Change over Time of Triphenyltetrazolium Measured Infarct Size in Rat Isolated Hearts
Authors: Cleveland William J , Douglas Hunter F , Salzman Michele M , Balzer Claudius , Riess Matthias L 
Published: June 2021

Introduction

Infarct size (IS) is a valuable measure used to assess damage caused by experimental ischemia/reperfusion (I/R) injury. Accurate quantification of IS is vital in determining the efficacy of treatments, and their ability to confer cardioprotection. Thus, a consistent means of assessing IS becomes necessary, in order to avoid unintentionally biased assessment of experimental parameters. Variable times at which IS is measured following 2,3,5‐triphenyltetrazolium chloride (TTC) staining may have a confounding effect on accurate measurements. The objective of this study was to determine the optimal time to measure IS following TTC staining in rat isolated hearts.

Methods

Langendorff‐prepared hearts from adult male Brown Norway rats (n=17) utilized for studies of cardioprotection in I/R injury were used. For this analysis, treatments were divided into groups based on: cardioprotection (n=3), control (n=9), or abolishment of cardioprotection (n=5). Following the experimental procedure, hearts were sliced transversely in 3mm slices and stained using 1% TTC solution, which stains viable cells red. Stained heart slices were then stored in 4% formalin at 4°C, then measured repeatedly in 24‐hour increments using cumulative planimetry. IS% was plotted over time to assess the temporal effects of measurement. Regression and correlation analyses were used to determine the relationship of IS% and time, with regards to the experimental treatments used. Data are expressed as mean±SEM and r2. Statistics: one‐way Anova, p<0.05, * vs control, † vs cardioprotective.

Results

Hearts showed a linear increase in measured IS% over time for all treatments. Hearts treated with a cardioprotective abolisher had higher IS% at 24‐hours (26±2.1%*†) than control (14±1.7%) and cardioprotective (10±3.5%). Additionally, hearts from the cardioprotective abolisher group had faster increases in measured IS% per day (4.3±3.4%, r2=0.91) than control (1.8±0.1%, r2=0.84) and cardioprotective (1.6±0.6%, r2=0.75).

Conclusion

These data show that measured IS has a positive correlation with time when TTC staining and cumulative planimetry are used. Additionally, the data indicate that certain treatments (e.g., cardioprotective abolishers) may cause accelerated increases in measured IS% over time. Care should be taken to measure IS at a set time ~24 hours after the experiment and TTC staining to avoid biasing data. Further experiments will need to look at more narrow time points to assess measures taken under 24 hours to determine whether this will provide more optimal measures.

Support or Funding Information

This work was supported, in part, by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.