マウス洞房結節の高分解能光学マッピング

The overall goal of this experimental protocol is to perform optical mapping of the mouse sinoatrial node from an intact Langendorff-perfused heart or an isolated atrial preparation. This method can help answer key questions in the sinoatrial node electro-and pathophysiology field about the mechanisms of pacemaker abnormalities, sick sinus syndrome, and various diseases associated atrial arrhythmias. The main advantage of this technique is that it enables the investigation of multiple parameters in the intact tissues with very high spatial and atemporal resolution.

After confirming the appropriate level of anesthesia by toe pinch, use curved 5.5 inch Mayo scissors and 5.5 inch Kelly Hemostatic Forceps to make a one centimeter incision on the front of the thorax. Using four inch curved Iris Forceps, quickly grasp the lung tissue and use four thirds inch Iris scissors to cut out the lung, thymus, and heart together, along with the pericardium. Wash the tissues in oxygenated 37 degree Celsius modified Tyrode solution.

Then use curved three inch Vannas Tubingen scissors and four thirds inch number five forceps to remove the lung, thymus, and fat tissue from the heart. Next, use a custom made 21 gauge cannula and two curved 4.3 inch number five B forceps to cannulate the aorta and simultaneously perfuse and superfuse the heart with 37 degree Celsius Tyrode solution directing the solution through an inline 11 micron nylon filter prior to the perfusion to prevent clogging of the coronary circulation. Then connect a pressure transducer to a Luer lock on the perfusion line via a three-way stopcock to monitor the aortic pressure adjusting the perfusion speed to maintain the aortic pressure between 60 to 80 millimeters of mercury as necessary.

For optical mapping of the sinoatrial node from a Langendorff-perfused heart, position the organ in a horizontal orientation with the posterior side facing up in a glass tissue organ bath chamber and use 0.1 millimeter diameter pins to fix the ventricular apex to the silicone-coated bottom of the chamber to prevent stream-induced movement during the experiment. Insert a small silicone tube into the left ventricle via the pulmonary vein, the left atria, and the mitral valve and use a 40 silk suture to fix the tube to the surrounding connective tissue. Next, use another 40 silk suture to noose the superior and inferior vena cavae and pin the edge of the right atrial appendage to the bottom of the chamber.

Stretch and pin the other end of the suture to the bottom of the chamber and place a custom-made pacing electrode on the edge of the right atrial appendage. Then place two electrocardiogram 12 millimeter needle monopolar electrodes near the base of the right and left ventricles and place the ground electrocardiogram electrode near the ventricle apex. To stain the tissue, inject the diluted dye into an inline perfusion port of the coronary perfusion line for delivery over a five to seven minute period.

After 20 minutes of stabilization, add 0.5 milliliters of 37 degree Celsius blebbistatin into the perfusate followed by the injection of 0.1 milliliters of blebbistatin diluted in 1 milliliter of 37 degree Celsius Tyrode solution into the coronary perfusion line over another five to seven minutes using an inline Luer lock injection port. Blebbistatin should be used with caution. To prevent precipitation of the inhibitor, first dissolve the blebbistatin in medium that has been warmed to a clear 37 degrees and vigorously stir the mixture.

To optically map the sample, first fix a small covered glass onto the surface of the solution over the tissue to reduce any motion artifacts from the vibrating solution. Then using a flexible bifurcated light guide with an excitation light provided by a constant current, low noise, halogen lamp, direct the filtered light beam onto the tissue and image the resulting fluorescent signal. After cannulating the heart as just demonstrated, dissect the ventricles away from the anterior side and open the right atrium through the tricuspid valve along the tricuspid valve superior vena cava axis followed by an incision through the medial limb of the crista terminalis.

To open the anterior atrial free wall, make an incision from the midline of the medial limb incision to the edge of the right bottom corner of the right atrial appendage and pin the flattened atrial free wall to the bottom of a silicone-coated chamber. To open the left atrial appendage, cut through the mitral valve along the mitral valve upper corner of the left atrial appendage. Then pin the opened left atrium as it was previously done for the right atrium.

Then partially remove the ventricular tissue. Preserve a rim of ventricular tissue for pinning the preparation to prevent damage to the atria and pin the tissue to the bottom of a Sylgard-coated chamber. Now lift the final preparation by about 0.5 millimeters from the bottom of the silicone-coated chamber to allow superfusion from both the epicardial and endocardial surfaces and superfuse the sample with 37 degree Celsius Tyrode solution.

Then place a custom-made pacing silver chloride electrode on the edge of the dissected right atrial appendage. Place one electrocardiogram 12 millimeter needle mono polar electrode near each of the right and left atrial appendages, and place the ground electrocardiogram electrode near the atrial ventricular junction. To stain the heart tissue, use a 1 millimeter pipette to slowly release voltage-sensitive dye diluted in 37 degree Celsius Tyrode solution directly onto the tissue.

Then immobilize the sample with the direct application of diluted blebbistatin onto the tissue followed by the administration of another 0.5 milliliters of blebbistatin via the perfusate and optically map the sample as just demonstrated. The tissue staining and blebbistatin inhibition steps are critical requiring accurate implementation and a precise staining procedure so that the atrial physiological parameters including the heart rate, allocation of the leading pacemaker and atrial conduction are not affected. Here a typical right atrial activation contour map reconstructed for spontaneous sinus rhythm for a Langendorff-perfused mouse heart with two corresponding right atrial contour maps acquired at 1 and 0.5 millisecond sampling rates are shown.

The early activation point is located within the intercaval region near the superior vena cava where the sinoatrial node is anatomically defined. In this experiment, after right atrial appendage pacing for at least one minute at 10 to 12 Hertz, the electrical stimulation was stopped and the sinoatrial node recovery time was calculated as the time interval between the last captured action potential and the first spontaneous action potential. Activation of the isolated atrial preparation during spontaneous sinus rhythm allows the precise area of the leading pacemaker location to be identified.

If the surgery and dye loading procedures are followed appropriately, no significant changes in the physiological characteristics of the atrium should be observed. Although arterial staining may require a larger amount of dye, the fluorescent signal in the sinoatrial node area tissue appears to be more stable by this loading method with a signal intensity decay time after coronary staining almost twice as long as that after surface staining. Once mastered, this technique can be completed in 30, 40 minutes if it's performed properly.

While attempting this procedure, take care to locate atrial anatomy before making a cut to avoid damaging the atrial tissue and the sinus node. Following this procedure, other measures like conventional glass mitral electrode recordings can be performed to answer additional questions about commutative transmembrane potential characteristics. After its development, this technique paved the way for researchers in the field of intellect physiology to explore atrial arrhythmias including sinoatrial node dysfunction in the heart.

After watching this video, you should have a good understanding of how to perform high resolution optical mapping of a mouse sinus node preparation.