Male Sprague-Dawley rats (Sam Tako Inc., Osan, South Korea) weighing between 220 and 250 g were used for this study. All animals were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health in 1996. The protocol was approved by the Animal Research Committee.

Animal preparation and surgery were performed as previously described [14]. Animals received general anesthesia with an intramuscular injection of 15 mg/kg of tiletamine/zolazepam (Zoletil50®, Virbac Lab., Carros, France) and 8 mg/kg of xylazine (Rompun®, Bayer, Seoul, South Korea). Additionally, 15 mg/kg of tiletamine/zolazepam and 8 mg/kg of xylazine was injected to maintain the level of anesthesia after 3 h reperfusion. If the tail moved (i.e. a sign of awakening), an additional 7.5 mg/kg of tiletamine/zolazepam and 4 mg/kg of xylazine was injected to maintain the level of anesthesia. The trachea was intubated, and then lungs were mechanically ventilated (tidal volume, 8 ml/kg; respiratory rate, 80 strokes/min) with ambient air using a volume-controlled rodent ventilator (Harvard Rodent Ventilator Model 607, Harvard, Holliston, MA, USA). The arterial blood pH and gases were maintained within normal physiological limits (pH, 7.35-7.45; PaCO₂, 30-40 mmHg; PaO₂, 60-100 mmHg).

A paramedian sternotomy was performed, and the pericardium was opened to expose the heart. For the I/R experiments, a 4-0 black silk was passed around the left coronary artery (LCA) territory to induce regional myocardial ischemia. The coronary artery was occluded by pulling a snare through a small vinyl chloride tube and clamping it with a mosquito haemostat. Reperfusion was achieved by releasing the clamp. Myocardial ischemia was confirmed by the appearance of regional epicardial cyanosis over the myocardial surface and by arrhythmia. After 30 min of ischemia, the myocardium was reperfused by loosening the snare, which was maintained for 6 h of reperfusion. Successful reperfusion was confirmed by the disappearance of epicardial cyanosis and the production of epicardial hyperaemia and arrhythmia after releasing the snare. Body temperature was monitored with a rectal thermometer (Sirecust 1260, Siemens Medical Electronics, MA, USA) and maintained at 36-38℃ with an electrical heating pad.

The animals were divided randomly into two sets of experiments. In the first set of experiments, animals were divided into three groups as follows: (1) I/R + Normal Saline (NS) group - rats in which 1 ml NS was administered intraperitoneally 30 min before the ischemic insult (n = 10), (2) I/R + U50 group - rats in which 50,000 U/kg ulinastatin (Ulistin®, Hanlim, Seoul, South Korea) diluted with NS was administered intraperitoneally 30 min before the ischemic insult (n = 10), and (3) I/R + U100 group - rats in which 100,000 U/kg ulinastatin diluted with NS was administered intraperitoneally 30 min before the ischemic insult (n = 10). Rats underwent a 30 min ischemia followed by reperfusion of the LCA territory. Hemodynamic measurements were recorded serially (30 min, 1 h, 1 h 30 min, 2 h, 6 h) during the 6 h of reperfusion, and myocardial infarct size was measured.

In the second set of experiments, animals were divided into five groups as follows: (1) sham + NS group - rats in which 1 ml NS was administered intraperitoneally 30 min before the sham operation (n = 6), (2) sham + U100 group - rats in which 100,000 U/kg ulinastatin diluted with NS was administered intraperitoneally 30 min before the sham operation (n = 6), (3) I/R + NS group - rats in which 1 ml NS was administered intraperitoneally 30 min before the ischemic insult (n = 6), (4) I/R + U50 group - rats in which 50,000 U/kg ulinastatin diluted with NS was administered intraperitoneally 30 min before the ischemic insult (n = 6), and (5) I/R + U100 group - rats in which 100,000 U/kg ulinastatin diluted by NS was administered intraperitoneally 30 min before the ischemic insult (n = 6). All animals were exposed to 30 min ischemia followed by 6 h of reperfusion. The sham group had paramedian sternotomy and pericardiotomy performed without the LCA occlusion. The thoracic cavity was opened to expose the heart for sampling of 3 ml blood from the right ventricle. Blood samples were collected in heparin containing tubes or conical tubes to measure cardiac troponin-I (cTnI), creatine kinase (CK) concentration, tumor necrosis factor (TNF)-α, and myeloperoxidase (MPO) activity. The entire sample was centrifuged, and serum and/or plasma were collected and frozen at -70℃ until analysed.

The right common carotid artery was cannulated with a 2-Fr Millar catheter (Model SPR-407, Millar Instruments Inc., Houston, TX, USA) and advanced into the left ventricle (LV) to measure baseline hemodynamic function (heart rate [HR], left ventricular end diastolic pressure [LVEDP], the maximum rate of intraventricular pressure increase [+dP/dt_max], and the maximum rate of intraventricular pressure decrease [-dP/dt_max]). Hemodynamic measurements were recorded serially (30 min, 1 h, 1 h 30 min, 2 h, 6 h) during the 6 h of reperfusion.

After cardiac function analysis, the LCA was occluded again and 2 ml of 1% Evans blue dye was injected into the pulmonary artery to facilitate area at risk (AAR) determination as described in a previous report [14]. The perfused myocardium was stained with Evans blue dye, whereas the occluded vascular bed was not. The atrium, right ventricle, and major vessels were removed from the heart, and the AAR was separated from the nonischemic area. The AAR was cut into small pieces and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37℃ for 20 min. The non-infarcted myocardium stained a deep red color with TTC, and the infarcted area (IA) remained pale yellow. The sections were fixed for 24 h in 2% formalin, and the LV, AAR, and IA weights were determined. The AAR and IA were expressed as a percentage of the LV and AAR weights, respectively.

Serum levels of cTnI were determined by an enzyme linked immunosorbent assay (ELISA) kit from Life Diagnostics (cat. no. 2010-2-HS, Life Diagnostics Inc., Wester Chester, PA, USA) as described previously [14]. The cTnI activity was detected by spectrophotometry (Infinite® F200, Tecan Group Ltd., Mannedorf, Switzerland) at 460 nm. Serum levels of CK were determined by EnzyChrom™ creatine kinase assay kit (ECPK-100, BioAssay Systems, CA, USA). CK activities were detected by colorimetric determination at 340 nm.

Plasma level of MPO were determined by rat MPO ELISA test kit (Hbt HK 105, Hycult biotechnology b.v., Uden, Netherlands) according to the manufacturer as described previously [15]. MPO activities were detected to determine neutrophil accumulation by spectrophotometry (Infinite® F200, Tecan Group Ltd., Mannedorf, Switzerland) at 460 nm after reaction of the plasma sample at MPO antibody-coated plate. Plasma concentrations of immunoreactive TNF-α were determined with an ELISA kit (RAT00, R&D Systems inc., Minneapolis, MN, USA) according to the manufacturer's protocol as described previously [15]. This assay employs the quantitative sandwich enzyme immunoassay technique. Plasma was reacted with the assay reagents in the TNF-α kit and analyzed by spectrophotometry (Infinite® F200, Tecan Group Ltd., Männedorf, Switzerland) at an absorbance of 460 nm.

All values are expressed as the means ± SD. All statistical analyses were performed using SPSS software (ver. 12.0 for Windows, SPSS Inc., Chicago, IL, USA). The differences between groups were evaluated using a Friedman one-way analysis of variance and a Tukey's multiple comparison post hoc test. Statistical significance was defined as P < 0.05.

The hemodynamic measurements recorded serially during the 6 h after reperfusion revealed substantial LV dysfunction as demonstrated by decreased ±dP/dt_max after I/R injury in the I/R + NS group compared to the basal hemodynamic function (Fig. 1). The LVEDP in the I/R + U100 group (12 ± 2 mmHg, P = 0.012) showed significant difference compared to the I/R + NS group (15 ± 2 mmHg ) after 6 h of reperfusion. The ±dP/dt_max in the I/R + U100 group (+dP/dt_max: +3,088 ± 362 mmHg; P < 0.001, -dP/dt_max: -2,157 ± 290 mmHg; P = 0.011) showed significant difference compared to the I/R + NS group (+dP/dt_max: +3,972 ± 227 mmHg/sec, -dP/dt_max: -3,412 ± 248 mmHg) after 6 h of reperfusion. The HR was not significant difference between the groups.

The AAR/LV induced by occluding the LCA territory was not significantlt different between the three groups. The IA/AAR in the I/R + NS group (49 ± 7 %) was significantly different compared to the I/R + U50 (40 ± 7 %, P = 0.016) and I/R + U100 groups (33 ± 7 %, P < 0.001) (Fig. 2).

I/R induced a significant increase in the serum level of CK after 6 h reperfusion. The elevation of serum CK concentration in the I/R + NS group (200 ± 40 U/L) was significantlt inhibited in the I/R + U50 (149 ± 45 U/L, P = 0.012) and I/R + U100 (114 ± 35 U/L, P < 0.001) groups after regional I/R injury (Fig. 3). I/R induced a significant increase in serum levels of cTnI after 6 h reperfusion. The elevation of serum cTnI concentration in the I/R + NS group (20 ± 4 ng/ml) were significantly higher than in the I/R + U50 (16 ± 3 ng/ml, P = 0.043) and I/R + U100 (14 ± 4 ng/ml, P = 0.012) groups after regional I/R injury (Fig. 4).

I/R induced a significant increase in the plasma level of MPO after 6 h reperfusion. The elevation of plasma MPO concentration in the I/R + NS group (3,760 ± 60 ng/ml) were significantly higher than the I/R + U50 (3,572 ± 84 ng/ml, P = 0.041) and I/R + U100 (2,848 ± 120 ng/ml, P < 0.001) groups after regional I/R injury (Fig. 5). I/R induced a significant increase in the plasma level of TNF-α after 6 h reperfusion. The elevation of plasma TNF-α in the I/R + NS group (18 ± 1.2 ng/ml) was significantly greater than the I/R + U50 (16 ± 0.9 ng/ml, P = 0.042) and I/R + U100 (13 ± 0.6 ng/ml, P < 0.001) groups after regional I/R injury (Fig. 6).