Evaluating Microvascular Hydrogen Peroxide

In vessels, Mito Peroxy Yellow 1 (MitoPY1)^{22 (link)} was used to evaluate microvessel generation of _mtH₂O₂. As previously described,^{12 (link)} after cannulation in a warmed chamber (37°C) containing HEPES buffer at pH 7.4, arterioles were perfused intraluminally with MitoPY1 (5 μmol/L, 1 hour) at low levels of flow, below the threshold for dilation, until the luminal surface was bathed in MitoPY1 containing buffer. Next the transvascular pressure gradient was changed from zero to 100 cm H₂O, and fluorescence measured after 5 minutes. Experiments were performed in the presence or absence of PEG-catalase (500 U/mL). Fluorescence was evaluated with a Nikon Eclipse TE200 microscope using a krypton/argon laser at excitation wavelength of 488 nm and measured emission between 530 and 590 nm. All comparisons were made using vessels studied at the same session with constant microscope image display settings. In cultured cells, mitoSox was used to measure superoxide generation. Cells were incubated for 30 minutes with 10 μmol/L mitoSox. Relative average fluorescence intensity was normalized to background fluorescence and presented as percent change from baseline.