This method is reliable to study the generation of myocardial infarction and an excellent platform to use for investigating cardiac recovery after, for example, stem cell transplantation. The main advantage of this procedure is that it can be used to generate uniform infarct sizes in a quick, easy, and reproducible manner. Novel experimental interventions, such as pharmacological, or the tissue engineered based strategies, can be tested in this model.
And successfully test that options can be further confirmed in large animal models. After confirming a lack of response to pain reflex in an anesthetized 27 gram, 14 week old mouse, use a hair trimmer to remove the fur of the chest and neck of the animal and place the mouse in a supine position on a heated pad. Tape the hind and forelimbs in a spread eagle position and use sequential povidone-iodine and 80%ethanol scrubs to disinfect the exposed skin.
Make a midline skin incision from the lower third of the sternum to the chin, and use curved forceps to carefully separate the muscles around the neck to expose the trachea. Use micro scissors to perform a tracheotomy between the second and third cartilage rings. And set the ventilator to a ventilation frequency of 110 per minute with a tidal volume of 0.5 milliliters.
Then, insert a plastic, 20 gauge cannula connected to the ventilator into the trachea. Next, use cautery to detach the right pectoralis muscle from its sternal origin between the third and seventh ribs. And use side angled spring scissors to cut the fourth to six ribs as close as possible to the sternum.
Dissect the underlying connective tissue to obtain a clear view into the chest cavity, cauterizing the mammary artery to stem bleeding as necessary. Insert a mini gold stain retractor to spread the ribs and keep the chest cavity open. And use blunt forceps to open the pericardium to expose the heart.
Use a blunt rod to lift the heart from the thoracic cavity and decrease the tension of the retractor to reduce the chest opening and to keep the heart from falling back into the abdominal cavity. Pre-cool a three millimeter diameter cryoprobe for 10 seconds, and apply the cryoprobe on the interior left ventricle wall for 10 seconds to generate a left ventricular, cryoinjury infarct. Detach the cryoprobe from the left ventricular wall with room temperature saline irrigation and reenlarge the chest opening with the retractor.
Use the rod to gently return the heart to the thoracic cavity and remove the retractor. Use a 6-0 suture to connect the sternotomy with a single knot. And use a running 6-0 suture to close the chest cavity.
Use a 10 milliliter syringe to evacuate any remaining air from the chest before tying the knot and adapt the skin at the caudal edge before using a running 5-0 suture to close the tissue to the point of the tracheal opening. After reducing the isoflurane and the animal gains spontaneous breathing remove the tracheal catheter and close the tracheal incision with one 8-0 suture. Replace the ventral neck muscles back to their anatomical position over the trachea and complete the skin suture.
Then, add metamizole to the drinking water for pain analgesia for three days with daily animal monitoring for eight weeks. In cryoinjured hearts echocardiography reveals a significantly reduced ejection fraction and fractional area change, with functional impairment observed from day seven post-surgery until the experimental endpoint at 56 days. As assessed by pressure-volume loop analysis, cryoinfraction leads to an impaired left ventricle function which is reflected as a decrease in stroke volume, stroke work, cardiac output, and pre-load adjusted maximal power.
Ex-vivo optical mapping of fluorescent voltage-sensitive dyed hearts demonstrates a blockage of electrical conduction at the border of injury, indicating a local electrical uncoupling. Histological staining with Masson's trichrome reveals transmural fibrotic tissue formation at the site of injury. The infarct size can be calculated by measuring the infarct scar area or midline scar length.
In addition, immunofluorescent staining against a cardiomyocyte marker and Collagen-I confirm the presence of fibrotic remodeling and the loss of cardiomyocytes at the site of injury. It's important to maintain the same contact duration of the cryoproblem each animal as different duration may lead to incongruent infarct sizes. Once mastered this technique can be performed in 20 minutes if it is performed properly.
After watching this video you should have a good understanding how to perform the cryoinjury model. Good luck with your experiments.
