This video presents an experimental model of cervical heart transplantation to investigate ischemia reperfusion injury with syngeneic mice, or to investigate graft rejection with allogeneic animals. The first step of the procedure is to harvest the heart from the donor mouse. The graft is stored in Cardioplegic solution on ice for up to four hours, depending on the experimental setting.
The next step is the preparation of the right cervical vessels of the recipient mouse, the vessel wall of the divided external jugular vein and carotid artery, or averted over a cuff and fixed with a ligature. The final step of the procedure is the implantation of the graft into the recipient mouse. The ascending aorta of the graft is pulled over the cuff with the carotid artery and the pulmonary trunk is anastomosis with the external jugular vein.
Ultimately, the clamps are removed and reperfusion begins. Normally, the heart develops sinus rhythm within one minute Compared to abdominal graft implantation to cervical heart transplant. Modal is less invasive and animals recover faster.
Also by using cals for anastomosis, suturing of small vessels can be completely avoided, making this a technically more feasible model. After pre sedating the mouse with isof fluorine, anesthetize it with an intraperitoneal injection of anesthetics, confirm surgical tolerance by checking for the pedal withdrawal reflex. Then secure the mouse in a supine position on the operative field.
Then disinfect the skin three times with chlorhexidine. Now perform a midline abdominal incision and retract the intestines to the left using a wet gauze to expose the inferior ven cva. Then into the IVC inject 50 units of heparin diluted in 0.4 milliliters of saline to prevent coagulation.
One minute later, inci the vessel, open the diaphragm and perform a bilateral thoracotomy to expose the heart, reflect the anterior chest wall superiorly and hold in place with a clamp. Next, remove the thymus and bluntly dissect the connective tissue between the aorta and pulmonary trunk incise the right superior VA cva. Then use a 30 gauge needle to puncture the mobilized as descending aorta at the brachiocephalic trunk and slowly perfuse the heart retro gradually using three milliliters of ice cold cardioplegic solution.
Do not apply excessive pressure. Do not inject any air. And to further enhance the cardiac arrest, drip ice cold saline onto the graft.
Then transect the aorta proximally to the origin of the brachiocephalic trunk and ligate the inferior and both superior vein cavi close to the heart. Using ATO silk After the ligatures are placed, transect each vessel distally to the ligatures. Next, free the pulmonary trunk as distally as possible and ligate the pulmonary arteries close to the bifurcation.
With ose silk also transect these vessels distally to the ligatures. The last vessels to ligate are the pulmonary veins, which can be ligated together with a single ose silk ligature. Then transect these veins and remove the heart from the donor.
Store the heart in ice cold cardioplegic solution until it is needed to begin under the microscope. Prepare the cuffs for anastomosis by cutting the polyamide tubing with scissors or a number 11 scalpel blade. After preparing the recipient for surgery as done with the donor, provide a subcutaneous injection of 0.25 milliliters of warm saline solution for fluid replacement.
Next, shave the hair of the right cervical region and position the mouse supine on a warm pad, tape down the legs, but do not overstretch the front limbs, which can compromise respiration. Then apply ophthalmic ointment and disinfect the surgical site three times with several chlorhexidine scrubs. Now start the surgery by making a transverse skin incision from the right mandibular angle to the jugular notch.
Then bluntly mobilize the external jugular vein and cauterize the side branches with a bipolar forceps and transect them. Divide the external jugular vein between the ligatures where there is a confluence with the vein from the submandibular gland. Once isolated, pull the external jugular vein through the cuff.
Then occlude the vessel proximally by placing a vascular clamp on the extension of the cuff. Now remove the ligature and irrigate the vessel lumen with one to 10 heparinized saline. Complete attachment to the cuff by averting the vessel wall over it and fixing the vessel with a preset circumferential ose silk ligature.
Now remove the superficial part of the sternocleidomastoid muscle. Next, mobilize the right common carotid artery. Then below the carotid bifurcation, place two ligatures and transect the vessel between them.
Now pull the carotid artery through the cuff and put a al clamp on the extension proximal to the cuff. To occlude the vessel, remove the ligature and irrigate the vessel lumen with one to 10 heparinized saline. Then gently dilate the lumen with a vessel dilator.
And as before, complete the vessel attachment to the cuff. The most difficult part of the procedure is to avert the vessel volume of the carted artery over the cuff because it easily flips back. It is therefore important to completely mobilize the artery to select an appropriate cuff size and to gently dilate the vessel lumen before aversion.
In preparation for the graft, remove the right lobe of the submandibular gland and keep the operative field moist. Place the graft in the recipient cervical region upside down with the aorta oriented medially and the pulmonary artery laterally. Then pull the aorta of the graft over the cuff with the carotid artery and secure the anastomosis with a circumferential eight O silk ligature.
Next, incise the pulmonary trunk on its anterior aspect and pull it over the cuff with the external jugular vein. Use another ligature to secure this anastomosis. Now remove the clamp on the external jugular vein, and then on the carotid artery, the heart should quickly fill with blood to enhance the reperfusion drip warmed saline onto the graft.
Normally the heart will start a sinus rhythm within a minute. Now, remove the cuff extension at the venous site and check over the graft. The anastomosis must be tension free and vessels should not show any torsion.
Do not cauterize vessels of the skin. Since this may impair wound healing, the bleeding should stop spontaneously close the skin with a single continuous suture. After the surgery, inject the animal with a quarter of a milliliter of warm saline subcutaneously.
Next, inject a drug cocktail subcutaneously for the narcotics to be antagonized. Let the animal recover on a warm pad until it can maintain stern recumbent. Observe the animal for at least an hour before deeming it fully recovered.
Once recovered, the mouse should be housed alone for at least three days. Provide analgesia for a minimum of 72 hours. Also weigh the animal daily to check for severe weight loss and monitor the animal's general health.
Using the described protocol, syngeneic Black six mice were transplanted for histopathological. Examination grafts were stained with hemat, toil and eoin and serious red. Non-transplant Animal sections were used as controls compared to controls.
Prolonged ischemia. Reperfusion resulted in a strong inflammatory reaction with tissue edema. Hyperemia infiltrating leukocytes as indicated with stars and microvascular occlusion.
Serious red staining showed marked interstitial collagen deposition indicated by the arrows. Such collagen depositions were not seen in the controls. This transplantation model is interesting tool in cardiovascular and transplant research.
It is especially useful to investigate myocardial ischemia reperfusion injury before starting with experiments. Be sure that you can perform this technique with reproducible operative results as this may otherwise be a major bias anastomosis and therefore warm ischemia time should be limited to less than 20 minutes.
