Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

The overall goal of this procedure is to investigate the cellular and molecular mechanisms underlying neo tissue formation, valve thickening, and stenosis development within a tissue engineered heart valve. This is accomplished by first harvesting the pulmonary heart valve from donor mouse heart tissue. In the second step, the entire heart is harvested from a second donor.

The donor pulmonary heart valve is then implanted onto the donor heart and the heart with the new heart valve is hetero topically transplanted into a recipient mouse. Ultimately, the function of the transplanted heart and blood flow through the pulmonary valve can be monitored with a high frequency ultrasound system in the pulse wave Doppler mode. The primary advantage of the use of the mouse model over other large animal models is that the use of a murine model enables us to take advantage of a wide range of molecular reagents that enable us to study the cellular molecular mechanisms underlying the formation of vascular neo tissue in the development of stenosis and tissue engineered heart valves, Generally individuals new to this method will struggle because of the complexity of the survival.Microsurgery.

Visual demonstration of this method is critical as the proper osmosis on the transplantation sites are difficult to learn and because it is difficult to perform osmosis on the small caliber vessels without hemorrhage To harvest the donor pulmonary heart valve, first clip the chest area of a six to eight week old female donor, C 57 black six mouse, and place the mouse in a dorsal recumbent position. After making a thoracotomy, expose the heart, make a small cut on the right atrium and perfuse the left ventricle with ice cold saline. Next blunt, dissect the pulmonary artery from the ascending aorta and then cut out the pulmonary valve along with a two millimeter cuff of the pulmonary artery and store the pulmonary valve in a cold, heparin and saline solution for up to two hours before transplantation onto the donor heart to harvest the donor heart.

After performing a thoracotomy on a euthanized six to eight week old female donor, C 57 black six mouse as just demonstrated bluntly, separate the heart, inferior vena cva, superior vena cva, ascending aorta, pulmonary artery, and pulmonary vein. Peruses the inferior vena CVA with ice, cold, sterile saline, and then ligate the inferior vena cva, superior vena cva and pulmonary vein with 6.0 silk suture, then cut superior to the ligatures, cut the aorta and pulmonary artery with a two millimeter cuff and cut out the pulmonary valve to transplant the pulmonary heart valve onto the donor heart. First, place the valve into the donor heart and orient the tissue.

Next, use 10 oh monofilament sutures on tapered needles to place a stitch on the right side of the valve to secure it. Then place five to six continuous stitches along the other side of the pulmonary valve. After finishing the front of the valve, rotate the heart horizontally and suture the back of the pulmonary valve onto the donor heart.

Then ligate the pulmonary vein of the donor heart and extract the heart. Then place the heart in a cold heparin saline solution for up to two hours before implantation into the recipient mouse. Now confirm sedation of a six to eight week old female recipient, C 57 black six mouse by toe pinch.

And then after clipping the abdominal hair and lubricating the eyes with sterile ophthalmic ointment, place the mouse in a dorsal recumbent position, disinfect the abdomen with Betadine and alcohol pads, and cover the mouse with a sterile drape leaving the incision area only exposed. Next, make a midline laparotomy incision from below the xiphoid to the supra pubic region. Then insert a self retaining retractor and wrap the intestines in saline moistened gauze.

Bluntly, define the infrarenal aorta and vena cva. Then place two six oh silk sutures and ligate proximally and distally around the aorta and inferior vena cva. To restrain the blood circulation, use a 30 gauge needle to make an AOR autotomy in the abdominal aorta and ven otomy in the inferior vena cva.

Extend the opening with scissors to the size of the donor aorta and pulmonary artery. Place the donor heart on the right side of the abdominal aorta and cover it with moisturized sterile gauze and moisturize it. Secure the donor aorta with one stitch on the proximal end of the opening in the abdominal aorta, and then place four to five continuous stitches from the distal end of the abdominal aorta.

Flip the heart to the left side. Cover it with saline infused gauze and suture the distal end of the abdominal aorta with four to five more continuous sutures. Secure the donor pulmonary artery with one stitch on the proximal end of the opening in the inferior vena cva, and then place four to five continuous stitches from the distal end of the inferior vena cava.

Then flush the inferior vena CVA lumen with heparin and saline solution. Close the right wall of the donor pulmonary artery and recipient inferior vena CVA with continuous sutures to the distal end. Now remove the distal ligature and apply a topical absorbable sterile hemostat agent to control the hemorrhage.

When the hemorrhage stops completely, remove the proximal suture and control the second hemorrhage in the same way the transplanted heart will start to beat within five minutes after successful anastomosis. Then return the intestines and use six oh sutures to close the abdominal musculature and skin in two layers. Finally, subcutaneously, inject 0.5 milliliters of saline and place the mouse in a recovery cage on a warming pad until the animal is fully mobile.

Upon recovery, place the mouse alone until fully recovered in a new cage with paper bedding and administer pain medication. Drinking water for 48 hours. In these images, the implanted heart in the abdominal space right after heart transplantation and five minutes after transplantation are shown.

Upon removing the sutures on both sides of the aorta and inferior vena cva, the heart begins to beat within one to two minutes and becomes pinker as the blood begins to circulate. Note the greater dilation in the right atrium. As the heartbeat gradually grows stronger and becomes stable after 24 hours, the blood flow through the implanted pulmonary valve can be measured per percutaneously 10 days after implantation.

Using a high frequency ultrasound system with a pulsed wave doppler mode, the locations of the aorta right ventricle implanted pulmonary valve and pulmonary artery in B mode can be observed. The yellow sample volume overlay is located on the implanted PV as illustrated in the schematic. As indicated in the graph, the donor heart QRS wave is detected rhythmically and independently of the recipient heart wave with a peak velocity of around 100 millimeters per second.

While attempting this procedure, it is important to remember to completely stop any hemorrhage at the pulmonary valve transplantation site or at the an osmosis on the donor aorta to the recipient abdominal aorta Following this procedure. Other method like transplantation of tissue engineered heart valves onto transing, mice can be performed to answer additional questions about the source of the cells populating the diseased heart valve. After watching this video, you should have gained a good understanding of how to harvest the pulmonary heart valve, how to perform a heart valve replacement and heterotopic heart transplantation in a mouse model.