Marcello Alecci

Whole rat electron paramagnetic resonance imaging of a nitroxide free radical by a radio frequency (280 MHz) spectrometer

Low frequency (280 MHz) electron paramagnetic resonance spectroscopy has been used to follow uptake, distribution and reduction of the nitroxyl spin label PCA in the rat. No difference of half life was found in seven rats submitted to three administrations of PCA (11.3 +/- 0.4; 11.0 +/- 0.6 and 11.5 +/- 0.7 min). Transversal two-dimensional images of PCA distribution in the rat body were obtained over 6 min by means of field gradients. PCA was observed in three regions by projections along the longitudinal axis of the rat. PCA accumulation was found in the lower abdomen 12 min after the start of the PCA injection.

pH-sensitive imaging by low-frequency EPR : a model study for biological applications

The use of pH-sensitive nitroxides, in conjunction with low-frequency EPR, offers a unique opportunity for non-invasive assessment of pH values (in the range 0 to 14) in living animals. In the present study, we have investigated the potential use of pH-sensitive nitroxide free radicals in conjunction with EPR imaging techniques at low and very low frequencies (280 MHz-2.1 GHz). In particular, we have measured the hyperfine splitting (hfs) of a pH-sensitive probe at three different EPR frequencies: 280 MHz, 1.1 GHz and 2.1 GHz. We have also developed EPR imaging experiments with phantoms simulating in vivo conditions, using pH-sensitive probes at 280 MHz (spatial-spatial) and 1.1 GHz (spectral-spatial). Finally, we discuss the actual sensitivity/resolution limits of the EPR imaging techniques at low frequencies. Practical applications of this method in the biomedical field are suggested for the continuous and non-invasive localization of pH in vivo.

Hypoxia induces peroxisome proliferator-activated receptor α (PPARα) and lipid metabolism peroxisomal enzymes in human glioblastoma cells.

Glioblastoma multiforme (GBM) represents the most severe type of glioma, the most common brain tumor. Their malignancy shows a relationship with an increased proliferation and a poorly organized tumor vascularization, an event that leads to inadequate blood supply, hypoxic areas and at last to the formation of necrotic areas, a feature of glioblastoma. Hypoxic/necrotic tumors are more resistant to chemotherapy and radiation therapies, thus it is crucial to formulate new therapeutic approaches that can render these tumors more sensitive to the action of conventional therapies. It has been demonstrated that under hypoxia, gliomas accumulate lipid droplets and that this event is positively correlated with the degree of malignancy, glioblastoma being the most endowed with lipid droplets. We have previously demonstrated in ex vivo glioma specimens a grade‐dependent lipid metabolism perturbation. Here we studied the lipid pathways and the presence of stemness markers in glioma primary cultures, obtained from surgical specimens of patients affected by glioma at different grade of malignancy, GBM primary cultures cultured under both hypoxic and normoxic conditions, as well as normal human astrocytes. The results obtained demonstrate that hypoxia plays a crucial role in regulating the expression of lipid metabolism peroxisomal enzymes, the lipid droplets accumulation as well as the transcription factor PPARα. J. Cell. Biochem. 112: 3891–3901, 2011.

Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling

In breast cancer it has been proposed that the presence of cancer stem cells may drive tumor initiation, progression and recurrences. IL-8, up-regulated in breast cancer, and associated with poor prognosis, increases CSC self-renewal in cell line models. It signals via two cell surface receptors, CXCR1 and CXCR2. Recently, the IL-8/CXCR1 axis was proposed as an attractive pathway for the design of specific therapies against breast cancer stem cells. Reparixin, a powerful CXCR1 inhibitor, was effective in reducing in vivo the tumour-initiating population in several NOD/SCID mice breast cancer models, showing that the selective targeting of CXCR1 and the combination of reparixin and docetaxel resulted in a concomitant reduction of the bulk tumour mass and CSC population. The available data indicate that IL-8, expressed by tumour cells and induced by chemotherapeutic treatment, is a key regulator of the survival and self-renewal of the population of CXCR1-expressing CSC. Consequently, this investigation on the mechanism of action of the reparixin/paclitaxel combination, was based on the observation that reparixin treatment contained the formation of metastases in several experimental models. However, specific data on the formation of breast cancer brain metastases, which carry remarkable morbidity and mortality to a substantial proportion of advanced breast cancer patients, have not been generated. The obtained data indicate a beneficial use of the drug combination reparixin and paclitaxel to counteract brain tumour metastasis due to CSC, probably due to the combined effects of the two drugs, the pro-apoptotic action of paclitaxel and the cytostatic and anti-migratory effects of reparixin.

Post-processing noise removal algorithm for magnetic resonance imaging based on edge detection and wavelet analysis

A post-processing noise suppression technique for biomedical MRI images is presented. The described procedure recovers both sharp edges and smooth surfaces from a given noisy MRI image; it does not blur the edges and does not introduce spikes or other artefacts. The fine details of the image are also preserved. The proposed algorithm first extracts the edges from the original image and then performs noise reduction by using a wavelet de-noise method. After the application of the wavelet method, the edges are restored to the filtered image. The result is the original image with less noise, fine detail and sharp edges. Edge extraction is performed by using an algorithm based on Sobel operators. The wavelet de-noise method is based on the calculation of the correlation factor between wavelet coefficients belonging to different scales. The algorithm was tested on several MRI images and, as an example of its application, we report the results obtained from a spin echo (multi echo) MRI image of a human wrist collected with a low field experimental scanner (the signal-to-noise ratio, SNR, of the experimental image was 12). Other filtering operations have been performed after the addition of white noise on both channels of the experimental image, before the magnitude calculation. The results at SNR = 7, SNR = 5 and SNR = 3 are also reported. For SNR values between 5 and 12, the improvement in SNR was substantial and the fine details were preserved, the edges were not blurred and no spikes or other artefacts were evident, demonstrating the good performances of our method. At very low SNR (SNR = 3) our result is worse than that obtained by a simpler filtering procedure.

Two-dimensional 220 MHz Fourier transform EPR imaging

In the last decade radiofrequency continuous-wave EPR spectrometers have been developed to detect and localize free radicals in vivo. Only recently, pulsed radiofrequency EPR spectrometers have been described for imaging applications with small samples. In the present work, we show the first two-dimensional image obtained at 220 MHz on a large phantom (40 ml) that simulates typical conditions of in vivo EPR imaging. This pulsed EPR apparatus has the potential to make the time required for three-dimensional imaging compatible with the biological half-life of normally used paramagnetic probes.

First imaging results obtained with a multimodal apparatus combining low-field (35.7 mT) MRI and pulsed EPRI

Nuclear magnetic resonance imaging (MRI) provides excellent images of organs and is an essential diagnostic tool in the medical field. Electron paramagnetic resonance imaging (EPRI) is being increasingly used in the biomedical field because of recent hardware advances. We present the first images obtained with a low-field (35.7 mT) multimodal apparatus that combines MRI and pulsed EPRI. For this purpose, the sample is composed of two sections, one sensitive to MRI and the other sensitive to EPRI. The MRI section of the sample is composed of three tubes containing 7 ml of a 10 mM CuSO4 water solution. The EPR section of the sample is composed of two tubes containing 350 mg of lithium phthalocyanine. The EPR image represents the two-dimensional projection of the whole sample and is reconstructed from 32 one-dimensional projections by using the Fourier reconstruction method. The MRI image is obtained by selecting a sample slice, 10 mm in thickness, by using a spin-echo sequence and the two-dimensional fast Fourier transform. The experimental results obtained with this apparatus show that the spatial resolution is better than 1 mm for the MRI section and better than 7 mm for the EPRI section. The measured SNR of the MRI and EPRI images were about 60 and 160, respectively. A detailed description of the hardware, pulse sequences and image reconstruction techniques is reported.

Optimization of axial RF field distribution in low-frequency EPR loop-gap resonators

A novel coupling method that optimizes the axial RF distribution of low-frequency EPR loop-gap resonators is presented. It consists of a resonant coupling loop positioned at the centre of a two-section loop-gap resonator. This arrangement ensures a symmetrical distribution of the radio frequency field along the axis of the resonator. The design of a central coupling system suitable for EPR resonators operating at about 220 MHz is described. Experimental results show that with the central coupling system the RF field is symmetrical and has a very good axial homogeneity (100% of the resonator length).

Resonant inductive coupling of RF EPR resonators in the presence of electrically conducting samples

A new method of matching for radio frequency EPR resonators is proposed. It uses resonant inductive coupling and is particularly suitable for studies with lossy conducting samples. The equivalent circuit of the resonant inductive coupling method is presented and a theoretical analysis of the equations that produce the critical coupling coefficient is discussed. A prototype resonant inductive coupling suitable for loop gap resonators operating at about 200 MHz has been developed and tested. Experimental results obtained with saline solutions show the advantages of resonant inductive coupling in terms of the coupling coefficient, RF magnetic field amplitude and field homogeneity. This matching method has been especially designed for CW EPR applications, in which the adoption of critical coupling is important for optimization of sensitivity. Moreover, resonant inductive coupling should also produce benefits for pulsed EPR studies.

VALIDATION OF NUMERICAL APPROACHES FOR ELECTROMAGNETIC CHARACTERIZATION OF MAGNETIC RESONANCE RADIOFREQUENCY COILS

Numerical methods based on solutions of Maxwells equations are usually adopted for the electromagnetic characterization of Magnetic Resonance (MR) Radiofrequency (RF) coils. In this context, many difierent numerical methods can be employed, including time domain methods, e.g., the Finite-Difierence Time-Domain (FDTD), and frequency domain methods, e.g., the Finite Element Methods (FEM) and the Method of Moments (MoM). We provide