Experimental protocols.

Adult male Sprague-Dawley (SD) rats, 8 weeks of age were used. Experimental protocol 1: At the time of reperfusion, animals were divided into 3 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC alone (FITC 0.33 mg in saline 3.3 mL/kg), or 3) FITC-NP (PLGA 8.3 mg containing 0.33 mg FITC in saline 3.3 mL/kg). Three hours after reperfusion, animals were sacrificed. The left lower panel shows representative stereomicrographs of heart sections double-stained with Evans blue and TTC: the MI area (TTC negative, white), non-MI area within AAR (TTC positive/Evans blue negative, red), non-ischemic area (TTC positive/Evans blue positive, purple) and AAR (Evans blue negative). Experimental protocol 2: At the time of reperfusion, animals were divided into 4 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC-NP (PLGA 8.3 mg/kg in saline 3.3 mL/kg), 3) pitavastatin (1.0 and 10 mg/kg in saline 3.3 mL/kg), or 4) pitavastatin-NP (PLGA containing of 0.1 and 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 3: Animals were divided into 3 groups receiving administration of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) vehicle (saline 3.3 mL/kg) after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia, 3) pitavastatin-NP (PLGA containing of 1.0 mg/kg pitavastatin in saline 3.3 mL/kg) after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia. Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 4: To examine the effects of Pitavastatin-NP on left ventricular function after IR, animals were divided into 3 groups that received intravenous injection of the following drugs at the time of reperfusion: 1) vehicle (saline 3.3 mL/kg), 2) pitavastatin alone (1.0 mg/kg in saline 3.3 mL/kg) or 3) Pitavastatin-NP (PLGA containing 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Echocardiography and measurement of systolic blood pressure and heart rate by using tail-cuff method were performed at baseline and 2-day, 1-week, 2-weeks and 4-weeks after reperfusion.