Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz...

Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease.

Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology.

The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×10¹⁴ CP• and (9.9 ± 1.0) ×10¹⁴ DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells.

The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.