2.4. Langendorff Heart Perfusion System

A total of 55 adult male Sprague-Dawley rat (350–450 g) were anesthetized with urethane (1.5 g/kg, i.p.), then the hearts were perfused in a Langendorff system. After 30 min of stabilization, hearts were subjected to 30 min of global no-flow ischemia by stopping the perfusion. Reperfusion was followed with Krebs Henseleit (KH) buffer and GAA together for 2 h. A thermoregulated chamber kept the heart at 37 °C throughout the experiment. Control hearts were not subjected to I/R. The heart slices were sectioned at a thickness of 2 mm and stained with triphenyltetrazolium chloride (25 mg/100 mL) for 10 min and then fixed with 4% formaldehyde solution for 48 h to enhance color contrast. Unstained areas were measured using ImageJ software (Version 1.50; National Institutes of Health, Bethesda, MD, USA) to calculate the percentage of grayish-white areas.