G. Gualtieri

Whole mouse nitroxide free radical pharmacokinetics by low frequency electron paramagnetic resonance

The in vivo uptake distribution and reduction of the oxygen-sensitive nitroxide spin label PCA in the mouse monitored by low frequency electron paramagnetic resonance (EPR) spectroscopy are reported. Spectra were obtained from the head and liver regions of pentobarbital anesthetized mice during different circulatory and ventilatory conditions. Identical clearances were found in these regions during normoxia. Moderate hypoxia (10% O2-90% N2) did not significantly affect the spin label reduction rate.

Extremely low frequency electromagnetic fields (ELF‐EMFs) induce in vitro angiogenesis process in human endothelial cells

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.

Inhibition of Angiogenesis Mediated by Extremely Low- Frequency Magnetic Fields (ELF-MFs)

The formation of new blood vessels is an essential therapeutic target in many diseases such as cancer, ischemic diseases, and chronic inflammation. In this regard, extremely low-frequency (ELF) electromagnetic fields (EMFs) seem able to inhibit vessel growth when used in a specific window of amplitude. To investigate the mechanism of anti-angiogenic action of ELF-EMFs we tested the effect of a sinusoidal magnetic field (MF) of 2 mT intensity and frequency of 50 Hz on endothelial cell models HUVEC and MS-1 measuring cell status and proliferation, motility and tubule formation ability. MS-1 cells when injected in mice determined a rapid tumor-like growth that was significantly reduced in mice inoculated with MF-exposed cells. In particular, histological analysis of tumors derived from mice inoculated with MF-exposed MS-1 cells indicated a reduction of hemangioma size, of blood-filled spaces, and in hemorrhage. In parallel, in vitro proliferation of MS-1 treated with MF was significantly inhibited. We also found that the MF-exposure down-regulated the process of proliferation, migration and formation of tubule-like structures in HUVECs. Using western blotting and immunofluorescence analysis, we collected data about the possible influence of MF on the signalling pathway activated by the vascular endothelial growth factor (VEGF). In particular, MF exposure significantly reduced the expression and activation levels of VEGFR2, suggesting a direct or indirect influence of MF on VEGF receptors placed on cellular membrane. In conclusion MF reduced, in vitro and in vivo, the ability of endothelial cells to form new vessels, most probably affecting VEGF signal transduction pathway that was less responsive to activation. These findings could not only explain the mechanism of anti-angiogenic action exerted by MFs, but also promote the possible development of new therapeutic applications for treatment of those diseases where excessive angiogenesis is involved.

Oxygen-dependent reduction of a nitroxide free radical by electron paramagnetic resonance monitoring of circulating rat blood

The effects of fraction of inspired oxygen (FiO2) on the reduction of a nitroxide free radical were studied by X-band electron paramagnetic resonance (EPR) monitoring of circulating rat blood. The decay half-life of the metabolism/elimination phase increased significantly by 24 +/- 8% during hyperoxia and decreased significantly by 16 +/- 4% during hypoxia.

New experimental procedures for in vivo L-band and radio frequency EPR spectroscopy/imaging

Electron paramagnetic resonance spectroscopy/imaging experiments are currently being performed on small animals and isolated organs. The exogenous paramagnetic probes commonly used are nitroxide (aminoxyl) free radicals. Nitroxide reduction and clearance were evaluated to understand in vivo distribution and metabolism. Pharmacokinetics data were collected with the same nitroxide at three different frequencies in an external rat blood circuit, in a mouse head and in a whole rat body. A 280 MHz spectrometer made it possible to obtain images of a whole rat body after nitroxide intravenous injection. At L-band (1500 MHz), a synchronization technique was used to obtain images of a dynamic phantom utilized to mimic the model of the isolated perfused rat heart. Different experimental procedures are discussed and the problems which affect sensitivity and resolution are evaluated.

Dielectric resonators in a TE102 X‐band rectangular cavity

A method for simply coupling a dielectric resonator (DR) to a standard TE102 rectangular cavity is described. The DR/TE102 resonator is easily realized with minor changes to existing rectangular cavities. In comparison with other resonant structures based on dielectric resonators, coupling of the microwave power to the dielectric is much easier. Its use in electron paramagnetic resonance spectroscopy results in a high filling factor and a high value of the H1 field inside the resonator. It can be particularly useful for pulsed application, where high filling factors are required.

An EPR study of lipid vesicles as paramagnetic agent vectors

Paramagnetic nitroxides have been proposed as probes in electron paramagnetic resonance (EPR) imaging and in clinical diagnosis. However, nitroxides are rapidly reducedin vivo to hydroxylamines, diamagnetic EPR-inactive species. Reduction occurs in blood via soluble agents such as ascorbic acid, as well as in the cells via enzymatic and non-enzymatic endocellular systems. To prevent the reduction, a water soluble nitroxide, i.e., potassium peroxylamine disulfonate, is entrapped in reverse phase evaporation vesicles. The loaded liposomes have a high entrapment capacity, and vesicles with the encapsulated agent are stable for days, even at room temperature. The vesiclesin vitro can almost completely prevent the reduction of the entrapped nitroxide by ascorbic acid. In blood of a rat, enriched with a homogenate of rat liver proteins, the vesicles are able to greatly prolong the life time of the nitroxide. In particular, the encapsulated nitroxide has a half-life of more than one hour, compared to two minutes for free nitroxide under the same conditions. Due to these protective effects, the lipid vesicles might be useful as a delivery system for paramagnetic agents.

Integration of D‐shaped gradient coils in a Bruker TM circular cavity for X‐band electron spin resonance imaging

A very compact design for the coil assembly of an X‐band imaging apparatus is described. The design is based on D‐shaped coils obtained by cutting a circular current distribution by two parallel chords. The coils make the best possible use of the space available within the particular geometry of a cylindrical Bruker TM110 cavity. Its compact design results in a maximum width of 45 mm, which is available in most electromagnets used for electron paramagnetic resonance (EPR) spectroscopy. Using nitroxide free radicals of 60 mT in linewidth and field gradients of 1.5 mT/cm, a resolution of 100 μm was obtained.

EPR study of Fremy’s salt nitroxide roduction by ascorbic acid; influence of the bulk pH values

EPR and UV spectroscopy were used to investigate the efficiency of ascorbic acid in reducing Fremy’s salt. Our data indicates that the first proton electron transfer from ascorbate occurs within the mixing time. Even after the disappearance of the UV signal of the ascorbate, EPR measurements showed that the reaction goes forward, indicating a biphasic redox process. The slower time-course of this second phase was related to the initial concentrations of the reductant. Experiments performed at four different pH values demonstrated that the reduction was a function of the bulk solution pH. At the lower pH, after a fast initial reduction, the Fremy’s salt EPR signal remained constant, while at physiological or higher pH a further reduction was found.The reaction rates demonstrate that the reducing power of ascorbic acid towards Fremy’s salt strongly depends on its dissociation state.

Static magnetic field effect on the Fremy's salt-ascorbic acid chemical reaction studied by continuous-wave electron paramagnetic resonance.

Static magnetic field effect in the framework of the radial pair mechanism (RPM) theory was studied on the biologically significant chemical reaction between ascorbic acid and Fremys salt. The data indicate that the reaction rate depends on the applied magnetic field strength. The time scale of the studied reaction and the improved continuous-wave electron paramagnetic resonance system allowed for the first time the direct comparison of the amplitude differences between exposed and control samples in the strictly same boundary conditions. Until now the RPM was studied in a different time scale, focusing only on faster reactions by time-resolved techniques or by spectrophotometer measurement. The magnetic field effects presently measured can not be extended tout court to living systems; however the understanding of magnetic field sensitivity in basic chemical reaction in vitro could help clarifying the underlying basic step of interaction between magnetic fields and biological systems.