The Rat Model of Myocardial Ischemia/Reperfusion Injury

1.1. Animal Preparations

Male Sprague Dawley rats, weighting 250-280 g, were used for the study.

This study was approved by the Institutional Animal Care and Use Committee of National Defense Medical Center, Taiwan. All animals obtained from the National Laboratory Animal Breeding and Research Center of the National Science Council, Taiwan and were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes

of Health (NIH Publication No. 85-23, revised 1996).

Rats were anesthetized with intraperitoneal (i.p.) pentobarbital sodium (60 mg/kg) and urethane (300 mg/kg). The use of rat preparations as models of myocardial ischemia and infarction induced by left coronary occlusion is common (Kane et al., 1980; Clark et al., 1980; Manning et al., 1984; Curtis et al., 1987).The animal preparations and surgical procedures to induce ischemia and reperfusion were performed as described previously (Chung et al., 2010).

Left coronary artery was occluded for 45 min followed by 2 h of reperfusion to induce an irreversible ischemia/reperfusion injury.

37

1.2. Experimental Groups

The animals were assigned to one of five treatment groups. (1) Control group: rats received the vehicle, dimethyl sulfoxide (i.p., 0.03 ml) 40 min prior to occlusion (n = 30); (2) Pre-Wog 5 group: wogonin (Biotic Chemical, Taiwan) 5 mg/kg was given i.p. 40 min prior to occlusion (n = 15); (3) Pre-Wog 10 group: wogonin 10 mg/kg was given i.p. 40 min prior to occlusion (n = 30); (4) Pre-Wog 20 group: wogonin 20 mg/kg was given i.p. 40 min prior to occlusion (n = 10); (5) Post-Wog 10 group: wogonin 10 mg/kg was administered i.p. 15 min after occlusion (n = 15). The blood pressure, heart rate and electrocardiograms were continuously monitored throughout the experimental period.

1.3. Ventricular Arrhythmias

Diagnosis and quantification of arrhythmias conformed with the guidelines of the Lambeth Conventions (Walker et al., 1988).Ventricular arrhythmias were recorded by the time to onset of first arrhythmia, incidence of ventricular tachycardia and ventricular fibrillation (all types), incidence of sustained ventricular fibrillation, and arrhythmia score (Johnston et al., 1983). Sustained ventricular fibrillation was defined as ventricular fibrillation lasting continuously for more than 120 sec (the incidence of ventricular fibrillation

38

provides a measure of susceptibility to ventricular fibrillation initiation, and the incidence of sustained ventricular fibrillation provides a measure of ventricular fibrillation maintenance in this model) (Curtis & Hearse, 1989; Tsuchihashi &

Curtis, 1991).All arrhythmias were scored on a 0-8 arrhythmia scoring scale for 0-30 min post-ligation period (Johnston et al., 1983). The value 0 was given for 0-50 ventricular premature contractions with no ventricular tachycardia or ventricular fibrillation over the observation period; 1, for 50-500 ventricular premature contractions only; 2, for > 500 ventricular premature contractions, or one episode of spontaneously reversible ventricular tachycardia or ventricular fibrillation; 3, for one or more episodes of spontaneously reversible ventricular tachycardia and/or ventricular fibrillation lasting less than 60 sec; 4, for reversible ventricular tachycardia and/or ventricular fibrillation episodes lasting 60-120 sec; 5, for ventricular tachycardia and/or ventricular fibrillation episodes lasting more than 120 sec; 6, fatal ventricular fibrillation starting at > 15 min after occlusion; 7, fatal ventricular fibrillation starting at between 4 min and 14 min 59 sec after occlusion; 8, fatal ventricular fibrillation within 4 min. The mortality in each group was also evaluated.

1.4. Area at risk and infarct

39

At the end of 2-h reperfusion, the left coronary artery was re-occluded and 0.3 ml Evens blue (3 %) was injected intravenously to denote the area at risk.

The heart was then excised and frozen for 90 min (-20 ^oC). The entire ventricular area was sectioned into four 3 mm thick slices from the apex to the base and incubated in 1 % triphenyl tetrazolium chloride (phosphate buffer, pH 7.4) for 20 min (37 ^oC). The surviving tissue turns a deep red, while the infarct portion is white. The slice was fixed in 10% formalin overnight. The areas of risk and infarct were taken with digital camera. The areas were then measured and analyzed using Image-Pro plus analysis software. Infarct size is presented as a percentage of area at risk (infarct: area at risk).

1.5. Plasma Creatine Kinase-muscle-brain Fraction, Lactate

Dehydrogenase and Tissue Necrosis Factor- Levels Analysis

Acute ischemia/reperfusion injury was assessed with the measurement of plasma creatine kinase-muscle-brain and lactate dehydrogenase levels 60 min after reperfusion (Moss et al., 2007). Plasma levels of creatine kinase-muscle-brain and lactate dehydrogenase were measured using an analyzer of Fuji DRI-CHEM FDC 3000 (Fuji Photo Film, Japan).The tissue necrosis factor- level was determined by an enzyme-linked immunoadsorbent

40

assay (rat TNF- Immunoassay Kit, R&D Systems, USA) according to the manufacturer’s instructions.

1.6. Superoxide Anion Production in Ischemic Myocardium after

Reperfusion

Superoxide anion production in ischemic cardiomyocytes after ischemia/reperfusion was measured by modified lucigenin-enhanced chemiluminescence, as described previously (Chen et al., 2006). In brief, myocaridium samples (3 × 3 mm) taken from the ischemic regions 30 min after reperfusion. Scintillation plates containing Krebs-HEPES buffer with lucigenin (1.25 mM) were placed into a microplate luminometer (Hidex, Microplate Luminometer, Finland). Counts were obtained in duplicate at a 15-sec interval.

Plates containing all components with the exception of organs were counted as background, and these blank values were subtracted from the chemiluminescence signals obtained from the organ samples. All samples were dried in a 90-^oC (16 h) oven for expressing results on a milligram myocardium dry weight basis. These results were expressed as count per second / milligram of myocardium dry weight.

1.7. Western Blot Analysis

41

To elucidate the effect of wogonin on the protein expression of MCP-1, activation of NF-κB, and p38 MAPK signaling pathway, and apoptosis elicited by ischemia/reperfusion, Western blot analysis was used. After 45-min ischemia and 2-h reperfusion, the ischemic region of myocardium was isolated and immediately frozen in liquid nitrogen, and stored at -80 °C until processed.

Primary antibodies probed in this experiment were mouse monoclonal anti-phospho-IκBα antibody (Cell signaling, USA; 1:1000), mouse anti-phospho-p65 antibody (Epitomics, USA; 1:1000), mouse monoclonal anti-phospho-p38 MAPK antibody (Cell Signaling; 1:1000), mouse polyclonal anti-MCP-1 antibody (eBioscience, USA; 1:1000), and rabbit monoclonal anti-caspase-3 (active) antibody (Epitomics, USA; 1:500), respectively. The ratios of phospho-IκBα, phospho-p65, phospho-p38 MAPK, MCP-1 or active caspase-3 to α-actin were calculated for statistical analysis to standardize densitometry measurements between individual samples.