This study describes a customizable method to create a myocardial function model in Miro by precision ligation of the left anterior descending coronary artery through micro manipulation. This protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival. Use a rectangular OT with a heating pad pre-warmed to 37 degrees Celsius throughout the surgical procedure.
Before the procedure, disinfect the board with ultraviolet light and 70%alcohol. Begin by placing the mouse on the OT in a supine position with gauze under the head to avoid overheating the eyes. Apply ophthalmic ointment to the eyes to prevent them from drying out.
Use a fur removal cream on the thorax and massage evenly with a sterile cotton swab for one minute. Wipe the excess fur with gauze. Use povidone iodine followed by 70%alcohol to clean the area, and cover the thorax with gauze.
Use a 4-O suture under the upper incisors and secure it to the anchor point to keep the mouth slightly open and facilitate cannulation. Pull the tail to keep the body straight, and secure it to the OT using tape. Secure the four limbs with tape and tighten them on the anchor points.
Do not overstretch the front limbs to avoid respiratory compromise. Use forceps to open the jaw and lift the tongue. Then use an illuminator to visualize the throat and glottis.
Gently insert a 22 gauge cannula with a blunt truncated needle through the mouth into the trachea, about one centimeter down the throat. Use one hand to hold the tongue and move it slightly upwards with blunt forceps. Simultaneously use the other hand to gently insert the tube into the trachea.
Be careful not to insert the tube into the esophagus. Carefully remove the needle. Before connecting to the ventilator check the intubation by placing the tube into the water for bubbles to form.
Connect the endotracheal tube to a ventilator set to 120 per minute, and tidal volume adjusted to 250 microliters. Verify intubation by checking bilateral symmetrical chest expansion. Then fix the connection to the OT with tape to avoid it falling off.
Place ECG electrodes on the paws and connect them to the ECG recorder. Monitor cardiac electrophysiology throughout the procedure. Remove the gauze on the thorax.
Disinfect the incision area using three scrub cycles with 70%alcohol. To reduce contamination of the surgical site, cover the mouse with a sterile surgical drape with a hole over the surgical field. After making an oblique skin incision with a scalpel, expose the ribs by carrying out a blunt dissection of the subcutaneous tissues.
Be careful not to injure the vessels, ribs, and lungs. Use sterile cotton applicators to stop the bleeding. Identify the third intercostal space and make an incision of about six to eight millimeters.
Then open the chest cavity by carrying out blunt dissection of tissues in the intercostal space. Be careful not to injure the internal thoracic artery. Span the intercostal space using forceps.
Insert pre sterilized homemade retractors into the ribcage, and pull back to spread the incision to approximately six millimeters in width. Attach the retractors to the OT with rubber bands. Carefully remove the surrounding tissues to expose the heart entirely.
Pull off the pericardium gently with curved forceps without injuring the heart. Use a dissecting microscope and direct a focused and appropriate light for LAD visualization. Gently press the site below the chosen ligation position to temporarily enlarge the LAD, and then recheck the LAD.
Under a dissecting microscope, use a tapered needle to pass an 8-0 silk ligature underneath the LAD. Ensure that the needle is not deep enough to enter the left ventricle and not too shallow to avoid damaging the LAD. Tie the ligature with a loose double knot.
Place a two to three millimeter PE-10 tubing into a loop parallel to the artery. Tighten the ligature loop gently until it is around the artery and tubing. Then secure the loop with a slip knot.
Take care not to damage the myocardial wall with excessive tightening pressure. A pallor coloration in the anterior wall of the left ventricle after ligation confirms cessation of blood flow in the LAD. If permanent ligation is required, proceed as described in the text manuscript.
Remove the retractors from the incision. Then close the wound temporarily with a bulldog clamp. Ensure that the mouse continues to be connected to the ventilator.
When the period of ischemia ends, remove the bulldog clamp and insert the retractors to open the incision and expose the heart and the ligation site. Untie the slip knot and remove the PE-10 tubing. Change in the pale coloration to pink-red confirms blood flow restoration.
Simultaneously, to detect reperfusion, observe the ECG for a potential dissolution of ST elevation. Leave the 8-0 ligature in situ if performing subsequent Evans blue and TTC staining. Otherwise, remove the suture at this step.
Remove the retractors and close the incision by suturing the third and fourth ribs with a 4-O silk suture. Be careful not to injure the lung. Press the chest gently while tying the suture knots to push out any air that might be trapped in the chest cavity.
Close the muscle layers with continuous 4-O silk sutures. The standard images of the experimental process were acquired from endotracheal intubation, skin incision, thoracotomy, LAD identification, and ligation, to reperfusion. The representative ECG images verified myocardial ischemia and reperfusion with significant ST elevation after ligation.
And dissolution of ST elevation after the slip knot was untied. Before the thoracotomy, it is very important to keep the airway open, and the most important thing is to make sure that the LAD is firmly ligated and avoid damage. This procedure will be followed by echocardiography to evaluate the cardiac function as well as TTC to assess the myocardial injury.
