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Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance

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Abstract

Purpose

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.

Procedures

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.

Results

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.

Conclusion

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

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Data Availability

Data are available from the authors on request.

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Funding

This work was supported in part by NIH NCI RO1CA1262159 (GRE) and R33 HL157907 (ESN and SSE).

Author information

Author notes

  1. Hanan B. Elajaili and Lukas B. Woodcock contributed equally to this work.

Authors and Affiliations

  1. Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, University of Colorado Anschutz Medical Campus, 12700 E. 19th Ave., B131, Aurora, CO, 80045, USA

    Hanan B. Elajaili, Nathan M. Dee & Eva S. Nozik

  2. Department of Chemistry and Biochemistry, University of Denver, Denver, CO, 80210, USA

    Lukas B. Woodcock, Tanden A. Hovey, George A. Rinard, Samuel DeGraw, Autumn Canny, Sandra S. Eaton & Gareth R. Eaton

  3. Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA

    Joseph P. Y. Kao

Authors
  1. Hanan B. Elajaili
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  2. Lukas B. Woodcock
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  3. Tanden A. Hovey
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  4. George A. Rinard
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  5. Samuel DeGraw
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Contributions

Elajaili, Hanan B: designed and performed mouse experiments, analyzed data, interpreted results, and wrote manuscript. Woodcock, Lukas B: designed, modified, and tested imager; performed mouse imaging experiments; analyzed data; interpreted results; and wrote manuscript. Hovey, Tanden A.: designed, modified, and tested imager; performed mouse imaging experiments; analyzed data; interpreted results; and wrote manuscript. Rinard, George A.: modified imager and designed performance tests, designed and built rapid scan resonator, and edited manuscript. DeGraw, Samuel: designed and implemented method of automated fitting of nitroxide spectra. Canny, Autumn: designed and implemented software for analyzing spectra of probes in lung, analyzed data, edited manuscript. Nathan, Dee M.: performed mouse experiment and edited manuscript. Kao, Joseph P. Y.: designed spin probe experiments, interpreted results, and edited manuscript. Nozik, Eva S.: designed experiments, interpreted results, and edited manuscript. Eaton, Sandra S.: designed experiments, analyzed data, interpreted results, and wrote manuscript. Eaton, Gareth R.: improved imager, designed experiments, analyzed data, interpreted results, and wrote manuscript. All authors (except for DeGraw) reviewed the final manuscript and approved submission.

Corresponding author

Correspondence to Gareth R. Eaton.

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The authors declare no competing interests.

Additional information

Dedicated to Samuel DeGraw who passed away during the preparation of the study

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Elajaili, H.B., Woodcock, L.B., Hovey, T.A. et al. Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance. Mol Imaging Biol (2023). https://doi.org/10.1007/s11307-023-01860-3

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  • DOI: https://doi.org/10.1007/s11307-023-01860-3

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