Abstracts Biological Sciences

Real-Time Acquisition and Analysis of Endothelial Mitochondrial Superoxide Radical Production and Membrane Potential During In Vitro Ischemia/Reperfusion

by Randy James Giedt

The focus of this project was to elucidate the effects of ischemia/reperfusion on mitochondrial superoxide production by cultured endothelial cells in a parallel plate flow chamber in real time. To accomplish this, we used the mitochondria-targeted superoxide-specific fluorescent probe mitoSOX to determine parameters for its most effective use in a system composed of a parallel-plate flow chamber with cultured endothelial cells, a Nikon epifluorescence microscope, and digital image processing software. Based on the literature, the probe’s mitochondrial specificity is linked with the cell mitochondrial membrane potential. This made it necessary to study the effects of ischemia/reperfusion on mitochondrial membrane potential because of the possibility for mitoSOX to leak out of the mitochondria upon loss of mitochondrial membrane potential. The fluorescent probe rhodamine 123 was used in a similar manner as mitoSOX to accomplish this goal. The combination of the novel flow system and mitoSOX enabled us to obtain data comparable to that of a confocal microscope, as verified by the use of a variety of positive controls (both static endpoint images and real time analysis). Experiments using mitoSOX during ischemia/reperfusion showed a higher rate of mitochondrial superoxide production than that of shear alone, while the same experiments using rhodamine 123 showed a loss of mitochondrial membrane potential during ischemia and a partial recovery upon reperfusion. These studies may help us understand the mechanisms of injury on the mitochondria of coronary artery endothelial cells following cardiac ischemia/reperfusion.