Sequoia c512

The surgical protocol used to induce cardiac IR injury was performed according to the methods of a previous study.³² In brief, 100 mg/kg of pentobarbital was used for anesthesia. After occlusion of the left anterior descending coronary artery using 7 to 0 silk suture (US Surgical Corp, Norwalk, CT) for 45 minutes, the snare was released, and reperfusion was achieved for 4 hours (n = 6/group). Low-dose melatonin (10 mg/kg) and high-dose melatonin (20 mg/kg) were administered intraperitoneally 24 hours before cardiac IR injury, according to the methods of a previous study.¹³ After IR injury was induced, hearts were isolated, and the infarct areas were stained using 4% Evans blue and 2,3,5-triphenyltetrazolium chloride based on the methods of a previous report.³² In addition, blood was collected to analyze the concentrations of cardiac injury markers including CK-MB, troponin T, and LDH. Mice that underwent the surgical protocol without IR injury served as a sham group. Cardiac function was measured through echocardiography (14.0 MHz, Sequoia C512; Acuson, Berlin, Germany), according to the methods described in a previous study (n = 6/group).³²

Echocardiography was performed in all mice 6 h after the reperfusion according to our previous studies [8] . An echocardiogram (14.0 MHz, Sequoia C512; Acuson, Germany) was used to detect both 2-dimensional and M-mode images.
Electron microscopy was conducted as previously described. The whole heart was immediately fixed at 4 °C with 2% glutaraldehyde in a 0.1 mol/L sodium cacodylate buffer and postfixed for 1 h on ice with 1% osmium tetroxide after IR injury. After making the slices, the samples were stained with lead citrate and uranyl acetate and observed under a Hitachi H600 Electron Microscope (Hitachi, Japan) [23] (link). At least 30 cells in a minimum of 5 randomly selected fields were observed.

Echocardiography was performed using echocardiogram (14.0 MHz, Sequoia C512; Acuson, Germany) to monitor changes in cardiac function [14] (link). Myocardial contrast echocardiography (MCE) was performed using a 14 MHz linear transducer (Acuson Sequoia C512 system) with the constant infusion of microbubbles [10% Perflutren lipid microspheres (Definity, Lantheus Imaging) diluted tenfold in sterile saline] at 20 mL/min as previously described by our group [1] .