Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts

Abstract

Introduction Although the chemical mechanism of the triphenyltetrazolium (TTC) reaction, for macroscopic detection of myocardial infarction, has been described previously, literature reports on correct tissue preparation and the use of this technique in intact large animals are lacking. Methods We investigated the special requirements for TTC staining in blood-perfused porcine hearts, validated the various handling steps and provided detailed information for precise and easy use of this histochemical method. The left anterior descending coronary artery was occluded for 45 min followed by 6 h of reperfusion in an open chest preparation using anesthetised domestic pigs. The hearts were excised and the organ-handling steps and TTC-staining procedure validated. Results The protocol includes ( i) intracoronary saline perfusion, ( ii) pressure-controlled determination of the non-ischemic region by Evans blue dye, ( iii) a freeze-thaw cycle, ( iv) a triphenyltetrazolium incubation period, and ( v) a bleach cycle with 4% paraformaldehyde. The TTC-staining results were confirmed by histology of transitional regions of the infarct area, area-at-risk and non-risk-region. Discussion If some special features associated with blood-perfused porcine hearts are considered carefully, reliable results for subsequent infarct size calculations can be obtained and large potential errors excluded.