Establishment of In Vivo MIRI and Assessment of Myocardial Infarct Size

Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 80 mg/kg. After being anesthetized, the mice were placed in a supine position and secured on a dissection board, and an ECG monitor was installed. The left thoracic fur of the mice was shaved, and the shaved area was disinfected with alcohol-soaked cotton balls. A meticulous incision, about 1 cm in length, was delicately made on the area of skin where the pulsations of the heart were most distinctly palpable. The muscle layer was exposed, and the pectoralis major and pectoralis minor muscles were separated without damaging them. The pleura was then breached through the intercostal space between the third and fourth ribs, and then the heart was quickly squeezed out. Using 1–2 mm below the edge of the left atrial appendage and 0.5 mm beside the pulmonary artery cone as landmarks, a 6–0 silk suture was selected to ligate the left anterior descending (LAD) coronary artery. The needle was inserted to a depth of approximately 1 mm and a width of 1–2 mm to avoid piercing the heart. The ligature was tied in a slip knot, the proximal needle tip was trimmed short, and the distal end of the thread was left 3–4 cm outside the chest cavity. After ligation, the ventricular wall below the ligated area turned dark red or gray, and the heart was slowly pushed back into the chest cavity. Air was expelled from the chest cavity (to prevent pneumothorax), and the chest wall incision was closed with a purse-string suture, leaving the end of the ligation thread outside the chest for potential coronary recanalization. The ECG was closely monitored for changes within 30 min after ligation. Upon reaching the ischemic time, the ligature was slowly removed to restore the myocardial blood flow for 120 min.

The area of myocardial infarction was determined by Evans-Blue dye (Solarbio, China) and 2,3,5-triphenyltetrazolium chloride (TTC, Solarbio, China) double staining. The area in blue was considered as non-risk area. The area unstained by Evans Blue dye was identified as the area at risk (AAR), and the area unstained by TTC was considered as the infarcted tissue. Myocardial infarct size (IS) was calculated as the percentage of infarcted tissue divided by the AAR.