Direct detection of intracellular superoxide generation from freshly isolated rat aorta: a novel approach
Although superoxide (O2?) plays an important role in vascular diseases, direct measurement of O2? in viable tissue remains a challenge. The aim of this study was to design and validate a procedure to measure O2? generation using freshly isolated aorta. Method: The 8 mm fresh aortic segments from male and female rats were cut longitudinally in Krebs buffer at 37°C and mounted on the silicon elastomer surface with the lumenal surface facing upward. The segments were incubated with the nitric oxide probe 4,5-diaminofluorescein diacetate (DAF-2DA, 5 ?M) or O2? probe dihydroethidium (DHE, 2.5 ?M), followed by fluorescent recoding at longitudinal edge using Leica DMIRE2 microscope. Acetylcholine-induced increased DAF fluorescence was taken as evidence for the endothelial integrity. To examine the effect of O2? generator, DHE fluorescence was quantified before and after incubation of segments with phorbol myristate acetate (PMA, 0.1 ?M), or PMA in the presence of O2? scavenger (tempol, 100 ?M) for 30 min. Results: The extent of PMA-induced O2? increase was greater in female tissues (9.2 fold) than that in males (2.8 fold), and this was prevented by the presence of tempol. This data was comparable to our recent report on gender difference in PMA-induced O2? production in rat aorta using isometric tension study. Conclusion: This procedure will allow us to detect O2? generation in translucent viable vessels. (Supported by NHLBI)
Direct detection of intracellular superoxide generation from freshly isolated rat aorta: a novel approach.
FASEB Journal, 22(Supp 1),