This procedure begins by anesthetizing a mouse, preparing it for aseptic surgery and placing it in a supine position atop a heating pad. Endotracheal intubation is performed using PE 90 tubing, which is then connected to a rodent ventilator. Next, a partial thoracotomy to the second rib is performed and the sternum retracted.
The transverse aorta is identified and subsequently ligated between the innominate and left carotid arteries. The rib cage is closed, followed by the skin with a continuous suture pattern. Finally, the endotracheal tube is removed and the mouse is allowed to recover.
Hi, I'm Angela Dal Meda from the laboratory of Dr.Zander Wars in the Department of Molecular Physiology and biophysics at Baylor College of Medicine. Today we will show you procedure for transverse aortic constriction in mice. We use this procedure in our laboratory to study mechanisms underlying cardiac hypertrophy and heart failure development.
So let's get started. Prior to beginning transverse aortic constriction or tax surgery, disinfect the operating field with 75%isopropyl alcohol. Turn on the heating pad and set it at the right temperature.
The system we recommend is a gamer circulating water pump connected to a heat therapy pad that is maintained at 37 plus or minus one degree Celsius. Sterilize surgical tools in a hot bead sterilizer before surgery. For the tax surgical procedure, the following surgical instruments are needed.
A pair of blunt scissors, two coarse curved forceps to find 45 degree angled forceps, A pair of angled spring scissors, a chest retractor, and a needle holder. Cotton applicators should also be on hand in case of bleeding. With the operating field prepared, we can proceed to intubation of the mouse.
In preparation for intubation, the mouse is anesthetized in an induction chamber and hair clippers are used to shave the fur from the neck line to mid chest level. Place the mouse in a supine position atop the heating pad. It is important to maintain normal body temperature during surgery to avoid a rapid decrease in heart rate and possible sudden death.
Apply pressure on the mouse nail bed to confirm the mouse is fully anesthetized. Place a rubber band over the animal's front teeth to extend the neck Using curved forceps in one hand, gently manipulate the tongue to the side with the other hand. Insert PE 90 tubing into the trachea.
Next, connect the endotracheal tube to a Harvard volume cycled rodent ventilator. The ventilator should be cycling at 125 to 150 breaths per minute and a tidal volume of 0.1 to 0.3 milliliters depending on the body weight of the mouse, disinfect the skin inside the surgical field with Betadine solution followed by 70%alcohol. Repeat this procedure three times to prevent contamination of the surgical field during the operation place a sterile drape over the mouse leaving only the operation field exposed.
We are now ready to perform ligation of the transverse aorta during this surgical procedure. Anesthesia is maintained at 1.5 to 2%isof fluorine with 0.5 to one liter per minute, 100%oxygen. Verify the correct level of anesthesia by applying pressure on the mouse nail bed.
To confirm absence of the token reflex. Begin the surgery by performing a partial thoracotomy. Cut the sternum starting at the cranial end down to the second rib level.
Using a surgical microscope, retract the sternum using a chest retractor. Next, use fine tip 45 degree angled forceps to gently separate the thymus and fat tissue from the aortic arch. After identifying the transverse aorta, place a small piece of 6.0 silk suture between the an denominate and left carotid arteries.
Tie two loose knots around the transverse aorta and place a small piece of a 27 and a half gauge blunt needle. Parallel to the transfer aorta. Quickly tie the first knot against the needle, followed by the second knot, and then promptly remove the needle in order to yield a constriction of 0.4 millimeters in diameter.
Once the transverse aorta has been ligated, remove the chest retractor and pinch off the outflow of the ventilator for two seconds. To carefully reinflate the lungs, close the ribcage using a 6.0 proline suture with an interrupted suture pattern. Finally, close the skin using a 6.0 proline suture with a continuous suture pattern.
For postoperative analgesia. Inject the mouse intraperitoneal with buprenorphine. If there are signs of dehydration after surgery, 0.3 to one milliliter of sterile saline is given intraperitoneal.
Gradually lower the anesthesia to the off position. When signs of spontaneous breathing occur, remove the endotracheal tube, then move the mouse to the prone position and allow it to recover on a heating pad. One week after transverse aortic constriction, the mouse is re anesthetized to determine the degree of pressure overload induced by lation of the transverse aorta.
Anesthesia and body temperature are maintained as shown previously. Place a 20 megahertz Doppler probe on the left and right sides of the neck at a 45 degree angle. To detect carotid artery flow velocities, we use a computer-based Doppler signal processor to display and store doppler signals.
Depending on the degree of pressure overload required for the experimental protocol, only mice with a right carotid or RC over left carotid or lc flow ratio within a certain range are included for further analysis. For example, a moderate degree of pressure overload leads to a ratio of five to eight, whereas a tighter constriction resulting in severe pressure overload leads to a ratio of eight to 10. A sham animal, however, is expected to have a ratio of about one.
The typical surgical survival following TAC in wild type mice is about 80 to 90%successful. Surgical ligation of the transverse aorta will lead to a doppler flow velocity ratio between right and left carotid artery or RC over lc of five to 10 compared to sham operated mice. Mice with pressure overload are expected to develop cardiac hypertrophy within one to two weeks and cardiac dilatation after six to eight weeks depending on the tightness of the constriction.
We've just shown you how to perform transverse aortic constriction in mice, but performing this procedure is important to maintain normal body temperature and minimize blood loss. So that's it. Thanks for watching and good luck with your experiments.
