Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining

• Published in: Journal of pharmacological and toxicological methods Vol. 55; no. 2; pp. 201 - 208
• Main Authors: Pitts, Kelly R., Stiko, Ann, Buetow, Bernard, Lott, Fred, Guo, Ping, Virca, Duke, Toombs, Christopher F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2007
• Subjects: Animals; Chromatography, High Pressure Liquid; Coloring Agents - chemistry; Electrophoresis, Polyacrylamide Gel; Heme; Hemeproteins - chemistry; Hemeproteins - metabolism; Hemorrhage; Hemorrhage - etiology; Hemorrhage - metabolism; Hemorrhage - pathology; Infarct; Methods; Myocardial Infarction - complications; Myocardial Infarction - metabolism; Myocardial Infarction - pathology; Myocardial Reperfusion Injury - complications; Myocardial Reperfusion Injury - metabolism; Myocardial Reperfusion Injury - pathology; Myocardium - chemistry; Myocardium - metabolism; Myocardium - pathology; Rat; Rats; Rats, Sprague-Dawley; Sequence Analysis, Protein; Staining and Labeling - methods; Tetrazolium Salts - chemistry; TTC; TTC; Infarct; Rat; Hemorrhage; Methods; Heme
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2006.06.005

Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 °C for up to 3 weeks. Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INF. However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury.