Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Our protocol presents a practical guide on surgery for creating aortic regurgitation in the mouse, and provides clinically relevant characteristics of volume overload induced hypertrophy. This surgery-based technique provides a more reliable and reproducible, clinically relevant volume overload model in the mouse than any other known technique. The indications of this technique can stand toward the study of molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

This method could provide insight into comparative studies, differentiating structural, functional, and signal characteristics of volume overload from pressure overload. Anybody who is trying this technique for first time should be skillful in echocardiography or can closely collaborate with technicians who can well operate an ultrasound machine. Begin by starting the ultrasound machine, connected to a 30-megahertz probe.

Set the temperature-controlled ultrasound animal platform for the aortic arch view in which the right side of the mouse is tilted up. Connect a micromanometer to the data acquisition device and analog to digital converter. Immerse the micromanometer's calibration covette in saline for saline calibration.

Prepare the necessary surgical tools, including various forceps and scissors. After making a median incision in the neck of the anesthetized animal, bluntly dissect the left and right parts of the thyroid gland. With the curved fine-thumb forceps, expose the RCCA for as long as possible by separating the stylohyoideus muscle and fat tissue in the right paratracheal region.

Avoid injury of the vagal nerve, as this can cause hypotension, bradycardia, and death. Pass two 6.0 silk threads under the vessel. Using one thread, ligate the distal RCCA with a tight knot.

Then fix the two ends of the tight knot next to the head of the animal to maintain light tension on the RCCA. Using the second thread, place a loose knot on the proximal RCCA to fill the sealed region of the RCCA with blood, making it easy to incise. Now using small pinch scissors, open the RCCA by making a one to two-millimeter wedge-shaped opening proximal to the tight knot.

Ensure that the incision is neither too small to insert a catheter nor too large to snap during insertion. Prepare a plastic catheter containing a metal wire, and stretch the incision with long-handed, curved tying forceps. Insert the plastic catheter containing the metal wire into the RCCA, then relieve the loose knot to advance the catheter and wire around two centimeters and move towards the loose knot.

Under ultrasound guidance, carefully advance the catheter and the wire through the RCCA and ascending aorta. Collect basal ultrasound data in color and pulse wave Doppler modes before the plastic catheter and metal wire reach the aortic orifice. Using ultrasound, visualize the ascending aorta, the LV outflow tract, the catheter, and the wire.

Once the catheter and the wire reach the aortic orifice, protrude the tip of the wire from the catheter, and puncture the aortic valves. After puncturing the aortic valves, slightly retreat the catheter and wire from the aortic orifice. Collect post-perforation ultrasound data in color Doppler mode and pulse-wave Doppler mode.

In the color Doppler mode, the regurgitant flow appears red during cardiac diastole, and can be quantitatively confirmed in pulse-wave Doppler mode. A peak diastolic aortic flow velocity between 300 to 500 millimeters per second is satisfactory. After confirmation of successful perforation of the aortic valves, carefully withdraw the plastic catheter with the central metal wire before ligating the RCCA.

Close the skin using a 5.0 silk suture in a continuous suture pattern, and apply the povidone iodine solution to the suture site. Administer meloxicam subcutaneously for analgesia into the mouse. After four weeks of AR, use echocardiographic B mode, color Doppler mode, and pulse-wave Doppler mode to assess the blood flow of the aortic arch in the aortic arch view, and measure PDVA as demonstrated earlier.

Use echocardiographic B mode and M mode to assess LV dimension and contractility in the parasternal long-axis view. After the echocardiographic imaging, performed the invasive hemodynamic measurement. Since the RCCA was permanently ligated during the AR surgery, insert the micromanometer into the left common carotid artery.

Record the maximal contraction and relaxation velocity. The color Doppler spectrum of the aortic arch showed regurgitant flow in AR mice immediately post-operation. Pulsed wave Doppler recordings of aortic arch flow demonstrated strikingly elevated regurgitant flow in AR mice.

Invasive hemodynamic measurement demonstrated significantly lower AEDP in AR mice, however, pulse pressure was promoted in AR mice, relative to sham animals. The mortality in the perioperative period within two hours after surgery is 15%and in the postoperative period of four weeks, it is around 20%Macroscopic photographs of the LV outflow tract indicated intact valves in sham mice and perforated aortic valves in AR mice. M-mode echocardiographic images of sham and AR hearts are shown here.

AR-induced enlargement of the LV cavity displayed strikingly increased LVEDD and LVESD, along with slightly elevated LVPWTD. On the contrary, AR caused a reduction in LVEF and LVFS. Invasive hemodynamic measurement also demonstrated impaired cardiac function, as shown by a remarkable reduction in maximal contraction and relaxation velocity.

Cutting open the right common carotid artery is the most crucial step. This technique paves the way for researchers to better understand the difference between volume overload and pressure overload cardiac hypertrophy by comparative studies.