Abstract 11838: Real-Time Flourescence Imaging of Cardiac Tissue Slices Provides Novel Insight into Mechanisms of Myocardial Protection
OBJECTIVE: There are limited models available for the assessment of myocardial protection. Here we provide the first report of the use of mammalian cardiac tissue slices for the real time analysis of molecular events critical to ischemia-reperfusion (I-R) injury.
METHODS: Hearts from wild type mice were extracted and sliced into 400 micron thin sections using an automated Leica tissue slicer. Cardiac slices were loaded onto a stainless steel airtight chamber mounted above an inverted microscope for real-time fluorescence analysis. Simulated ischemia-reperfusion injury was achieved by perfusing the slices for 60 minutes with ischemic media (Buffered salt solution, BSS, 0.5% O2, 20% CO2) followed by 3 hrs of normoxic BSS (21% O2, 5% CO2). For cell death analysis, wild type slices were treated with calcein viability dye (1uM) for 30 min prior to I-R.
RESULTS: 5-6 tissue slices were obtained from each preparation and full thickness contractions at a rate of 65 + 4 bpm were noted for as long as 16 hours post extraction. Cell death was 0.39 + 0.5%, 8.8 + 1.7%, and 54.9 + 8 % after baseline, ischemia, and reperfusion, respectively. Tetramethylrhodamine-ethyl ester (TMRE) staining and Nicotinamide adenine dinucleotide (NADH) fluorescence were used to evaluate mitochondrial injury during ischemia. A significant decrease in mitochondrial potential was noted during ischemia prior to reperfusion. To assess the role of oxidant stress during ischemia, slices were obtained from transgenic mice overexpressing a mitochondrially targeted ratiometric green fluorescent protein (ro-GFP) prior to simulated I-R. There was a significant increase in the oxidant signal by 13% after 60 min of ischemia compared with normoxic controls. Cell death after I-R was significantly attenuated by incubating the slices with the chemical antioxidant, N-acetyl cysteine (NAC).
CONCLUSIONS: Cardiac tissue slices provide a novel method for the analysis of myocardial cell death and oxidant stress. In our model, oxidant stress and a loss of mitochondrial potential occurred during ischemia and were required for reperfusion-mediated cell death.
- © 2011 by American Heart Association, Inc.