This protocol presents an improved method to obtain transient myocardial hypertrophy, using absorbable sutures, which stimulates left ventricular hypertrophy decrease after removing pressure overload. Using this protocol, researchers can easily mastered the model with a lower operation mortality. Demonstrating the procedure will be Xiaoxia Huang, a technician from my laboratory.
Begin by pinching off the tip of a 25 gauge needle with a needle holder and blunting it. Then, pass a 5/0 absorbable suture through this needle and curve the blunt needle to a 90 degree angle using the holder. Pinch off the tip of another 25 gauge needle, then curve it to a 120 degree angle and smoothen the tip with a holder to be used as a spacer in the ligation step.
Prepare sterilized surgical instruments, including one ophthalmic scissors, one micro scissors, two microsurgical elbow tweezers, one needle holder, and one micro needle holder. To begin the surgery, confirm complete anesthesia of the mouse with the pedal withdrawal reflex. Keep the mouse in the supine position by fixing the incisors with a suture and fixing the limbs with adhesive tape.
Remove the hair from the neck and disinfect the area with iodine and 75%alcohol. Start the surgery by making an incision over 10 millimeters at the midline position between the suprasternal notch and chest. Then separate the skin and the superficial fascia.
Make an incision in the first intercostal space as close as possible to the sternum and bluntly penetrate to open this space with elbow tweezers. Gently separate the parenchyma and the thymus until the transverse aortic arch is visible. Pass a 5/0 absorbable suture under the aortic arch between the brachiocephalic artery and the left common carotid artery with a latch needle.
Place the previously prepared spacer on the transverse aorta and make a double knot on the spacer with the suture. Gently, but quickly remove the spacer and cut the ends of the suture. Close the first intercostal space and skin using 5/0 nylon sutures.
Disinfect the skin again with 75%alcohol. Seven days after surgery, place the mouse in the supine position, maintaining it at 37 degrees Celsius, and tape its limbs to the electrode. Remove chest hair and apply ultrasonic coupling agent to the mouse's chest.
Then access TAC with a 30 megahertz probe. Tilt the platform to the far left. Keep the probe in the vertical position and lower it on the chest along the right parasternal line.
Adjust the X and Y-axis under B-mode until the aortic arch and constriction are clearly visible. Using Doppler mode, measure the peak velocity, dimensions, and contractility of the left ventricle. Select the mice with a velocity of more than 3000 millimeters per second as the TAC group.
Using a 30 megahertz probe, assess the left ventricular dimensions and contractility. Reset the platform to the horizontal position, keeping the probe at 30 degrees counterclockwise to the left parasternal line. Obtain a short axis view of the heart by manipulating the X and Y-axis in B-mode.
Then, press M-mode to show the indicator line and acquire images with Cine Store and Frame Store for later measurement of the LV chamber dimension, fractional shortening, and LV wall thickness. Pulsed-wave Doppler imaging performed after 14 days of TAC showed that the blood flow velocity at the constriction was greater than 3000 millimeters per second even though an absorbable suture had been used to constrict the aortic arch. The pressure gradient of blood flow after 14 days of TAC was maintained above 40 millimeters of mercury.
Interestingly, there was no constriction in the fourth week after surgery, indicating that the absorbable suture had been completely absorbed. The left ventricular parameters in the silk suture group and the absorbable suture group on days zero, 14, and 28 after TAC, on M-mode imaging, are shown here. On day 14 after TAC, the left ventricular posterior wall thickness at end-diastole increased, while the left ventricular internal diameter at end-diastole slightly decreased.
The ejection fraction of the left ventricle was unaffected by the use of absorbable sutures. The heart weight to body weight ratio comparison of the silk suture group, absorbable suture group, and the sham group is shown here. Histological slices of the heart showed that cardiomyocytes significantly enlarged from day 14 to day 28 in the silk suture group, but mostly regressed on day 28 in the absorbable suture group.
When attempting this protocol, it is most important to not damage the parental pleura to avoid pneumothorax. Make sure to blunt the tip of the needle as much as possible and perform the procedure gently. There are many potential application for this method, such as establishing the model of myocardial hypertrophy preconditioning, exploring the mechanism of hypertrophy regression, and investigating the time of left ventricular reversible remodeling.
