Heterogeneity of regional redox status and relation of the redox status to oxygenation in a tumor model, evaluated using electron paramagnetic resonance imaging.

It is widely accepted that redox status, along with the partial pressure of oxygen (pO(2)), determines the efficacy of some therapeutic methods applied to treat tumors, including radiation. Redox status, evaluated by the reduction of a nitroxyl probe, was reportedly heterogeneous in a mouse tumor model. However, neither variation of heterogeneity of the redox status among mice nor the relation of the redox status to pO(2) in tumors has been characterized sufficiently. In this study, the regional reduction status in a mouse radiation-induced fibrosarcoma tumor model was evaluated using sequential three-dimensional electron paramagnetic resonance (EPR) imaging after i.v. injection of a tissue-permeable nitroxyl probe, HM-PROXYL. The regional decay of HM-PROXYL signal obeyed first-order kinetics, and the amplitude of the reduction rate and extent of its heterogeneity in a tumor varied among six mice. The tissue pO(2) was measured using EPR oximetry with lithium phthalocyanine (LiPc) microcrystals implanted within the tumor. The location of LiPc was determined with EPR imaging. A sequential image was obtained following the injection of HM-PROXYL, even after LiPc implantation, by choosing an HM-PROXYL signal peak which does not overlap with the signal of LiPc. The relationship between pO(2) and the reduction rate at the region of pO(2) measurement was found to be low (r = 0.357) in 13 tumor-bearing mice, indicating that the extent of oxygenation does not necessarily affect the redox status under air-breathing conditions. The results strongly indicate the necessity of measurements of both redox status and oxygenation in every tumor to characterize tumor physiology.

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy.

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.

In vivo oxygen radical generation in the skin of the protoporphyria model mouse with visible light exposure: an L-band ESR study.

Although oxygen radicals are thought to play a key role in the skin injury that is caused by protoporphyria, there is no direct evidence of generation of these radicals in vivo. This study measured the generation of oxygen radicals caused by visible light non-invasively in the skin of griseofulvin-induced protoporphyria model mice, using an in vivo electron spin resonance spectrometer equipped with a surface-coil-type resonator that could detect radicals within about 0.5 mm of the skin surface. A durable nitroxyl radical was administered intravenously as a probe. Light irradiation enhanced the decay of the nitroxyl signal in griseofulvin-treated mice, whereas light irradiation did not enhance the signal decay in control mice. The enhanced signal decay was completely suppressed by intravenous administration of hydroxyl radical scavengers, superoxide dismutase or catalase, or the intraperitoneal administration of desferrioxamine. The enhanced signal decay with illumination was reversible, and quickly responded to turning the light on and off. These observations suggest that the hydroxyl radical is generated via an iron-catalyzed reaction in the skin. This paper demonstrates, for the first time, the specific generation of oxygen radicals in response to light irradiation of the skin of protoporphyria model mice.

Nonequivalent spectra of unpaired electrons in field and frequency modulation.

We report a difference in the spectral lineshapes of continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy between field and frequency modulation. This finding addresses the long-standing question of the effect of modulation in EPR absorption. We compared the first-derivative EPR spectra at 1.1 GHz for lithium phthalocyanine crystals, which have a single narrow linewidth in the EPR absorption spectrum, using field and frequency modulation. The experimental findings suggest that unpaired electrons have different behaviors under perturbation due to field and frequency modulation.

Detection of electron paramagnetic resonance absorption using frequency modulation.

A frequency modulation (FM) method was developed to measure electron paramagnetic resonance (EPR) absorption. The first-derivative spectrum of 1,1-diphenyl-2-picrylhydrazyl (DPPH) powder was measured with this FM method. Frequency modulation of up to 1.6 MHz (peak-to-peak) was achieved at a microwave carrier frequency of 1.1 GHz. This corresponds to a magnetic field modulation of 57microT (peak-to-peak) at 40.3 mT. By using a tunable microwave resonator and automatic control systems, we achieved a practical continuous-wave (CW) EPR spectrometer that incorporates the FM method. In the present experiments, the EPR signal intensity was proportional to the magnitude of frequency modulation. The background signal at the modulation frequency (1 kHz) for EPR detection was also proportional to the magnitude of frequency modulation. An automatic matching control (AMC) system reduced the amplitude of noise in microwave detection and improved the baseline stability. Distortion of the spectral lineshape was seen when the spectrometer settings were not appropriate, e.g., with a lack of the open-loop gain in automatic tuning control (ATC). FM is an alternative to field modulation when the side-effect of field modulation is detrimental for EPR detection. The present spectroscopic technique based on the FM scheme is useful for measuring the first derivative with respect to the microwave frequency in investigations of electron-spin-related phenomena.

1.1-GHz continuous-wave EPR spectroscopy with a frequency modulation method.

Continuous-wave EPR spectroscopy using a frequency modulation (FM) scheme was developed. An electronically tunable resonator and an automatic tuning control (ATC) system were used. Using the FM scheme instead of magnetic field modulation, we detected EPR absorption at the first derivative mode. We used a microwave frequency of 1.1 GHz in the present experiment. Similar signal-to-noise ratios were obtained with conventional field modulation and the FM method, and a low-quality factor EPR resonator was not necessary to suppress the significant microwave reflection from the resonator. The FM method with a tunable resonator may be an alternative solution to achieving phase-sensitive detection, when the side-effects of magnetic field modulation, such as microphonic noise and mechanical vibration, are detrimental for EPR detection.

Experimental investigation of RF magnetic field homogeneity in a bridged loop-gap resonator.

The relationship between an RF magnetic field and an electric shielding plate called a bridge in a bridged loop-gap resonator (BLGR), which is used for EPR spectroscopy and imaging, is examined. The magnetic field in a BLGR is perturbed due to discontinuity of the conductive loop and electric fringing fields near the gaps. The magnetic field homogeneity in the BLGR can be improved with a bridge. The optimum angle for such bridges can be found by measuring the RF magnetic field. In this study, the RF magnetic field was measured for BLGRs 70 mm in diameter, with a resonance frequency of 302 +/- 5 MHz. The field uniformity on a circle with a radius of 27 mm was improved by 8% with bridges at optimal angles. Improved RF magnetic field homogeneity associated with a BLGR is demonstrated.