EGCG and adenosine receptor Vol. 63, No. 4, October 2012 thick transverse slices using a rat heart slice matrix (Zivic Instru- ments, Pittsburgh, PA, USA). The slices were incubated in Statistical analysis 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich Data are presented as means ± SEM. Data analysis was per- Chemical, St. Louis, MO, USA) in sodium phosphate buffer (pH formed with a personal computer statistical software package = 7.4) at 37oC for 20 min and subsequently immersed in 10% (SPSS for windows, Release 17.0; SPSS Inc, Chicago, IL, USA). formalin to enhance the contrast. The left ventricle (LV) was Data were analyzed using one-way analysis of variance (ANOVA) removed from the remaining tissue. The myocardial AR in the LV with Tukey’s HSD post-hoc testing. Null hypotheses of no was identified by illuminating with UV light. The necrotic area difference were rejected if P values were less than 0.05. (AN, unstained with TTC) and AR (nonfluorescent under UV light) were traced on a clear acetate transparent sheet (Fig. 2B and 2C) and quantified with UTHSCSA Image Tool, version 3.0 Results (Department of Dental Diagnostic Science at The University A total of 96 rat hearts were used for infarct measurement of Texas Health Science Center, San Antonio, Texas, USA). The experiment. Four rats were excluded during the stabilization areas were converted into volumes by multiplying them by slice thickness. The AN volume was expressed as a percentage of the period because of a CF > 18 ml/min or < 8 ml/min (2), LVDP < 80 mmHg (1), or HR < 250 beats/min (1). A further two hearts AR volume. All morphometric measurements were performed were excluded due to irreversible post-ventricular fibrillation in a blinded fashion by a separate technician. pump failure (1 in control and 1 in EGCG + ZM241385). There- Fig. 2. (A) Regional ischemia was induced by pulling the snare at the level of the proximal left coronary artery, (B) The area at risk of left ventricle slice was identified by UV light illumination as the tissue without fluorescence. (C) The area of necrosis was identified by unstained area by TTC (closed circle) in area at risk. Table 1. Morphometric Data (each group n = 9) Group CON EGCG EGCG + NAL EGCG + SPT EGCG + DPCPX EGCG + ZM241385 EGCG + MRS1706 EGCG + MRS1334 BW (gm) 315.6 ± 5.7 316.7 ± 5.7 315.6 ± 4.7 311.5 ± 5.7 318.8 ± 5.6 316.7 ± 5.7 313.3 ± 4.2 314.9 ± 5.3 HW (gm) 1.58 ± 0.03 1.61 ± 0.05 1.60 ± 0.03 1.61 ± 0.05 1.61 ± 0.04 1.60 ± 0.07 1.59 ± 0.05 1.59 ± 0.03 HW/BW (%) 0.50 ± 0.01 0.51 ± 0.01 0.51 ± 0.01 0.52 ± 0.02 0.51 ± 0.01 0.51 ± 0.03 0.51 ± 0.01 0.51 ± 0.01 LV volume (cm3) 0.708 ± 0.027 0.717 ± 0.023 0.698 ± 0.018 0.692 ± 0.020 0.723 ± 0.008 0.738 ± 0.023 0.740 ± 0.032 0.693 ± 0.031 AR volume (cm3) 0.492 ± 0.023 0.527 ± 0.035 0.461 ± 0.037 0.465 ± 0.032 0.468 ± 0.020 0.487 ± 0.031 0.468 ± 0.018 0.467 ± 0.037 Values are means ± SEM. CON: untreated control heart, EGCG: polyphenol (-)-epigallocatechin gallate, NAL: nonspecific opioid receptor anta- gonist naloxone, SPT: nonspecific adenosine receptor (ADR) antagonist 8-(p-sulfophenyl)theophylline hydrate, DPCPX: A1 ADR antagonist, ZM241385: A2a ADR antagonist, MRS1706: A2b ADR antagonist, MRS1334: A3 ADR antagonist, BW: body weight, HW: heart weight, LV: left ventricle, AR: area at risk. There were no significant differences among groups. 342 AR/LV (%) 69.6 ± 2.6 73.7 ± 4.8 65.7 ± 4.4 67.3 ± 4.1 64.8 ± 2.8 66.3 ± 4.6 63.5 ± 1.7 66.9 ± 2.4 www.ekja.org