The overall goal of this procedure, is to perform transverse aortic constriction, in a murine closed chest model, inducing left ventricular myocardial hypertrophy. This minimal invasive technique, helps to reduce respiratory problems, and minimize the inflammatory response. This method can help us answer key questions, in the field of immunology, for example, the response of cardiac macrophagia in the development of left ventricular hypertrophy.
The main advantage of this technique is, that the rib cage remains intact, and therefore the trauma is minimized. Since spaces and tidal volumes are adapted to body weight, this protocol can be used in different sized mice. It also reduces surgical trauma, and decreases deviation in measurement values.
After anasthetizing a mouse, according to the text protocol, move the mouse to a temperature controlled operating table, and detach the nosecone that is connected to the anesthesia induction box to maintain narcosis. With a nylon suture, fix the upper incisors of the mouse. Pinch the tail to insure the absence of a reflex.
Use adhesive tape to fixate the extremities. After inserting a rectal probe, apply depilatory cream to the throat and upper chest, according to the manufacturer's instructions, and allow it to sit for one minute. Then, wipe off the cream and repeat if necessary.
With the ventilator settings adjusted to physiological parameters, and the animal under a surgical microscope, incise the skin at the mid line about three millimeters under the mandibular down to the second rib. Identify the midline and connective tissues of the submandibular gland. Then, use two angled forceps to gently and bluntly divide the gland at the midline, and explore the tracheal muscle.
Gently prepare the trachea, by bluntly pulling the paratracheal muscles apart. Use forceps to pull on the tongue to straighten the throat for easier intubation, and gently insert an intubation canula inside the trachea. Confirm the intubation by direct visualization of the tube inside the trachea, and by checking for proper chest movement.
Prepare both carotid arteries, which lay adjunct to the trachea, by using forceps to gently pull the connective tissue apart. Apply ultrasound gel to the tip of the 20 hertz doppler probe, and place the tip of the probe on the right and the left carotid artery, at an angle of less that 45 degrees. Slowly rotate the probe to move it lateral and medial to find a doppler signal, and then tilt the probe to optimize the signal.
Using a fresh set of sterile gloves and scissors, expand the skin incision down to the second intercostal space. Then, with the tip of the forceps, open the second intercostal space, and insert the retractors. At 200%magnification, identify midline and connective tissues.
Then, use angled forceps to gently divide the thymus. Remove the fatty tissue until the aortic arch can be clearly seen. With angled thumb forceps, prepare a tunnel under the transverse aorta, between the brachiocephalic artery, and left common carotid artery.
Then, using fine forceps, hold a 6-0 suture, and pass the thread under the aortic arch. With the second forceps, take the thread from the other side of the arch. Carefully place the spacer parallel to the transverse aorta.
Prepare a loose double knot on the spacer and insure optimal placement of the spacer in parallel to the aorta. Then, tie the first knot, and quickly make a second contrary knot, before promptly removing the spacer. The most critical step of the whole procedure is to tie both knots quickly and tightly, and remove the spacer immediately.
Close the second intercostal space, with a 6-0 polypropylene suture, and pay special attention to the subclavian vessels when ligating. Then, using a 6-0 polypropylene suture, stitch the skin in a continuous suture pattern. Confirm ligation, and carry out post operative care, according to the text protocol.
These doppler flow velocity measurements validate a successful TAC, which causes an augmented blood velocity in the right carotid artery, due to elevated pressure in the left ventricle and aorta, while causing post stenotic attenuated blood flow velocity in the left carotid artery. As shown in this graph, heart weight and body weight ratios significantly increased in TAC mice, compared to non-banded mice, six days after surgery. This ratio was nearly constant after 21 days.
Heart rate has a significant effect on left ventricular contractility and cardiac output. This graph reveals that there were no differences in the heart rates of aortic banded and non aortic banded mice, after 21 days. As demonstrated here, a constant banding of the aorta, was proven by an increased systolic blood pressure, measured via a left ventricular catheter after 21 days.
C57 black six mice, are commonly known to develop eccentric hypertrophy with systolic dysfunction after TAC. An increase of left ventricular diameter was found, which also appears significant in pressure volume measurements. As a result, end systolic and diastolic volumes increased significantly.
Once mastered, this technique can be performed in under 10 minutes, if it's done properly. Finally, this procedure provides an optimized analysis of the immune response in left ventricular hypertrophy, because the minimal surgical trauma restricts the unspecific induction of the immune system. After watching this video, you should have a good understanding of how to perform standardized, minimal invasive transverse aortic constriction.
