simultaneous fibrillary conduction toward the LAA. The data correlates with a frequency gradient between the PLA and the LAA at nine and 6.4 hertz respectively. Rotors are generally more frequent in the PLA than in the LAA, suggesting that reentry to the PLA has an essential role in maintaining the arrhythmia.
Once the surgical and technical methods are mastered. This experiment can be complete in two, three hours. There are several key points for a successful experiment.
First, it is critical to prevent air from entering the hard coronary system. Second, it is important to identify all the vein, artifices, and seal then off properly. And third, it is important to employ the least amounts of motion and coupler fluorescence die and reduce the exposure to excitation light as much as possible.
The, The development of the endocardial epicardial mapping approach shown here paves the way for researchers to explore the dynamics of wave propagation in greater details than before, and in highly relevant animal models of complex cardiac arrhythmias.
