Projections for 2D spectral-spatial images were obtained by continuous wave and rapid-scan electron paramagnetic resonance using a bimodal cross-loop resonator at 251 MHz. The phantom consisted of three 4 mm tubes containing different 15N,2H-substituted nitroxides. Rapid-scan and continuous wave images were obtained with 5 min total acquisition times. For comparison, images also were obtained with 29 s acquisition time for rapid scan and 15 min for continuous wave. Relative to continuous wave projections obtained for the same data acquisition time, rapid-scan projections had significantly less low-frequency noise and substantially higher signal-to-noise at higher gradients. Because of the improved image quality for the same data acquisition time, linewidths could be determined more accurately from the rapid-scan images than from the continuous wave images.
Electron spin relaxation times at 295 K were measured at frequencies between 250 MHz and 34 GHz for perdeuterated 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (PDT) in five solvents with viscosities that result in tumbling correlation times, τR, between 4 and 50 ps and for three 14N/15N pairs of nitroxides in water with τR between 9 and 19 ps. To test the impact of structure on relaxation three additional nitroxides with τR between 10 and 26 ps were studied. In this fast tumbling regime T2−1 ∼ T1−1 at frequencies up to about 9 GHz. At 34 GHz T2−1 > T1−1 due to increased contributions to T2−1 from incomplete motional averaging of g-anisotropy, and T2−1 − T1−1 is proportional to τR. The contribution to T1−1 from spin rotation is independent of frequency and decreases as τR increases. Spin rotation dominates T1−1 at 34 GHz for all τR studied, and at all frequencies studied for τR = 4 ps. The contribution to T1−1 from modulation of nitrogen hyperfine anisotropy increases as frequency decreases and as τR increases; it dominates at low frequencies for τR > ∼ 15 ps. The contribution from modulation of g anisotropy is significant only at 34 GHz. Inclusion of a thermally-activated process was required to account for the observation that for most of the radicals, T1−1 was smaller at 250 MHz than at 1 to 2 GHz. The significant 15N/14N isotope effect, the small H/D isotope effect, and the viscosity dependence of the magnitude of the contribution from the thermally-activated process suggest that it arises from intramolecular motions of the nitroxide ring that modulate the isotropic A values.
Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T1 ~ T2 in the range of 0.50 to 1.1 μs at ambient temperature. Both spin-lattice relaxation T1 and spin-spin relaxation T2 are longer for 15N- than for 14N-nitroxides. The dominant contributions to T1 are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T1 on nitrogen nuclear spin state mI was observed for both 14N and 15N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band.
Bleomycin is a commonly used cancer therapeutic that is associated with oxidative stress leading to pulmonary toxicity. Bleomycin has been used in animal studies to model pulmonary fibrosis, acute respiratory distress syndrome, and pulmonary hypertension secondary to interstitial lung disease. The toxicity with bleomycin is initiated by direct oxidative damage, which then leads to subsequent inflammation and fibrosis mediated by generation of both extracellular ROS and intracellular ROS. While most studies focus on the intracellular ROS implicated in TGFβ signaling and fibrosis, the changes in the extracellular redox environment, particularly with the initiation of early inflammation, is also critical to the pathogenesis of bleomycin induced injury and fibrosis. In this review, we focus on the role of extracellular redox environment in bleomycin toxicity, with attention to the generation of extracellular ROS, alterations in the redox state of extracellular thiols, and the central role of the extracellular isoform of superoxide dismutase in the development of bleomycin induced injury and fibrosis.
Acute lung injury (ALI) is an inflammatory lung disease, which manifests itself in patients as acute respiratory distress syndrome (ARDS). Previous studies have implicated alveolar-epithelial succinate in ALI protection. Therefore, we hypothesized that targeting alveolar succinate dehydrogenase SDH A would result in elevated succinate levels and concomitant lung protection. Wild-type (WT) mice or transgenic mice with targeted alveolar-epithelial Sdha or hypoxia-inducible transcription factor Hif1a deletion were exposed to ALI induced by mechanical ventilation. Succinate metabolism was assessed in alveolar-epithelial via mass spectrometry as well as redox measurements and evaluation of lung injury. In WT mice, ALI induced by mechanical ventilation decreased SDHA activity and increased succinate in alveolar-epithelial. In vitro, cell-permeable succinate decreased epithelial inflammation during stretch injury. Mice with inducible alveolar-epithelial Sdha deletion (Sdha loxp/loxp SPC-CreER mice) revealed reduced lung inflammation, improved alveolar barrier function, and attenuated histologic injury. Consistent with a functional role of succinate to stabilize HIF, Sdha loxp/loxp SPC-CreER experienced enhanced Hif1a levels during hypoxia or ALI. Conversely, Hif1a loxp/loxp SPC-CreER showed increased inflammation with ALI induced by mechanical ventilation. Finally, wild-type mice treated with intratracheal dimethlysuccinate were protected during ALI. These data suggest that targeting alveolar-epithelial SDHA dampens ALI via succinate-mediated stabilization 2 of 18 | VOHWINKEL Et aL.
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