To detect if nitric oxide (NO) is produced in rat forebrain ischemia, we applied an electron paramagnetic resonance (EPR) NO-trapping technique. We also performed a detailed characterization of the technique. Diethyldithiocarbamate (DETC) and Fe-citrate were used as NO-trapping reagents. Under controlled ventilation, forebrain ischemia was produced by occlusion of both carotid arteries combined with hemorrhagic hypotension at 50 mm Hg for 15 min. DETC and Fe were administered 30 min prior to the onset of ischemia. During ischemia, the cerebral cortex was removed, and EPR samples were prepared. At liquid nitrogen temperatures, the NO-Fe-DETC signal (a triplet signal centered at g = 2.039 with the hyperfine coupling constant aN of 13 G) was detected overlapping Cu-DETC signals. By perfusing various concentrations of an NO-generating agent, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, into the rat brains, the amount of the “trapped NO” was calibrated. The size of the NO-Fe-DETC signal was well correlated with the NO concentrations in the perfusate (correlation coefficient r = 0.998, p < 0.01). Based on this calibration curve, it was found that the amount of trapped NO during forebrain ischemia increased to seven times that of the control (control n = 5, forebrain ischemia n = 4, p < 0.005).
Electron Paramagnetic Resonance Characterization of Tetrahydrobiopterin Radical Formation in Bacterial Nitric Oxide Synthase Compared to Mammalian Nitric Oxide Synthase
