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Atti del III Convegno Nazionale Interazioni tra Campi Elettromagnetici e Biosistemi Napoli 2-4 luglio 2014 ISBN: 9788894008906 con il contributo di

Indice Effetti Biologici (Sessione 1) Improved protection against methylglyoxal and re-programming of energy metabolism as novel features of the proliferative response of human neuroblastoma cells to 50 Hz magnetic field S. Falone, S. Santini Jr, M. Cacchio, S. Di Loreto, F. Amicarelli

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A Low-Frequency Electromagnetic (LF-EMF) Exposure Scheme Induces Autophagy Activation to Counteract in Vitro Aβ-Amyloid Neurotoxicity F. Manai, L. Fassina, L. Venturini, F. Angeletti, C. Osera, N. Marchesi, M. Amadio, A. Pascale, G. Ricevuti, S. Comincini, S. Caorsi

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Pulsed electromagnetic field prevents H2O2-induced reactive oxygen species production by increasing MnSOD activity in SK-N-BE neuronal cells N. Marchesi, M. Amadio, S. Falone, C. Osera, L. Fassina, G. Magenes, S. Comincini, S. Caorsi, F. Amicarelli, G. Ricevuti, S. Govoni, A. Pascale

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Pulsed Electromagnetic Fields Stimulate Osteogenic Differentiation in Human Bone Marrow and Adipose Tissue Derived Mesenchymal Stem Cells A. Ongaro, A. Pellati, L. Bagheri, C. Fortini, M. De Mattei

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Effect of Magnetic Field (MF) on Alkaline Phosphatase Activity of Human Osteosarcoma Cells T. Rescigno, M. Caputo, B. Bisceglia, M. F. Tecce

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Exposure Of SH-SY5Y Cells To Inhomogeneous Static Magnetic Field (31.7-232.0 mT) During Cisplatin Administration C. Vergallo, M. Ahmadi, H. Mobasheri and L. Dini

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Human Fibroblasts Exposed to THz Radiation: Study of Genotoxic Effects by Micronucleus Assay A. Sgura, E. Coluzzi, C. Cicia, A. De Amicis, S. De Sanctis, S. Di Cristofaro, V. Franchini, E. Regalbuto, A. Doria, E. Giovenale, R. Bei, M. Fantini, M. Benvenuto, L. Masuelli, F. Lista, G. P. Gallerano

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Valutazione Esposizione Elettromagnetica Exposure by WLAN in an Indoor Environment L. Mondini, C. Sbrana, F. Arreghini

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Human exposure evaluation to mobile phone electromagnetic emissions G. d’Amore, L.Anglesio, A. Benedetto, M. Mantovan, M.Polese

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Indoor levels of ELF magnetic fields in buildings with built-in transformers S. Lagorio, L. Kheifets

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General public co-exposure to electromagnetic fields and radon in urban environment R. Massa, M. Pugliese, M. Quarto, V. Roca, S. Romeo, O. Zeni

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Applicazioni Medicali (Sessione 1) Dielectric and Thermal Properties of Tissues Undergoing Microwave Thermal Ablation Procedures L. Farina, V. Lopresto, R. Pinto, M. Cavagnaro

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Modelling of Deep Trascranial Magnetic Stimulation: Electric Field Distributions Induced in a Realistic Human Model by Different Coil Designs V. Guadagnin, M. Parazzini, S. Fiocchi, I. Liorni and P. Ravazzani

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Computational Modeling of Transcranial Direct Current Stimulation: Effect of Human Anatomical Variability M. Parazzini, S. Fiocchi, I. Liorni, V. Guadagnin, A. Priori, P. Ravazzani

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Non-invasive imaging of biological microstructures by VHF waves G. Ala, P. Cassarà, E. Francomano, S. Ganci, G. Caruso, P. D. Gallo

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Selective Targeting of Magnetic Nanoparticles for Contrast Enhanced Microwave Imaging: a Preliminary Study F. Costa, A. Sannino, O. M. Bucci

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Magnetic Nanoparticles Enhanced Microwave Breast Cancer Imaging: towards a First Experimental Assessment O. M. Bucci, G. Bellizzi, L. Crocco, R. Scapaticci, G. Di Massa, S. Costanzo, A. Borgia

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A Novel Electromagnetic Guiding System to Support Running Athletes Affected by Visual Diseases G. Cerri, A. De Leo, V. Di Mattia, G. Manfredi, V. Mariani Primiani, V. Petrini, M. Pieralisi, P. Russo, L. Scalise

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Effetti biologici (Sessione 2) Effects of ELF magnetic field tuned on “parametric resonance” conditions on single channel K+ currents in a human neural cell line I.Zironi, A. Virelli, E. Gavoçi, D. Remondini, B. Del Re, G. Giorgi, G. Castellani, G. Aicardi, F. Bersani

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Effects on Newborn Mice Irradiated with ELF Magnetic Fields and X-rays I. Udroiu, A. Antoccia, C. Tanzarella, L. Giuliani, A. Sgura

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Denaturation of Proteins Induced by Exposure to Electromagnetic Fields E. Calabrò, S. Magazù

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Millimeter wave radiation- induced movement in giant vesicles S. Romeo, R. Massa, A. Ramundo Orlando

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Protective effects of non-ionizing radiofrequency fields in mammalian cells damaged by mutagens: a possible involvement of DNA repair mechanism A. Sannino, O. Zeni, S. Romeo, R. Massa, Vijayalaxmi, M. R. Scarfì

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On the Ergodicity of Molecular Dynamics Simulations: a Single DNA Strand Under the Action of a Continuous Wave Electric Field. P. Marracino, A. Paffi, A. Bannò, F. Apollonio, M. Liberti, G. d’Inzeo

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Effetti Biologici (Sessione 2) Effects of 2,45 GHz Exposure on the Growth of Phaseolus vulgaris L. Plants N. D’Ambrosio, M.D. Migliore, R. Montuori, I. Petriccione, M. Somma, R. Massa

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The Orientation of Zebrafish (Danio rerio): Roles of Rheotaxis and Magnetotaxis A. Cresci, R. De Rosa, C. Agnisola

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Capillaroscopic and thermographic monitoring as a biological indicator in exposure to electromagnetic radiation R. Pennarola, R. Delia, A. A. Russo, E. Pennarola, R. Barletta

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MRI A numerical tool to evaluate occupational exposure in MRI S. Romeo, A. Sannino, O. Zeni, M. R. Scarfì, R. Massa, V. Cerciello, R. d’Angelo

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Implications of Directive 2013/35/UE on Protection of MRI Workers M. Tomaiuolo, R. Falsaperla, A. Polichetti

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Evolution of Subjective Symptoms in a Group of Operators Recently Engaged in Magnetic Resonance Imaging (MRI) G. Zanotti, F. Gobba

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Calculation of the ultimate intrinsic signal-to noise ratio in MRI for elliptical dielectric objects using Mathieu Functions B. Grivo, G. Carluccio,V. Angellotti, C. Ianniello, R. Massa, R. Lattanzi

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Electrical Characterization of Materials in the Frequency Range of MRI Applications V. Angellotti, G. Panariello, C. Ianniello, B. Grivo, M.D. Migliore, R. Massa, R. Lattanzi

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A Two-Compartment Tissue-Mimicking MRI Phantom for the Validation of Electrical Properties Mapping C. Ianniello, R., V. Angellotti, B. Grivo, R. Massa, R. Lattanzi

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Electromagnetic and Thermal Dosimetric Assessment of MRI Patients During the RF Exposure with Birdcage Coils N. Cesario, R. Massa

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Applicazioni medicali (Sessione 2) A numerical and experimental analysis of electroporation in mammalian cells exposed to ns pulsed electric fields P. Lamberti, S. Romeo, A. Sannino, M. R. Scarfì, V. Tucci, O. Zeni p. 73 Radar Detection Technique Based on Artificial Neural Network in Breast Cancer Diagnosis: Preliminary Assessment Study S. Caorsi, C. Lenzi

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Remote Breath Activity Monitoring by Using UWB and CW Radar Sensors S. Pisa, P. Bernardi, R. Cicchetti, R. Giusto, E. Pittella, E. Piuzzi, O. Testa

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Dosimetria numerica e sistemi espositivi A Meshfree Boundary Method for M/EEG Forward Computations G. Ala, G. E. Fasshauer, E. Francomano, S. Ganci, M. I. J. McCourt

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Numerical Dosimetry of In Vitro Biological Samples Exposed to an Induction Magnetic Field at 50 Hz C. Merla, V. Lopresto, C. Consales, B. Benassi, C. Marino, R. Pinto

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Numerical dosimetry inside an extremely-low-frequency electromagnetic bioreactor M. E. Mognaschi, P. Di Barba, G. Magenes, L. Fassina

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An exposure system for in vitro bioelectromagnetic studies under multiple-frequency scenarios S. Romeo, C. D'Avino, D. Pinchera, O. Zeni, M.R. Scarfì, R. Massa

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Design and development of a continuous wave functional near infrared spectroscopy system D. Agrò, R. Canicattì, M. Pinto, A. Tomasino, G. Adamo, L. Curcio, A. Parisi, S. Stivala, N. Galioto, C. Giaconia, A. Busacca

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A versatile, nanosecond electric pulse generation system for in vitro bioelectric research studies" S. Romeo, O. Zeni, A. Sannino, L. Zeni, M. R. Scarfì

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Valutazione del rischio ed implicazioni legali Evaluation of the occupational risk for workers exposed to the magnetic field of an induction heater L. Cerra

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Electromagnetic Radiation by the MUOS Satellite Earth Station in Sicily (Italy): Health Risk Assessment A. Polichetti, R. Pozzi

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Health Effects from Cell Phone Radiations. The Verdict of the Supreme Court of Cassation in Italy B. Bisceglia, V. Ivone, P. Persico

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Sessione di chiusura What is the state of present bioelectromagnetic research? Lesson from the activity in the editorial board of Bioelectromagnetics and from the School of Bioelectromagnetism “Alessandro Chiabrera” Riflessioni sulla ricerca in Bioelettromagnetismo: Conversazione di Ferdinando Bersani, Direttore Scuola Internazionale di Biolettromagnetismo-Alessandro Chiabrera p. 97

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Improved protection against methylglyoxal and re-programming of energy metabolism as novel features of the proliferative response of human neuroblastoma cells to 50 Hz magnetic field Falone S. *, Santini S. Jr*, Cacchio M.#, Di Loreto S.+, Amicarelli F.* *

University of L'Aquila, L'Aquila, Italy;#University “G. d'Annunzio”, Chieti, Italy; +Institute of Translational Pharmacology National Research Council (CNR), L'Aquila, Italy

programming. After an initial activation of the glycolytic pathway and a depression of mitochondrial aerobic activity (5 days), a decreased glycolytic flux and a near-significant increase of the mitochondrial pathway activity were revealed after 15-day exposure (Fig. 3), and this shift was confirmed by the analysis of the protein levels of peroxisome proliferatoractivated receptor-gamma coactivator-1alpha (PGC-1α) (Fig. I. INTRODUCTION 4). We also found that after an initial pro-oxidant effect, the Extremely low frequency magnetic fields (ELF-MFs) are ELF-MF treatment increased the amount of reduced GSH (Fig. currently classified as “possibly carcinogenic to humans” (1). 6), though not through an activation of GSSG recycling Human neuroblastoma cells chronically exposed ELF-MF pathway (not shown). Moreover, the long-term ELF-MF undergo a cytoproliferative response and express new proteins exposure reduced the lipid peroxidative damage (-40% vs. likely linked to enhanced antioxidant defense and CTR, Fig. 5) and increased catalase- and glutathione undifferentiated status (2). By overexpressing the glyoxalase peroxidase-based antioxidant activities (not shown), thus system, tumors are well protected against methylglyoxal (MG), confirming our previous findings (2). In chronically-exposed a dicarbonyl compound mainly produced by glycolysis, the cells we observed increased MG-targeting detoxification main energy source on which cancer cells rely (3). MG is capacities, along with improved removal of MG precursors cytotoxic through the promotion of dicarbonyl and oxidative and reduced MG-related protein damage; coherently, cells stress (4, 5). So far, ELF-MF-induced changes in energy exposed to MF for 15 days resulted much more resistant metabolism and in cellular defense towards MG are still against toxic concentrations of exogenous MG (Fig. 6). unknown. We investigated whether the strong proliferative However, 5-day ELF-MF treated cells exhibited increased response induced by ELF-MF on neuro-derived SH-SY5Y MG-related damage, reduced MG scavenging efficiency and cells could be linked to re-programming of cellular more vulnerability towards MG cytotoxicity (Fig. 6). metabolism and changes in the activity of detoxification systems against MG. Summary — A continuous exposure to a 50 Hz, 1 mT extremely low frequency magnetic fields (ELF-MF) induced a timedependent perturbation in cellular biology in human neuroblastoma cells, and promoted conditions commonly associated with more malignant phenotypes and probable chemoresistance.

II. MATERIALS AND METHODS The sinusoidal 50Hz, 1mT ELF-MF was generated through a copper-wired solenoid (2, 6). Cells were cultured and processed for viable cell counting as described previously (2). The concentration of exogenous MG (400µM) was chosen following concentration-response curves. Lipid peroxidation and GSH were measured photometrically (7, 8), enzymatic activities were measured spectrophotometrically (9-16), argpyrimidine, pCREB, pERK1/2 and b(III)-tubulin were measured through immunoblotting (17-19). ANOVA and appropriate post-hoc tests were used for inferential statistics (*P 100 °C), where complex phenomena related to both physical (e.g. water vaporisation) and morphologic modifications (e.g. tissue contraction) occur. Regarding the thermal properties of the biological tissues, their dependence from the temperature is settled. Yet the knowledge of the underlying mechanisms and related phenomena is limited: therefore more researches are essential to gain further insight.

[16]

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M. Ahmed, C.L. Brace, F.T. Lee FT, S.N. Goldberg. “Principles of and advances in percutaneous ablation”, Radiology, vol. 258, pp.351-69, 2011. L. Chin and M. Sherar, “Changes in dielectric properties of ex vivo bovine liver at 915 MHz during heating,” Phys Med Biol, vol. 46, pp. 197-211, 2001. C. Bircan and S.A. Barringer, “Determination of protein denaturation of muscle foods using the dielectric properties,” J Food Sci., vol. 67, pp. 202-205, 2002. C.L. Brace, “Temperature-dependent dielectric properties of liver tissue measured during thermal ablation: toward an improved numerical model,” in Conf. Proc. IEEE Eng. Med. Biol. Soc., 2008, pp 230–233. C.C. Johnson and A.W. Guy, “Non-ionising electromagnetic wave effects in biological materials and systems,” Proc. IEEE, vol.66, pp. 692–718, 1972. K.R. Foster, J.L. Schepps, R.D. Stoy and H.P. Schwan, “Dielectric properties of brain tissue between 0.01 and 10 GHz,” Phys. Med. Biol., vol. 24, pp. 1177-1187, 1979. K.R. Foster and H.P. Schwann, “Dielectric properties of tissues and biological materials: a critical review,” Crit. Rev. Biomed. Eng., vol. 17, pp. 25-104, 1989. S. Ryynanen, “The electromagnetic properties of food materials: a review of basic properties,” J. Food Eng., vol. 26, pp. 409-429, 1995. L. Chin and M. Sherar, “Changes in the dielectric properties of rat prostate ex vivo at 915 MHz during heating,” Int. J. Hyperth., vol. 20, pp. 517-527, 2004. P.R. Stauffer, F. Rossetto, M. Prakash, D.G. Neuman and T. Lee, “Phantom and animal tissues for modeling the dielectric properties of human liver,” Int. J. Hyperth., vol. 19, pp. 89-101, 2003. M. Lazebnik, M.C. Converse, J.H. Booske and S.C. Hagness, “Ultrawideband temperature-dependent dielectric properties of aninal liver tissue in the microwave frequency range,” Phys. Med. Biol., vol. 51, pp. 1941-1955, 2006. Z. Ji and C.L. Brace, “Expanded modeling of temperature-dependent dielectric properties for microwave thermal ablation,” Phys Med Biol, vol. 56, pp. 5249-5264, 2011. V. Lopresto, R. Pinto, G.A. Lovisolo, and M. Cavagnaro, “Changes in the dielectric properties of ex vivo bovine liver during microwave thermal ablation at 2.45 GHz,” Phys Med Biol, vol. 57, pp. 2309-2327, 2012. V. Lopresto, R. Pinto, and M. Cavagnaro, “Experimental characterization of the thermal lesion induced by microwave ablation,” Int J Hypertermia, vol. 30, pp. 110-118, 2014. J.W. Valvano, J.R. Cochran, K.R. Diller, “Thermal conductivity and diffusivity of biomaterials measured with self-heating thermistors,” Int J Thermophys., vol.6, pp. 301-311, 1985. D. Haemmerich, I. Santos, D.J. Schutt, J.G. Webster, D.M. Mahvi, “In vitro measurements of temperature-dependent specific heat of liver tissue,” Med Eng Phys., vol. 28, pp. 194-197, 2006. D. Yang, M.C. Converse, D.M. Mahvi, and J.G Webster, “Measurement and analysis of tissue temperature during microwave liver ablation,” IEEE Trans Biomed Eng, vol. 54, pp. 150-155, 2007. D. Yang, M.C. Converse, D.M. Mahvi, and J.G Webster, “Expanding the bioheat equation to include tissue internal water evaporation during heating,” IEEE Trans Biomed Eng, vol. 54, pp. 1382-1388, 2007. A. Bahattacharya and R.L. Mahajan, “Temperature dependence of thermal conductivity of biological tissues,” Physiol Meas, vol. 24, pp. 769-783, 2003. S.R. Guntur, K.I. Lee, D-G. Paeng, A.J. Coleman, and M.J. Choi, “Temperature-dependent thermal properties of ex vivo liver undergoing thermal ablation,” Ultrasound in Med & Biol, vol. 39, pp.1771-1784, 2013. M.J. Assael, K.D. Antoniadis and W.A. Wakeham, “Historical evolution of the transient hot-wire technique,” Int J Thermophys., vol. 31, pp. 1051-1072, 2010. S. Alvarado, E. Marin, A.G. Juarez, A. Calderon and R. Ivanov, “A hotwire method based thermal conductivity measurement apparatus for teaching purposes,” Eur J Phys., vol. 33, pp. 897-906, 2012.

Modelling of Deep Trascranial Magnetic Stimulation: Electric Field Distributions Induced in a Realistic Human Model by Different Coil Designs V. Guadagnin1, M. Parazzini1, S. Fiocchi1, I. Liorni1,2 and P. Ravazzani1 Istituto di Elettronica e di Ingegneria dell’Informazione e delle Telecomunicazioni, IEIIT-CNR, Piazza Leonardo da Vinci, 32, 20133 Milano, Italy 2 DEIB, Politecnico di Milano, Piazza Leonardo da Vinci, 32, 20133 Milano, Italy

1

Abstract—This paper investigates the induced electric field distributions in a realistic human head model due to 16 coil configurations. We used the scalar potential finite element method differentiating the brain structures. We found that, despite the presence of a depth-focality tradeoff, some coils are able to reach subcortical white matter tracts at effective electric field level.

constituted by the circular coil and the figure-8 coil, the other 14 are chosen because identified by the clinical literature as the most effective for DTMS [3].

I. INTRODUCTION Transcranial magnetic stimulation (TMS) is a non-invasive technique of neurostimulation based on the principle of electromagnetic induction of an electric field in the brain [1]. This field is able to modulate the transmembrane potentials of neuronal cells and, as a result, their activity. Recently, there has been an increasing interest in extending the TMS to deep brain region [2], with the consequences that new coils design have been developed [3]. In the meantime, several clinical studies showed that deep transcranial magnetic stimulation (DTMS) could be more effective than TMS in treating a very wide range of neurological, psychiatric and medical conditions, e.g. major depressive disorder, schizophrenia, bipolar depression, post-traumatic stress disorder, obsessivecompulsive disorder and substance addictions [4]. The region that is activated in the brain corresponding, approximately, with the areas in which the induced electric field (E) is maximum [3]. The location of these regions depends on the coil geometry and on its position on the scalp. Thus, the knowledge of the induced E distributions and the comparison between the different configurations of coils are indispensable in order to evaluate the experimental results and to optimize the therapeutic treatments. Despite the increasingly interest in this technique and the rising number of clinical applications [4], little has been done so far to quantify the induced E distributions in the brain due to different configuration of coil used for DTMS. Indeed, until now, these distributions have been estimated or using simplified head geometric models, such as sphere [3], or considering each coil separately and without a comparison among different coil configurations [5]. The aim of this study, therefore, consists in characterizing and comparing the induced E distributions in the brain of a realistic human head model by different configurations of coils for TMS and DTMS by mean of quantitative and comparative parameters. II. MATERIALS AND METHODS A. Coil designs Fig. 1 shows the 16 different coil configurations modelled in this study: two designs represent the references and are

Figure 1. 16 different coil configurations modelled divided into groups based on common geometric characteristics and placed on the head of the anatomical model “Ella”.

They are composed of a group of coils characterized by two pairs of figure-8 coil arranged according to different orientations on the scalp (group 1), of the category of large diameter circular coils (group 2), of the family of double cone coils (group 3), formed by two large adjacent circular windings fixed at different angles on the scalp, and of the group of H coils (group 4), consisting of complex threedimensional pattern of windings [5], [6]. B. Simulations The simulations were performed on the head of the realistic anatomical model “Ella” (a 26-years old female), of the Virtual Family [7]. The dielectric properties of each tissue were assigned based on the literature data at low frequency [8], [9]. Simulations were conducted using the magneto quasistatic low-frequency solver of the simulation platform SEMCAD X (by SPEAG, www.speag.com) [10]. With the purpose to compare the different coils configurations, in all the simulations a pulse current with amplitude of 1 A and a frequency of 5 kHz were used. C. Coil performance metrics For each coil, the E distributions were computed and analyzed in different brain structures. In each brain tissue, we estimated the percentage of volume that had E values equal to or greater than 10%-90% of the 99th percentile of the E distribution in the cortex. We also evaluated the E penetration depth by comparing the field values, normalized to the 99 th percentile of the cortical E, at the distances of 1 cm, 2 cm, 3 cm and 4 cm from the cortical surface both on frontal slices common to all the coils and on the frontal slices in which each coil reaches its maximum penetration depth. Moreover, we estimated the average distance from the cortical surface of the

50% of the 99th percentile of the E in the cortex of each coil on the frontal and on the sagittal planes. III. RESULTS Fig. 2 shows some examples of the E distributions in the brain due to some coils (i.e. the two reference coils and one for each group of DTMS coil configurations).

Figure 2. Surface distributions of the induced E in the cortex (1st row), white matter (2nd row) and cerebellum with deep brain tissues (3rd row) for the reference coils and for one coil of each group. The field amplitudes are normalized respect to the 99th percentile of the E distribution in the cortex.

In each panel the values are normalized with respect to the 99th percentile of the E distribution estimated on the cortex. These panels clearly show that varying the coil configurations induces variations in the E distributions in all the brain structures. More interestingly, it shows that only few coils (belonging to group 2 and 3) are able to reach deep brain structures (hippocampus, pons, midbrain, thalamus and hypothalamus), with E values ranging between the 20-30% of the maximum in the cortex. As to the E penetration depth, Fig. 3 shows the trend of E as a function of distance from the cortical surface for each coil configuration.

Figure 3. E values, normalized respect to the 99th percentile of the E distribution in the cortex, at increasing distance from the cortical surface, for the 16 coil configurations on the frontal slices in which each coil reaches its maximum penetration depth.

The E values are normalized respect to the 99th percentile of the E distribution in the cortex, evaluated for each coil configuration. It can be seen that the rate of decay of E with distance is significantly quicker for the reference coils, especially for the circular coil, and for the coils of the first group compared to the other configurations. In fact, the E values of these first coils at a depth of 4 cm are equal to 3040% of the maximum and to 20% of the maximum for the circular coil. The coils belonging to the second and to the

third group attenuate the degree of reduction of the induced E with the increase of distance: the E values at a depth of 4 cm are respectively equal on average to 55% and to 60% of the maximum. At last, the E decay profile as a function of distance from the cortical surface of the H coils (group 4) is more slower than the references and than the first group, but faster than the second and than the third group of coils. Indeed, the E values at a depth of 4 cm ranging between 47% and 55% of the maximum. In the case of the single circular coil, the average distance from the cortical surface of the 50% of the 99th percentile of the E evaluated in the cortex on different frontal and sagittal planes is approximately equal to 2 cm. For the figure-8 coil and for the coils belonging to the first group it is below 3 cm. The coils of the second group, on the other hand, increase the average depth of the 50% of the 99th percentile of the E in the cortex, up to 3.5 cm. The coils belonging to the third group are characterized by the highest depth values, ranging between 3.5 cm and 4.5 cm, on both planes. Finally, for the coils of the fourth group the average depth on both the planes ranges between 3 cm and 3.5 cm. IV. CONCLUSION This study shows that some configurations (particularly the H coils of group 4) result to be able to reach effective stimulation of subcortical white matter tracts. On the contrary, few coils are able to reach deeper brain structures with E values greater than 20% of the maximum. In conclusion, this study shows how the characterization of the E distributions could play an important role in the optimization of the geometry and of the position on the scalp of the coils and can help clinicians in the choice of the coil more suitable to fulfil the needs of a specific treatment. REFERENCES [1] A. T. Barker, R. Jalinous and I. L. Freeston, “Non-invasive magnetic stimulation of human motor cortex”, Lancet, vol. 1, pp. 1106-1107, 1985. [2] Y. Roth, F. Padberg, A. Zangen, “Transcranial magnetic stimulation of deep brain region: principles and methods”, Adv. Biol. Psychiatry, vol. 23, pp. 204-224, 2007. [3] Z. D. Deng, S. H. Lisanby, A. V. Peterchev, “Electric field depth-focality tradeoff in transcranial magnetic stimulation: simulation comparison of 50 coil design”, Brain Stimul., vol. 6, pp. 1-13, 2013. [4] F. S. Bersani, A. Minichino, P. G. Enticott, L. Mazzarini, N. Khan et al., “Deep transcranial magnetic stimulation as a treatment for psychiatric disorders: a comprehensive review”, Eur. Psychiatry, vol. 28(1), pp. 3039, 2013. [5] Y. Roth, A. Amir, Y. Levkovitz, A. Zangen, “Three-dimensional distribution of the electric field induced in the brain by transcranial magnetic stimulation using figure 8 and deep H-coils”, J. Clin. Neurophysiol., vol. 24, pp. 31-38, 2007. [6] A. Zangen, Y. Roth, B. Voller, M. Hallet, “Transcranial magnetic stimulation of deep brain regions: evidence for efficacy of the H-coil”, Clin. Neurophysiol., vol. 116, pp. 775-779, 2005. [7] A. Christ, W. Kainz, E. G. Hahn, K. Honegger, M. Zefferer et al., “The virtual family- development of surface-based anatomical models of two adults and two children for dosimetric simulations”, Phys Med. Biol., vol. 55, pp. N23-N38, 2010. [8] S. Gabriel, R. W. Lau, C. Gabriel, “The dielectric properties of biological tissues: II. Measurements in the frequency range of 10 Hz to 20 GHz”, Phys Med. Biol., vol. 41, pp. 2251-2269, 1996b. [9] C. Gabriel, A. Peyman, E. H. Grant, “Electrical conductivity of tissue at frequencies below 1 MHz”, Phys Med. Biol., vol. 54, pp. 4863-4878, 2009. [10] SEMCAD X v14.8 by SPEAG, Available at www.speag.com.

Computational Modeling of Transcranial Direct Current Stimulation: Effect of Human Anatomical Variability Marta Parazzini1, Serena Fiocchi1, Ilaria Liorni1,2, Vanessa Guadagnin1, Alberto Priori3,4, Paolo Ravazzani1 1

CNR Consiglio Nazionale delle Ricerche, Istituto di Elettronica e di Ingegneria dell’Informazione e delle Telecomunicazioni IEIIT, 20133 Milano, Italy; [email protected] 2 Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, 20133 Milano, Italy; 3 Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, 20122 Milano, Italy; 4 Centro Clinico per la Neurostimolazione, le Neurotecnologie ed i Disordini del Movimento, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122, Milano, Italy

Summary - This paper investigates how normal variations in anatomy may affect the current flow through the brain due to transcranial direct current stimulation. This was done by comparing the electric field and current density distributions within the tissues obtained in human models of different age and sex.

I INTRODUCTION Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that utilizes low intensity direct current to modulate brain excitability [1]. The increasingly widespread use of this technique and the rising number of clinical applications have boosted the interest in the estimation of the levels of the electric quantities in the brain tissues due to tDCS. As a consequences, there has been an increase of publications aiming to quantify the spatial distribution of the electric field (E) and of the current density (J) within the human brain tissues during tDCS, [for a review, see, 2-3]. Since the most of these studies are based on one single head model, it is still unknown how and if the human variability may affect the current flow through the tissues and particularly in the brain. Aim of this paper is to contribute in filling this gap in knowledge, assessing the effect of the anthropometric variables of the subject, of his/her morphological and anatomical characteristics on the spatial distribution of E and J within cortical and subcortical brain structures, considering different electrode montages. II MATERIALS AND METHODS Simulations were conducted using the simulation platform SEMCAD X (by SPEAG, www.speag.com), solving the Laplace equation to determine the electric potential distribution inside all the human tissues. Three realistic human models of the Virtual Family [4] (“Ella”: a 26-years-old female; “Duke”: a 34 years-old male; “Billie”: an 11 years old female) were used in the study. The dielectric properties of the tissues were assigned on the basis of the literature data at low frequency [5-6]. Four electrode montages were modeled: 1) Montage A: one electrode was placed on F3 and one on F4, as in [7]; 2) Montage B: one electrode was placed on T3 and the reference on the right arm, as in [8]; 3) Montage C: two electrodes were placed on C3 and C4, whereas the reference was on the right arm, as in [9]; 4) Montage D: one electrode was placed on Fz and the reference on the right tibia, as in [10]. In all the electrode montages described above, F3, F4, T3, C3, C4 and

Fz were referred to the 10-20 EEG system. The potential difference between the electrodes was adjusted to inject a total current of 1 mA in all the four electrode montages. For all the montages and the human models, the spatial amplitude distributions of E and J were analyzed in cortical and subcortical brain regions. For the gray and white matter, we also calculated the percentage of volume where the amplitude of E (or J) was greater than 70% or 50% of its peak. These percentages of volume will be named in the following sections as V70 or V50, respectively. To assess if there were any significant changes in the percentage of volume V70 or V50 due to the human anatomical variability, the Kruskal–Wallis one-way analysis of variance by ranks was performed. The human model was the only factor with three levels (“Ella”, “Duke” and “Billie”). If human model factor was significant, a Mann-Whitney non-parametric post-hoc multiple comparison test, with a Bonferroni adjustment to the criterion of significance, was also performed to assess which pairs of groups differ significantly from one another. The influence of the human variability on the field values was assessed evaluating the coefficient of variability (i.e., the ratio between the standard deviation and the mean, expressed in dB) among the models of both the peak and of the median of E (Epeak and Emedian) in the cortical and subcortical brain regions, for all the electrode montages. III. RESULTS Figure 1 shows the normalized amplitude distribution of E (or J) on different sections across the gray matter for the four electrode montages using “Billie” as an example. A qualitative comparison of these panels immediately indicates that varying the electrodes montage induces different spatial distribution of the amplitude of E (or J) in the gray matter. Interestingly for all the models, the symmetric electrode montages (Montage A and C) tend to have comparable amplitude of E/J in both hemispheres, while the lateralized electrode configuration (Montage B) generates higher amplitude of E/J asymmetrically in one hemisphere only. Similarly, configurations with anterior electrodes (Montage A and D) induce higher amplitude of both E (and J) in the anterior portion of the brain. The spread of the amplitude distribution over the gray and white matter, quantified as V70 or V50 resulted very similar across the human models. Indeed, statistical analysis on both V70 and V50 shows that the human model factor was not statistically significant (p-value>0.05) for all the electrode montages. The combination of human

model and electrode montage that shows the more focalized spatial distribution of both E (and J) is always the Montage A Montage A

Transversal Plane

TABLE I COEFFICIENT OF VARIABILITY (IN dB) OF THE PEAK AND OF THE MEDIAN OF E DUE TO THE VARIATION OF THE HUMAN MODEL IN DIFFERENT BRAIN TISSUES.

Sagittal Plane

CV of Epeak (dB)

CV of Emedian (dB)

Montage A

Montage B

Montage C

Montage D

Transversal Plane

Transversal Plane

Transversal Plane

Cortex White Cerebellum Pons Midbrain Medulla Thalamus

Coronal Plane


Coronal Plane


Sagittal Plane


Fig. 1 Brain sections across the gray matter of the spatial distribution of the amplitude of E/J for “Billie” for all the four electrode montages. The other tissues and brain regions have been masked of the images.

for any human model, whereas the electrodes montage characterized by a more widespread spatial distribution of the amplitude is Montage C. To quantify the variation due to the human variability on the field values, Table I reports the coefficient of variability among the models (CV, in dB) of both the peak and of the median values of E (Epeak and Emedian), in different brain regions for all the montages. The higher variability (up to almost 6 dB) was found for the Montage A, while for the Montages B-D the higher variability was about 3 dB (Montage D, thalamus, CV of Epeak). Only for the Montage A, the CV of Epeak is lower than the CV for Emedian, with the exception on the pons. On the contrary, for all the other Montages B-D, the variability of the peak is always higher than the one of the median, with the exception of the pons for all the montages. IV CONCLUSIONS Results of this study showed that the general trend of the spatial distributions of the field amplitude remains similar among the different human models, varying the electrode montages. However, the physical dimension of the subject and his/her morphological and anatomical characteristics somehow influence the detailed field distributions such as the field values.

3.96 4.27 5.47 5.54 5.04 4.76 5.83 Montage B 1.49 2.10 1.97 1.52 1.82 2.22 1.40 Montage C 1.23 1.50 0.75 1.14 2.19 2.21 2.37 Montage D 1.70 1.91 0.93 1.05 2.16 2.38 2.95

4.93 5.07 5.70 5.08 5.38 5.42 6.03 0.43 0.27 0.94 1.79 1.06 1.07 0.68 0.08 0.26 0.37 1.70 1.10 1.23 1.03 0.74 0.72 0.67 1.39 0.87 1.57 0.79

REFERENCES [1] [2]

[3] [4] [5] [6] [7] [8] [9]

[10]

Brunoni AR, Nitsche MA, Bolognini N, et al. Clinical research with transcranial direct current stimulation (tDCS): Challenges and future directions. Brain Stimul 2011 Apr 1. [Epub ahead of print] M Bikson, A Rahman A, Datta F Fregni, L Merabet. High-resolution modeling assisted design of customized and individualized transcranial direct current stimulation protocols. Neuromodulation. 2012 Jul;15(4):306-15. M. Bikson, A. Rahman, A Datta, Computational Models of Transcranial Direct Current Stimulation Clin EEG Neurosci, July 2012; vol. 43, 3: pp. 176-183 Christ A, Kainz W, Hahn GE, et al. The Virtual Family-development of surface-based anatomical models of two adults and two children for dosimetric simulations. Phys Med Biol 2010; 55: N23-N38. Gabriel S, Lau RW, Gabriel C. The dielectric properties of biological tissues: II. Measurements in the frequency range 10 Hz to 20 GHz. Phys Med Biol 1996; 41:2251-2269. Gabriel C. Comments on Dielectric properties of the skin. Phys. Med. Biol. 1997; 42: 1671-1674. B. Dell'osso, S. Zanoni, R. Ferrucci, et al., “Transcranial direct current stimulation for the outpatient treatment of poor-responder depressed patients,” Eur Psychiatry, 2011 [Epub ahead of print] M. Macis, F. Mameli, M. Fumagalli, et al., “On-line Transcranial Direct Current Stimulation (TDCS) in aphasia,” Neurol Sci; vol.31, pp. S37, 2010. F. Cogiamanian, S. Marceglia, G. Ardolino, S. Barbieri, A. Priori, “Improved isometric force endurance after transcranial direct current stimulation over the human motor cortical areas,” Eur J Neurosci;vol 26(1), pp. 242-249, 2007. Y, Vandermeeren J Jamart and M Ossemann 2010 Effect of tDCS with an extracephalic reference electrode on cardio-respiratory and autonomic functions BMC Neurosci. 11 38.

Non-invasive imaging of biological microstructures by VHF waves Guido Ala∗ , Pietro Cassarà† Elisa Francomano‡ , Salvatore Ganci∗ Giuseppe Caruso§ , Pio D. Gallo¶ , ∗ DEIM, Università di Palermo, Viale delle Scienze, Edificio 9, 90128 Palermo, Italy, [email protected], [email protected] † CNR, Istituto di Scienza e Tecnologie dell’Innovazione, Via G. Moruzzi 1, 56124 Pisa, Italy, [email protected] ‡ DICGIM, Università di Palermo, Viale delle Scienze, Edificio 6, 90128 Palermo, Italy, [email protected] § DIBIMEF, Università di Palermo, Via del Vespro, 129, 90127 Palermo, Italy, [email protected] ¶ DICHIRONS, Università di Palermo, Via L. GiuffrA¨, ˜ 5, 90127 Palermo, Italy, [email protected]

Abstract—The most of medical imaging methods involve ionizing waves and scanning of a wide human body area whether the area under investigation is large or not. In this paper, we propose a novel method to evaluate the shape of microstructures for application in the medical field, with a very low invasiveness for the patient. We focus our attention on the the tooth’s root canal shape estimation, which is a crucial step in the endodontic procedures. The proposed method makes use of a flexible thinwire antenna to be inserted into the tooth’s root canal and radiating non-ionizing Very High Frequency (VHF) waves. By measuring the spatial distribution of the magnetic field in the neighboring of the tooth, it is possible to reconstruct the tooth’s root canal shape by solving an inverse problem that involves the estimation of the shape of the antenna against the sensors panel. Simulation results show the validity and the robustness of the proposed approach.

I. I NTRODUCTION The medical imaging methods involve electromagnetic waves in a frequency range that spans from some Hz to GHz and over. Many of these methods are characterized by their invasiveness since they involve both ionizing waves and the scanning of wide areas even when only a focused investigation is needed. In particular, in this paper we address the tooth’s root canal shape reconstruction (Figure 1).

Fig. 1. Tooth’s root canal path (point red line).

The solution of such a problem is of great interests in endodontic procedures where rotary instruments, named “files”, are subjected to various types of mechanical stress. This stress is directly related to canal characteristics (i.e. angle, curvature radius, length, possible bifurcations) and modifies the instrument life and, consequently, its breakage [1]. Therefore, root canal reconstruction techniques have to be implemented. To this aim, as an alternative to established X-ray imaging,

we propose a novel method that is straightforward and can be applied many times on the patient, because of its low invasiveness. The proposed method works with non-ionizing low-power electromagnetic waves, in the Very High Frequency (VHF) range, and makes use of a system endowed with a microtransmitter and a sensors panel to acquire the spatial distribution of the magnetic field. The magnetic field component is selected since the physical domain can be characterized with about uniform magnetic permeability in the VHF range. The microtransmitter radiates the VHF waves by means of a flexible thin-wire microantenna, inserted in the root canal. Hence, the microantenna can be assumed to have the same shape of the analyzed structure. By measuring the spatial distribution of the magnetic field in the neighborhood of the thinwire antenna, it is possible to reconstruct the microstructure image by solving an electromagnetic inverse problem. II. M ETHODOLOGY The thin-wire antenna is supposed to be made by a sequence of linear segments: given a model for the characterization of the magnetic field at a set of points in space (forward problem) and given a set of measurements at these points, it is possible to solve the inverse problem in terms of the distances of the antenna axis points from the sensor panel. The forward problem can be solved in the frequency domain through a numerical model based on the point-matching Method of Moments (MoM) [2], [3]. The Levenberg-Marquart algorithm can be used in solving the inverse problem by means of minimization of the Euclidean distance between the measured field and the field generated by a given configuration of thinwire piecewise antenna [4]. The current distribution along the antenna is evaluated by solving an appropriate integral equation in frequency domain, derived from Maxwell’s equations. In particular, by introducing the relevant electromagnetic quantities (i.e., scalar electric and vector magnetic potentials) as functions of the unknown currents, the following integral equation in frequency domain, holds [4]: R R −jω L ~u · [~u0 Ω µIs (l0 )g(~r, ~r0 )dl0 ]dl+ R R R 0 (1) s (l ) + L ∂l∂tg [ jω1 ˙ Ω dIdl g(~r, ~r0 )dl0 ]dl = z˙s L Is (~r)dl 0 Equation (1) is a general relation that depends only on longitudinal current and on geometrical quantities related to

′

Exciting segment

has been reconstructed by assuming a two branch conductor or a three branch conductor, alternatively: if one considers common canal shapes and curvatures, both of these configurations can satisfactorily represent the canal shape. The behavior of the relative reconstruction error versus the SNR for different sensor panels and numbers of assumed antenna branches is shown in Figure 3. 0

10

2 branches, 24 sensors, flat panels 2 branches, 32 sensors, flat panels 2 branches, 32 sensors, round panel 3 branches, 24 sensors, flat panels 3 branches, 32 sensors, flat panels 3 branches, 32 sensors, round panel

-1

Relative error

the conductors constituting the thin-wire structures to be analyzed. In fact, Is is the longitudinal current flowing into the conductor, supposed concentrated on its axis (~u0 as the unit vector) because of the thin-wire assumption [2], ~u is the unit vector tangential to the conductor’s surface, as shown in Figure 2, z˙s is the per-unit-length surface impedance of the conductor, Ω is the length of the exciting conductor, the subscript tg indicates the component tangential to the wire surface, L is the length of the induced conductor, ~r and ~r0 are space position vectors of the observation and source points, ˙ r −~ −k|~ r0 | respectively; g(~r, ~r0 ) = e4π|~r−~r0 | is the Green’s function in an unbounded region. The quantity ˙ = + jωσ takes p into account the complex medium permittivity and k˙ = −ω 2 µ˙ is the wave number.

10

-2

10

′ O

Induced segment -3

10

III. N UMERICAL R ESULTS In order to validate the capabilities of the proposed method, numerical experiments concerning the shape estimation of a specific tooth’s root canal have been carried out. A NiTi (electrical conductivity 1.1 · 106 S/m) thin-wire antenna, 0.1 mm in radius and 2.3 cm in length, is assumed to be inserted in a typical root canal and fed by a sinusoidal current source with frequency equal to 100 MHz and the amplitude equal to 50 mA. It has to be underlined that this current value, as well as the selected frequency, can be well tolerated by the human tissues also depending on the time of application (i.e., a few seconds) [5]–[7]. In order to approximate the characteristics of a tooth, the medium around the canal is assumed to have zero electrical conductivity, relative permittivity equal to 15 and relative permeability equal to 1 [8]. The measurements are supposed to be affected by additive white Gaussian noise: the measured values of magnetic field at sensors locations have been calculated by means of the MoMbased forward solver described in Section II and then the noise has been added. Three different sensor panels has been tested, namely 24 or 32 sensors on two parallel flat panels and 32 sensors on a cylindrical panel. Moreover, in order to test the effect on reconstruction accuracy of an increasing number of variables involved in the optimization process, the shape of the antenna

30

35

40 45 SNR

50

55

60

dB

Fig. 2. General thin-wire geometric references.

The problem of finding the current distribution along the antenna, represented by equation (1), is numerically solved by splitting the thin-wire antenna into a finite number of linear segments. Once the currents are computed, the magnetic field components in the surrounding medium are given by the dipole theory, by superposing the effects of all segments [2].

25

Fig. 3.

Relative reconstruction error vs. SNR (dB).

Satisfactory accuracies has been achieved with all the configurations tested, even when the SNR is low. However, by approximating the antenna with a two branch conductor, a better robustness with respect to noise is observed. The prototype of the sensors panel and the related electronic equipment, is in progress. R EFERENCES [1] J. Wan, B.J. Rasimick, B.L Musikant, and A.S. Deutsch. A comparison of cyclic fatigue resistance in reciprocating and rotary nickel-titanium instruments. Journal Compilation Australian Society of Endodontology, 37:122–127, 2011. [2] G. Ala, P. Buccheri, E. Francomano, and A. Tortorici. Advanced algorithm for transient analysis of grounding systems by moments method. In IEE Conference Publication, pages 363–366, 1994. [3] G. Ala and M.L. Di Silvestre. Simulation model for electromagnetic transients in lightning protection systems. IEEE Transactions on Electromagnetic Compatibility, 44(4):539–554, 2002. [4] G. Ala, E. Cassarà, P. and Francomano, S. Ganci, G. Caruso, and P.D. Gallo. A numerical method for imaging of biological microstructures by VHF waves. Journal of Computational and Applied Mathematics, 259, Part B:805–814, 2014. [5] A. Barth, I. Ponocny, T. Gnambs, and R. Winker. No effects of short-term exposure to mobile phone electromagnetic fields on human cognitive performance: A meta-analysis. Bioelectromagnetics, 33(2):159– 165, 2012. [6] R.P. O’Connor, S.D. Madison, P. Leveque, H.L. Roderick, and M.D. Bootman. Exposure to gsm rf fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons. PLoS One, 5(7):1–16, 2010. [7] F. Sibella, M. Parazzini, A. Paglialonga, and P. Ravazzani. Assessment of sar in the tissues near a cochlear implant exposed to radiofrequency electromagnetic fields. Physics in Medicine and Biology, 54(8):135–141, 2009. [8] C. Gabriel. Compilation of the dielectric properties of body tissues at rf and microwave frequencies. Technical Report AUOE-TR-1996-0037, Physics Department, King’s College, London WC2R2LS, UK, June 1996.

Selective Targeting of Magnetic Nanoparticles for Contrast Enhanced Microwave Imaging: a Preliminary Study Federica Costa*, Anna Sannino*, Ovidio Mario Bucci*,# *

IREA - CNR, Via Diocleziano 328, 80124, Napoli; ( costa.f; sannino.a; bucci.om ) @irea.cnr.it; # DIETI - Università di Napoli Federico II, Via Claudio 21, 80125, Napoli; [email protected]

Summary –This communication summarizes our ongoing activity concerning the selective targeting of magnetic nanoparticles for contrast enhanced microwave imaging. In particular, we report the preliminary experimental results on the amount of targeting antibody which can be delivered in vitro in the most favorable conditions, thus providing an upper bound to the achievable nanoparticles concentration.

I.

INTRODUCTION

The use of magnetic nanoparticles (MNP), coupled to a proper differential measurement strategy, has been recently proposed to improve the capability of cancer microwave imaging (MWI) [1]. Since human tissues are nonmagnetic and MNP can be functionalized to selectively target cancerous cells, the use of MNP allows drastically reducing the occurrence of false positives and false negatives, which arises in standard MWI owing to the low electric contrast existing among cancerous tissues and healthy fibro-glandular ones. Indeed, an extensive, realistic, numerical study has shown that a reliable, quasi-real time, imaging of tumors of size smaller than 1 cm can be obtained, provided that a MNP concentration of the order of 10 mg/mL is achieved in the tumor [2]. A number of studies demonstrated the feasibility of conjugating nanoparticles with a monoclonal antibody against cancer cells receptors [3, 4], and targeting efficiencies even significantly larger than 10 mg/cm3 were reported, but the literature results are extremely variegated, differing by as much as two orders of magnitude. Due to the relevance of this point for the actual feasibility of the proposed imaging technique, we are performing an experimental campaign to provide a reliable upper bound to the achievable MNP concentrations in the most favorable conditions, namely:  cancer cells expressing an high number of receptors  highly selective targeting antibody Concerning the first point, several types of epithelial cancer cells overexpress Epidermal Growth Factor Receptor (EGFR) on their surfaces [5, 6]; in particular, the A431 line (Epidermoid Carcinoma Cell line) express 1-2 million molecules per cell [7], so that they are an ideal model of study. Concerning the antibody, Cetuximab (Ctx) is a clinically validated chimeric monoclonal antibody that targets EGFR with high affinity, abrogates ligand-induced EGFR phosphorylation [8] and initiates receptor endocytosis. Since each conjugated MNP harbors at least one attached antibody, the first step is the quantification of the amount of Ctx alone (i. e., without conjugating it to MNP) which can be targeted in vitro. This communication summarizes our ongoing activities on this topic and some of the obtained results.

II.

MATERIALS AND METHODS

The experiments were carried out on A431 and leukemia cell lines (HL-60), these latter used as negative controls for Ctx targeting because they do not expose EGFR. Cells were incubated with increasing concentrations of Ctx, (5, 10 and 20 μg/mL), using different incubation times (5, 30, 60, 120, 240 min), to determine the optimal concentration and treatment time which turned out to be 5 μg/mL Ctx and 1 h, respectively. Accordingly, the cells were treated with 5 μg/mL of Ctx for 1 h at 37°C or, in control experiments, 4°C (at this temperature the endocytosis is blocked). Then, the cells were washed with PBS for removing unbound Ctx, or with an acid buffer (50 mM Glycine-HCl pH 3.0, 0.1M NaCl) for removing also Ctx aspecifically bound to the cell plasmamembrane. Finally, the cells were fixed in paraformaldehyde, permeabilized with Triton X-100, blocked with bovine serum albumin (BSA), and stained with Rhodamine-linked anti-human IgG to detect and evaluate the Ctx specific targeting by indirect immunofluorescence. Cell nuclei were counterstained with SYTOX-green. The slides were analyzed with a Leica SP5 confocal microscope using an oil immersion objective (63X, NA 1.4). 543 nm HeNe and 488 nm Argon laser lines were used to excite Rhodamine and SYTOX-green, respectively. To determine the amount of targeting antibody, many Zstacks containing cells clusters, consisting of about 120 slices 0.1 µm thick slices, were collected. As the overall signal detected in a stack (the so called pixel sum) is proportional to the number of fluorophores (hence, of antibodies), by comparing such a signal with that from a known concentration of Ctx, the number of antibodies in the cell cluster could be evaluated in order to calculate the (mean) number of antibodies per cell.

III.

RESULTS

An example of the obtained results is shown in Figures 1-3. Figures 1 and 2 refer to Ctx targeting of A431 at 37°C, with PBS or acid buffer washing, respectively. As can be seen, a good deal of Ctx specifically targeted to the cells was actually internalized. The counter prove is shown in Figure 3, which refer to cells incubated at 4°C in order to block endocytosis. In this control experiment, only targeting of the plasmamembrane receptors was achieved. The very high selectivity of Ctx was confirmed by the results obtained with the HL-60 cells. In this case, not shown for brevity, no fluorescence on the cell membranes was detected, thus indicating absence of Ctx targeting.

The accurate determination of the mean number of targeting antibodies per cell is in progress. As a preliminary result, we here report that a first analysis on cells clusters provided a value of 1.6 million Ctx bound/internalized per cell, which matches very well with the above mentioned number of receptors found per cell. Further results will be provided during the oral presentation. IV.

Figure 1. Ctx binding/internalization in A431. Cells incubated with Ctx for 1 h at 37°C; PBS washing. Ctx labeled with Rhodamine-linked antihuman IgG (red). Nuclei were counterstained with SYTOX-green (green).

CONCLUSIONS

The use MNP, selectively targeted to cancerous tissues, could significantly improve the capability of cancer MWI. As a first step toward the determination of a reliable upper bound to the achievable MNP concentrations, we addressed the problem of quantifying the amount of targeting antibody which can be delivered in vitro. A431 Cell line has been adopted as target and Ctx as targeting antibody. The optimal incubation conditions have been determined, and the procedure for quantifying the uptake of antibody by confocal microscopy has been assessed. The first results of an extensive experimental campaign, which is in progress, indicate an antibody uptake of the order of 1.5*10 6 per cell, which is coherent with the known EGFR receptor expression level for the selected cell line. Apart from completing this preliminary step, which will be exploited to estimate a first upper bound, in the next future we will proceed to the delivery of Ctx conjugated MNP, in order to assess the actual loading possibilities on the tumor cells. REFERENCES

Figure 2. Ctx binding/ internalization in A431. Cells incubated with Ctx for 1 h at 37°C; acid washing. Ctx labeled with Rhodamine-linked antihuman IgG (red). Nuclei were counterstained with SYTOX-green (green).

[1] G. Bellizzi, O. M. Bucci , I. Catapano, “Microwave Cancer Imaging Exploiting Magnetic Nanoparticles as Contrast Agents”, IEEE Trans. on Biomedical Eng., Vol. 58, n. 9, pp.2528-2536, 2011 [2] O.M Bucci, G. Bellizzi, I. Catapano, L. Crocco, R. Scapaticci, “MNP Enhanced Microwave Breast Cancer Imaging: Measurement Constraints and Achievable Performances”, IEEE Antennas and Wireless Propagation Letters, Vol.11, pp.1630-1633, 2012. [3] C.L. Tseng, T.W. Wang, G.C. Dong, S. Yueh-Hsiu Wu, T.H. Young, M.J. Shieh, P.J. Lou, F.H. Lin, “Development of gelatin nanoparticles with biotinylated EGF conjugation for lung cancer targeting”, Biomaterials, Vol. 28, pp. 3996–4005, 2007. [4] S. Ke, X. Wen, M. Gurfinkel, C. Charnsangavej, S. Wallace, E.M. Sevick-Muraca, C. Li, “Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts”, Cancer Res., Vol. 63, pp. 7870–7875, 2003. [5] J. Mendelsohn, “The epidermal growth factor receptor as a target for cancer therapy”, Endocr Relat Cancer, Vol. 8, pp. 3-9,2001. [6] D.S Salomon., R. Brandt, F. Ciardiello, N. Normanno, “Epidermal growth factor-related peptides and their receptors in human malignancies”, Crit Rev Oncol Hematol, Vol. 19, pp. 183-232, 1995. [7] R.N. Fabricant, J.E. De Larco, G.J. Todaro, “Nerve growth factor receptors on human melanoma cells”, Proc. Natl. Acad. Sci. U.S.A., Vol. 74, pp. 565–569, 1977. [8] N.I. Goldstein, M. Prewett, K. Zuklys, P. Rockwell, J. Mendelsohn, “Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in human tumor xenograft model”, Clin. Cancer Res., Vol. 1, pp. 1311–1318, 1995.

Acknowledgements Figure 3. Ctx binding/ internalization in A431. Cells incubated with Ctx for 1 h at 4°C; acid washing. Ctx labeled with Rhodamine-linked antihuman IgG (red). Nuclei were counterstained with SYTOX-green (green).

This work was supported in part by the Italian Ministry of Research under the Program MERIT (Medical Research in Italy).

Magnetic Nanoparticles Enhanced Microwave Breast Cancer Imaging: towards a First Experimental Assessment Ovidio M. Bucci*, #, Gennaro Bellizzi*, Lorenzo Crocco#, Rosa Scapaticci#, Giuseppe Di Massa+, Sandra Costanzo+, Antonio Borgia+ *

#

DIETI - University of Naples Federico II, Via Claudio 28, 80125, Napoli, Italy, { bucci; gbellizz}@unina.it IREA- National Research Council of Italy, Via Diocleziano 328, 80124, Napoli, Italy, {crocco.l; scapaticci.r}@irea.cnr.it + DIMES, University of Calabria, Via Pietro Bucci, 87036, Rende (CS), Italy, {dimassa; costanzo} @deis.unical.it.

Abstract — Magnetic nanoparticles enhanced microwave imaging is an emerging technique for breast cancer screening, which is potentially capable to overcome the limitations of standard microwave based methodologies. In this communication, we describe the experimental system that has been realized to perform a first assessment of the basic principles underlying this novel technique.

I. INTRODUCTION In the last years, microwave imaging (MWI) has gained a significant attention as a potential tool for early stage breast cancer diagnosis. However, the expected breakthrough has not been achieved so far, and, despite the extensive research efforts, MWI is indeed currently not adopted in the clinical practice. The possible reason for this stalemate is the low contrast existing between healthy fibroglandular tissues and cancerous ones [1], which has indeed suggested that the typical practice of adopting agents to selectively increase the contrast should be pursued also in MWI in order to overcome such intrinsic limitations. The use of magnetic nanoparticles (MNP), already approved as contrast agents for magnetic resonance imaging, has been proposed in [2]. The power of this technique relies on the non magnetic nature of human tissues, which entails that properly targeted MNP can induce a specific and localized contrast variation into an otherwise magnetic homogeneous scenario. Such a circumstance means that the occurrence of false positives and false negatives is in principle avoided and dramatically reduced in practice. Extensive studies based on theoretical analysis and numerical simulations have demonstrated that this technique is feasible, as it requires devices whose dynamic range is comparable with those of standard MWI [3] and relies on imaging algorithms fed with a minimal amount of patient specific information [4]. However, to progress towards the stage of pre-clinical trials (which is the necessary next step due to the presence of a contrast agent), two issues must still be addressed. The first one is determining the actual concentration of MNP that can be targeted to cancerous cells, from which depends the amplitude of the useful signal for the imaging. The second one is the realization of a proof-ofconcept experiment to assess the technique when dealing with real antennas radiating in an actual coupling medium and interacting with MNP hosted in a biological environment. While the progresses concerned with the former issue are the topic of another communication presented at this

conference [5], in this work we describe the experimental system that has been realized to address the second aspect. II. DESCRIPTION OF THE EXPERIMENTAL SET-UP AND OF THE PROTOTYPE ANTENNAS One of the key features of MNP enhanced MWI is represented by the adoption of a polarizing magnetic field (PMF) to “drive” the MNPs, by turning on and off their response at microwaves [2]. As a matter of fact, this makes it possible to directly and effectively measure the diagnostically meaningful signal (the one backscattered by the targeted tumor), by means of a differential measurement strategy. This is far from being a detail since, the direct coupling of the antennas and the field backscattered by the breast would otherwise overwhelm the useful signal (due to the low amount of MNP that can be in any case targeted into cancerous cells). Accordingly, the proof-of-concept experiment herein presented aims to show the possibility to detect, with the expected dynamic range [3] and in a realistic situation, the variation of the scattering parameters due to a variation of the magnetic contrast induced modulating the amplitude of an externally applied PMF. For such a proof-of-concept demonstration, we have introduced a simplification consisting in considering only a “slice” of the actual device (transversal with respect to the breast), as this choice indeed allows to loosen the requirements in terms of the electromagnet’s performance, while keeping unaltered the meaningfulness of the experiment. The scheme of the resulting set-up is shown in Fig.1. Panel (a) shows the MWI portion of the device constituted by a copper box, of size 16x16x7cm, with four antennas mounted on the shorter sides. The antennas are connected through a switching matrix to a network analyser, so to allow the measure of four differential scattering parameters for each antenna position. The overall device is shown in panel (b), with the MWI part represented as the grey rectangular box placed between the poles of the electromagnet. This latter is in charge of producing (in a given portion of the copper box) the PMF whose amplitude modulation induces the variation of the magnetic contrast. The copper box is filled with a mixture of water and oil, whose properties allow to achieve a suitable coupling between the microwave fields and the breast, while keeping low losses (which would deteriorate the already low differential signal).

(b)

(a)

Fig. 1 Schematic representation of the proof-of-concept experiment. (a) Cross-section of the MWI system. The four antennas are placed at the sides of the metallic box, which is filled with the coupling medium. The sphere labeled MNP represents the magnetic nanoparticles embedded in a biological medium. (b) Cross-section of the whole system showing the electromagnet in charge of supplying the variable intensity PMF that drives the magnetic contrast’s variation. The box hosting the MNP is represented by the gray rectangle between the poles of the electromagnet.

While representing a simplified measurement set-up, it is worth noting that the design and the operation of such a simpler system already requires to address all the issues that that will have to be faced when realizing a full-scale prototype First of all, the magnetic part and the MWI one have not to interfere with each other so that the MWI sub-device has to be realized using diamagnetic metals. Second, a suitable intensity variation of the PMF has to be supplied to drive the variation of the magnetic contrast (this will provide information on the electromagnet required in the final prototype). Third, a real and proper coupling medium is exploited. Taking into account the above considerations, the design and the realization of the antennas needs to face two important aspects. The first one is that the probe has to exhibit the most effective radiating behavior in correspondence of MNP resonance frequencies [3,5], when operating in the adopted (dispersive and lossy) coupling medium. The second is that the antenna has to be designed in such a way to reduce as much as possible its dimension, in order to allocate all the probes actually required in the full scale prototype. Finally, besides being realized in diamagnetic material, the antenna should be immune to external fields, so it should radiate only in the broadside direction. For imaging purposes, the most convenient incident field is a magnetic field polarized along the axial direction of the slice (that is, parallel to the short side of the box) [3]. Accordingly, a cavity-backed microstrip slot antenna able to radiate into a dense medium (εr = 15-25; σ = 0.1-1S/m at around 2.3GHz) has been realized. The antenna, shown in Fig.2, is printed on a dielectric substrate having thickness equal to 0.762mm and relative permittivity εr = 10. The cavity is delimited by 1.5mm spaced via-holes, all having diameter equal to 1mm. From preliminary simulation results, it has been observed that the antenna exhibits a good matching at the design frequency of 2.28 GHz, which is suitable to achieve a meaningful MNP response [3]. To realize this goal, the dimensions and positions of the radiating slot, together with the location of the feeding probe, have been properly optimized.

Fig. 2 Prototype antenna

At the conference, the ongoing experimental activity, concerning with both the characterization of the whole system and the proof of concept measurements will be presented. ACKNOWLEDGMENT This work was supported in part by the Italian Ministry of Research under the Program MERIT (Medical Research in Italy). REFERENCES [1]

M. Lazebnik et al., “A large-scale study of the ultra wideband microwave dielectric properties of normal, benign and malignant breast tissue obtained from cancersurgeries,” Phys. Med. Biol., vol. 52, no. 20, pp. 6093–6115, Oct. 2007.

[2]

G. Bellizzi, O. M. Bucci, I. Catapano, “Microwave Cancer Imaging Exploiting Magnetic Nanoparticles as Contrast Agent,” IEEE Trans. On Biomed. Eng., vol. 58, no.9, pp.2528-2536, Sept. 2011.

[3]

O. M. Bucci, G. Bellizzi, I. Catapano, L. Crocco, R. Scapaticci,“MNP enhanced microwave breast cancer imaging: measurement constrains and achievable performance”, IEEE Antennas and Wireless Propagat. Lett, vol. 11, pp. 1630-1633, 2012.

[4]

R. Scapaticci, G. Bellizzi, I. Catapano, L. Crocco, O.M. Bucci, “An Effective Procedure for MNP Enhanced MWI”, IEEE Trans. on Biomed. Eng, vol. 61(4), pp. 1071-1079, 2014. F. Costa, A. Sannino, O.M. Bucci, “Selective Targeting of Magnetic Nanoparticles for Contrast Enhanced Microwave Imaging: a Preliminary Study,” Proceedings of ICEmB 2014, Naples, Italy, July 2014.

[5]

A Novel Electromagnetic Guiding System to Support Running Athletes Affected by Visual Diseases G. Cerri1, A. De Leo1, V. Di Mattia1, G. Manfredi1, V. Mariani Primiani1, V. Petrini1, M. Pieralisi1, P. Russo1, L. Scalise2 Dipartimento di Ingegneria dell’Informazione, Università Politecnica delle Marche, 60131 Ancona, Italy Dipartimento di Ingegneria Industriale e Scienze Matematiche, Università Politecnica delle Marche, 60131 Ancona, Italy 1

2

Summary – This contribution describes a novel electromagnetic guiding system for autonomous running of blind athletes. The system, consisting of a transmitting and a receiving unit, has been designed and realized using laboratory instrumentations. Finally it has been tested thanks to the collaboration of a blind volunteer with encouraging results.

I.

INTRODUCTION

In the world, 285 million people are estimated to be visually impaired: 39 million are blind and 246 have low vision [1]. It is commonly known that sport is an ideal mean to promote the integration of disabled people in general and the blind in particular. Sport can help people to overcome their disabilities by strengthening their self-esteem and their ability to face difficulties. It is equally true that the view plays an extremely important rule in almost every sport. For this reason the most popular sports among blinds are Goalball and Torball, which are specifically thought for people affected by visual diseases. The objective of the games is to roll a ball into the opposite goal while opposing players try to block the ball with their bodies [2]. The ball has minimal bounce and bells inside so as the players can track it listening for the sound produced. All players must wear opaque eyeshades at all times so everyone cannot see anything regardless of the degree of his/her visual impairment. Goalball was devised in 1946 in an effort to rehabilitate visually impaired veterans and in 1976 it was introduced at the Paralympic Games in Toronto. Torball was born around the 70s and it is quite similar to Goalball, but it is more versatile and can involve more people, as the size of the field is smaller, the ball is lighter and then better feasible even by women and children. Nevertheless, the number of blind and visually impaired people playing many other sports is increasingly growing. Some examples are athletics, judo, swimming, skiing and football. Visually impaired athlete can play many of them on their own, using suitable supports or following the vocal direction of a guide. Judo is not particularly difficult and the blind can compete on equal terms with sighted athletes because the rules require the constant physical contact between the two wrestlers. There are no particular difficulties even for swimming where, thanks to the floating lanes, the races can be played with all the athletes together. Unique special precaution is to allow the coach to touch the athlete on the head with a small stick to indicate the approach of the

pool-end and allow the preparation of the turn. As concerns skiing the modern technique used is based on a radio-guided communication: the instructor gives orders to the blind via a transceiver connected to a radio-headset placed on the head of the athlete. In other sports, such as running, visually impaired athletes still need to be physically linked to a sighted guide by means of a non-stretchable tether tied around their wrists or held between their fingers so as the distance between them does not exceed 50cm [3]. Although many guides are provided with an excellent preparation, the blind athlete’s performance could be limited by the presence of the sighted guide. This is mostly true for long races, as marathon, during which the blind athletes need to change more than one guide with evident disadvantages and limitations. In literature just few examples of technologies aimed to improve independency of blind athletes can be found. For example Balmer proposed a system based on the measurement of the magnetic field produced by a wire properly laid along the side of the track [4]. The field measured is then processed and compared with a reference signal in order to obtain information about athlete’s position respect to the correct track and to produce a suitable signal to alert the runner about the correct direction to follow. The main disadvantage of such system is the use of acoustic signals to communicate with the user. In fact it is commonly known that vibrations warning are to be preferred respect to auditory feedbacks, because the latter occupy user’s acoustic channel with the possibility of annoying him. Another recent equipment is the Remote Guide [5] that is able to guide the individual to the correct directions through auditory and vibrational sensors used to communicate the directions given remotely by a sighted guide. It is clear how this device is able to avoid the presence of the guide linked to the athlete during race, but it does not improve in a significant way the independence of blind runners and still present limitations due to the necessity of following the vocal directions of a coach. In this contest, an electromagnetic (EM) system has been designed and realized with the main aim of letting blind athletes run autonomously, at least during training. As Fig. 1 shows, the system is based on the generation of two EM walls drawing an invisible track that should be followed by the runner. In order to make sure that the athlete always remains inside the virtual hallway, it is necessary to generate a vibrotactile warning whenever he is getting closer to a borderline so as to encourage him to turn toward the central position,

where no more warning signals are produced.

Fig. 1 A schematic representation of the two EM walls created by the two transmitting antenna. The virtual hallway can guide the user, properly equipped with the receiving system, along whatever paths.

II.

vibrations produced by the system.

Fig, 2 Model of the transmitting antenna

SYSTEM DESCRIPTION

The EM guiding system proposed consists of transmitting and receiving units. The transmitting subsystem, to be placed on a car or a trolley running in front of the user, includes two radiating elements that generate the two EM walls and two amplitude (AM) modulators at two different frequencies in order to discriminate between left and right boundary. On the other side the receiving system is composed of a receiving antenna, an AM demodulator, two band-pass filters, a Micro Controller Unit for thresholds setting and further signal processing and two vibration transducers to communicate a warning signal to the blind athlete. An important issue is the design and the realization of the transmitting and receiving antennas working at about 10.47GHz. Since to generate an EM wall the radiation pattern should be narrow on the horizontal and wide on the vertical plane (fan shaped), a slot antenna has been chosen as radiating element. Fig. 2 and 3 show the antenna model and its radiation pattern respectively, while Table 1 resumes antenna radiation parameters calculated at the working frequency. As concerns the receiving element it has been designed in order to have a lightweight and wearable device with a radiation characteristic similar (but less restrictive) to those of the transmitting antennas. A microstrip array antenna of four elements has been modeled and realized. The combination of the transmitting and receiving radiation patterns leads to different levels of the received signals. As the position of the runner respect to the transmitting antennas changes, the magnitude of the received signal changes accordingly and it can be used to drive the warning signal. Thanks to the collaboration of a blind marathon athlete, the system performances have been tested on a series of paths set up in a big room of the faculty [6]. The blind volunteer has been equipped with the receiving unit while the trolley containing the transmitting system has been positioned at a safe distance in front of him. First tests have been carried out to let the user become confident with the new device. Then, the system has been tested on increasingly difficult paths and, most importantly, its performances have been compared with those of the traditional white cane with a clearly demonstration of the effectiveness of the system proposed. In particular, while using the cane the user needs to walk following a reference (as a ribbon delimiting the perimeter of the room), with the new system he can gradually go along the curves following no reference and being guided only by the

Fig. 3 Radiation pattern of the transmitting antenna calculated at 10.47GHz by CST Microwave Studio [7] T ABLE I R ADIATION P ARAMETERS OF TRANSMITTING ANTENNA AT 10.47GH Z

Gain Aperture Angle (-3dB), phi=0 Aperture Angle (-3dB), phi=90 III.

15dB 7deg (-23dB SLL) 76deg (-16.5dB SLL)

CONCLUSION

In this contribution an EM guiding system for autonomous running of marathon athletes has been designed and realized using laboratory instrumentation. Moreover, the system has been tested by a blind volunteer with encouraging results: the proposed EM device allows to guide a visually impaired or blind athlete along both easy and tricky paths; it ensures a fast pace that can be further increased simply installing the transmitting subsystem on a vehicle; according to user’s feedback he was completely confident while using the system, because it felt safe between the two EM walls without the need of looking for a reference to follow. REFERENCE [1] World Health Organization. Visual impairment and blindness – Fact Sheet 282. Available online: http://www.who.int/mediacentre/factsheets/fs282/en/ [2] http://www.paralympic.org/goalball [3] Paralympics 2012: the guide runners,” Sept. 2012, Available at: http://goo.gl/kCpHjy [4] Balmer, L. "A guidance device for a blind athlete." Medical and biological engineering 8.3 (1970): 301-307. [5] Ventura, Luiz Cláudio Locatelli, and P. R. Fernandes. "Remote guide for guiding the visually Impaired." Biosignals and Biorobotics Conference (BRC), 2011 ISSNIP. IEEE, 2011. [6] Test demonstration of the EM system. Available at: http://goo.gl/ddTTcE [7] https://www.cst.com

Effects of ELF magnetic field tuned on “parametric resonance” conditions on single channel K+ currents in a human neural cell line Isabella Zironi1, Angela Virelli1, Entelë Gavoçi1, Daniel Remondini1,2, Brunella Del Re2,3, Gianfranco Giorgi2,4, Gastone Castellani1,2, Giorgio Aicardi2,5, Ferdinando Bersani1,2 1

Department of Physics and Astronomy, University of Bologna, Italy Interdepartmental Center “L. Galvani”, University of Bologna, Italy 3 Department of Pharmacy and Biotechnology, University of Bologna, Italy 4 Department of Biological, Geological and Environmental Sciences, University of Bologna, Italy 5 Department for Life Quality Studies, University of Bologna, Italy 2

Unità ICEmB, Bologna. Viale Berti Pichat 6/2,40127 Bologna. e-mail: [email protected] SUMMARY

Despite the experimental evidence of biological effects of extremely low frequency magnetic fields (ELF-MF), the underlying mechanisms are still unclear. We studied the effect of different combinations of static and alternate ELFMF tuned on “parametric resonance” conditions for potassium (K+) on single channel K+ currents in differentiated human neuroblastoma BE(2)C cells. Present preliminary data obtained in four cells show a significant difference in open frequency of single K+ channels after exposure at 27 µT (rms), corresponding to the first maximum of the Bessel function. This observation needs to be confirmed in other cells.

have chosen a neuro-like cell line, namely the human neuroblastoma BE(2)C cells which, after 11-12 days of differentiation upon retinoic acid treatment, express various phenotypes of membrane K+ channels, which contribute to outward and inward currents. In a previous report, we have shown that the exposure to a combination of AC/DC magnetic field according to the “Ion Resonance Hypothesis” did not cause statistically significant changes in resting membrane potential, cell membrane capacitance and outward TEAsensitive K+ current density in whole-cell configuration [6]. Preliminary data obtained at single channel level are reported here. METHODS

The methods are reported in Figures 1, 2, and 3. INTRODUCTION

Several biophysical mechanisms have been proposed as a first step of the interaction of extremely low frequency magnetic fields (ELF-MF) with biosystems [1]. Among them the ion resonance models assume that resonant interactions may occur between specific ions in the biological material and a specific combination of static (DC) and low frequency alternate (AC) MF. The resonant frequency is assumed to coincide with the cyclotron frequency for the specific ion: f = (qBDC)/(2πm); where q is the charge of the ion, m is the mass of the naked ion and BDC is the magnitude of the DC magnetic field. The initial model of Liboff [2; 3] relates directly to movement of an ion through a channel across the cell membrane, while the “Paramagnetic Resonance Model” (PRM) of Lednev [4] and “Ion Parametric Resonance” (IPR) of Blanchard and Blackman [5] relate to stability of a chemical bond between an ion and a protein. These resonance theories do not predict directly specific biological effects, but simply a possible influence on ion dynamics at transduction sites which may eventually result in a biological change, probably many steps away from the primary interaction. In the present study, which is a second step of a larger project still in progress concerning the effects of MF on different ion channels, we investigated the effects of MF tuned on PRM conditions on neuronal K+ currents (IK), which plays a fundamental role in resting membrane potential maintenance and action potential repolarisation. For this investigation, we

Fig. 1. The exposure system consists of three large orthogonal pairs of square coils surrounding the patch-clamp setup allowing the manipulation of biological sample on the microscope stage as well as current recording during MF exposure. It is able to generate different combinations of DC and AC MF. A) Schematic representation of the exposure system geometry. B) The exposure system and the patch-clamp set-up: X, Y, and Z - the coils; A - the antivibration table; F- the Faraday cage.

Fig. 2. Plot of the Bessel function. Black dots indicate the points corresponding to different B1/B0 values. The corresponding values of DC and AC MF are reported in the Table below, for a cyclotron frequency of 8 Hz.

Table 1. Parameters of the magnetic field applied to the cells

DC magnetic field

AC magnetic field

B0 (µT)

B1 (µT) [rms]

Group no.

A

Freq.

Bessel function

fc (Hz)

J1(B1/B0) -

B0(x)

B0(y)

B0(z)

B1(x)

B1(y)

B1(z)

1

5

9.5

42

0

0

0

-

2

0

0

20.4

0

0

0

8

first zero

3

0

0

20.4

0

0

27

8

first maximum

4

0

0

20.4

0

0

55

8

second zero

5

0

0

20.4

0

0

77

8

first minimum

6

0

0

20.4

0

0

101

8

third zero

B

Fig. 3. Single channel inward K+ currents recorded in cell-attached configuration in differentiated human BE(2)C neuroblastoma cells. Representative example of single channel current traces recorded at – 100 mV of clamped voltage. Four open levels were often recorded. Cell differentiation was induced by adding to the medium 10 µM all-trans-retinoic acid (ATRA) for 11-12 days before data acquisition. Extracellular electrolyte solution contained (in mM): 123 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, 10 TEA. The micropipette solution contained (in mM): 145 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES. Sodium, calcium and chloride currents were always prevented by adding 100 nM TTX, 10 nM CdCl2 and 30 µM IAA in both solutions.

C

RESULTS

The results are reported in Figures 5 and 6. We found a significant (P < 0.05) difference between control and one treated group, i.e. the mean open frequency (both levels 1 and 2) of single K+ channels after exposure at 27 µT (rms), corresponding to the first maximum of the Bessel function (Fig. 6C). The exposure at 55 µT and 77 µT increased, although not significantly, the mean open time (Fig. 6A), probability (Fig. 6B) and frequency (Fig. 6C) of the level 2, and induced a larger variance compared to both control and first zero data. These observations need to be confirmed in other cells.

Fig. 6. The inward IK time of opening as a function of the MF points tuned on “parametric resonance” conditions for K+. Open states 1 and 2, defined in fig. 5, are elicited by voltages from -50 mV to -100 mV before and during exposure to MF (see Table 1). Data are presented as means of the values obtained in 4 cells by calculating: A) the time of opening divided by the number of openings (To/No); B) the time of opening divided by the time of recording (To/Ttot); C) the open frequency (number of openings divided by the total time of recording, No/Ttot) before (PRE; 0-4 min) and during (EXP, 4-8 min) exposure to MF. Then, a normalizing procedure was performed: each mean value was divided by the average of the values obtained during the PRE phase. The Student’s t test analysis was performed. REFERENCES

Fig. 5. The voltage-current relationship (I-V curve) for each open state (level) has been obtained from sample recordings.

[1] V.N. Bihni and A.V. Savin, Effects of weak magnetic fields on biological systems: physical aspects, Physics - Uspekhi 46: 259–291, 2003. [2] A.R. Liboff, Cyclotron resonance in membrane transport, In: A. Chiabrera, C. Nicolini, H.P. Schwan, editors. Interactions between electromagnetic field and cells. London: Plenum pp 281-296, 1985. [3] A.R. Liboff and B.R. McLeod, Kinetics of channelized membrane ions in magnetic fields, Bioelectromagnetics 9: 39-51, 1988. [4] V.V. Lednev, Possible mechanism for the influence of weak magnetic fields on biological systems, Bioelectromagnetics 12: 71-75, 1991. [5] J.P. Blanchard and C.F. Blackman, Clarification and application of an ion parametric resonance model for magnetic field interactions with biological systems, Bioelectromagnetics 15: 217-238, 1994. [6] E. Gavoçi, I. Zironi, D. Remondini, A. Virelli, G. Castellani, B. Del Re, G. Giorgi, G. Aicardi, F. Bersani, ELF magnetic fields tuned to ion parametric resonance conditions do not affect TEA-sensitive voltagedependent outward K+ currents in a human neural cell line. Bioelectromagnetics 34: 579-588, 2013.

Effects on Newborn Mice Irradiated with ELF Magnetic Fields and X-rays Ion Udroiu*, Antonio Antoccia*, Caterina Tanzarella*, Livio Giuliani+, Antonella Sgura* * Dipartimento + Centro

di Scienze, Università “Roma Tre”, V.le Marconi 446, 00146 Roma Ricerche Inail, Via Fontana Candida 1, 00040 Monte Porzio Catone (Roma) [email protected]

Abstract — Effects of ELF magnetic fields were studied exposing pregnant mice and their litters from day 12 post conception until weaning, with or without previous X-irradiation. Micronucleus test was performed on peripheral blood erythrocytes and reticulocytes of 101 pups. Preliminary data seem to indicate a genotoxic effect due to exposure to ELF magnetic fields.

breakage or spindle disturbance. The aim of this study was to investigate the possible synergistic effect of ELF magnetic fields and X-rays, during a very sensitive period such as the fetal and neonatal life, taking blood samples at different times. II. MATERIALS AND METHODS

I. INTRODUCTION Extremely low frequency (ELF) magnetic fields are almost ubiquitous in homes, cities and workplaces. However, there are few reports on in vivo genotoxic effects using micronucleus assays and the results are contradictory [1]. Svedenstal and Johanson [2] did not detect any increase of micronucleated erythrocytes in adult mice exposed for 90 days to a 14 µT magnetic field; the same result was obtained by Abramsson-Zetterberg and Grawé [3], using an identical field, both in adult and newborn mice. On the other hand, positive results were found using erythrocytes of adult rats exposed to 1 mT for 45 days [4], adult mice exposed to 5 µT for 40 days [5] and adult mice exposed to 200 µT for 7 days [6]. So far, only two works investigated genotoxic effects in newborn rodents, giving opposite results [3, 7]. Moreover, no study have been conducted in vivo on the genotoxic effects of ELF magnetic fields combined with X-rays.

Figure 1 – Solenoids used in the experiment

The micronucleus test [8] is a simple in vivo assay used to detect cytogenetic damage induced by chemical and physical mutagens. Micronuclei are produced after chromosome

Pregnant CD-1 Swiss (outbred) mice aging 6-8 weeks were divided into four groups, comprising two dams each. One group was unexposed and served as control (C, 27 pups); another group (E, 20 pups) was exposed to ELF magnetic fields from day 12 post conception (p.c.) until weaning, for a total of 30 days; another group (XE, 31 pups) was X-irradiated (1 Gy) on day 12 p.c. and immediately exposed to ELF magnetic fields until weaning (30 days in total); the last group (X, 23 pups) was X-irradiated (1 Gy) on day 12 p.c. The 50 Hz, 650 µT magnetic field was generated by a solenoid working 24 h per day. The solenoid (fig.1) was 0.8 m in length and 0.13 m in radius, with 552 turns of 2.5 mm 2 copper wire, wound in two layers in continuous forward-backward fashion around a cylinder of PVC. It was supplied by 50 Hz main power through a transformer. A voltage of 6.5 V (rms) has been applied to obtain a flux density of 650 mT (rms) at the centre of the solenoid. The field was uniform between ±5% in the volume where the mice were exposed. The solenoid was not shielded for the electric field, as the induced electric field was negligible due to the low voltage used. Exposure to X-rays was performed in a Gilardoni apparatus (Gilardoni, Italy; 250 kV, 6 mA, 3 mm Al filter) at a 0.5 Gy/min. The temperature and the relative humidity of the animal room were 22°C and 40% respectively. Artificial lighting was from 8 am to 8 pm and commercial pellets and tap water were available ad libitum throughout the experimental period. The temperature inside the coils was the same as in the room. Blood smears have been obtained puncturing the tail vein of the infant mice at birth and on day 11, 21, 40 and 140 after birth (the experiment is still in progress). The slides were fixed in absolute methanol and maintained at -20°C until staining. Smears were stained with acridine orange (20 µg/ml in pH 6.8 Sørensen buffer). Micronuclei were scored at 1000x magnification using a Zeiss Axiophot (Zeiss, Germany) fluorescence microscope (494 nm excitation filter, 523 nm barrier filter). Micronucleated

erythrocytes (MNE) and micronucleated reticulocytes (MNRET) frequencies were determined counting 1000 erythrocytes and 1000 reticulocytes per smear, respectively (fig.2). Since the frequencies were normally distributed, Welch’s t-test was used to compare the different groups. The level of significance was established at p0 N, tension force106), ruggedness, compact size, low cost, and reduced sensitivity with temperature, voltage fluctuations, and magnetic fields. These features make the SiPMs ideal candidates for CW-fNIRS applications and, furthermore, they could potentially increase the spatial resolution [4]-[8]. II. HARDWARE The following paper describes the work that has been carried out in order to develop an embedded CW-fNIRS system for monitoring haemodynamic signals during brain

activity [9]-[12]. The system has been designed in order to cover the entire skull surface, by employing a high number of optical components. Hence, it is capable to host up to 64 LED sources and 128 SiPM detectors. Additionally, in order to realize a compact and portable system, a multiplexing technique has been implemented, which allows to reduce the total power consumption for a battery-operated equipment. The system is realized as a scalable architecture, whose every elementary probe, built on a flexible stand, consists of 4 couples of bi-color LED (wavelengths equals to 735 and 850 nm) as light sources, 16 SiPMs as photo-detectors and a temperature sensor. The used SiPM are realized on n/p technology and they have been developed at the R&D lab at STMicroelectronics (Catania, Italy). Such SiPM are featured by a breakdown voltage of about 28 V and a 3.0× 3.0 mm2 active area (2500 microcells, 62% fill factor, and 60 µm cell pitch) enclosed in a 5.1 × 5.1 mm2 package. Each probe is linked to the main board, which hosts all the needed supply voltages and an ARM microcontroller (µC), capable to handle both the timing of the LEDs and the acquisition of the SiPMs. Finally, a UART interface transfers the digital data to a personal computer. In our CW-fNIRS system, the LED sources illuminate the tissue under investigation with a constant optical power. Light beam propagates inside the head, towards the grey matter, where it mostly bends. Then the SiPMs can capture the scattered and outgoing optical signal. The maximum depth of the photons injected inside the tissue depends on LED-SiPM distance. The time sharing polling schedule for the acquisition process is implemented as follows. The light sources are enabled one at a time and they radiate a constant optical power for 500 µs. During this time, SiPM selection is serially carried out. When a SiPM is selected, it is biased to the operating voltage and it produces a photocurrent proportional to the detected optical power. Such a current is then converted to voltage values by a signal conditioning circuit and it is finally acquired through the Analog to Digital Converter (ADC) integrated within the microcontroller. A dead time of 250 µs, in which all LEDs are off and no readout operations are performed, spaces the lighting of consecutive light sources. Moreover, the microcontroller implements a preliminary filtering action of data in view of a next post-processing and plotting. In any case, the system is able to handle LED-SiPM couples even if these optical components are positioned in different probes. This ability allows a user to optimize the handling of all the LED-SiPM couples, covering the head area under test. The main characteristic of the probes is their shape.

In fact, they could be joint together, thus covering the entire head surface of the patient. Each of the eight probes is driven by a dedicated circuit, as shown in Fig. 1. All the selecting circuits are controlled via the microcontroller. The current of the selected LED is adjusted via the voltage signal (VDAC) generated by the Digital to Analog Converter (DAC), integrated within the microcontroller, thus allowing a very fine tuning of the optical power emitted by each LED.

Fig. 2 oxygenated (red curves), de-oxygenated (blue curves) and total (yellow curves) haemoglobin levels coming from a LED-SiPM couple.

REFERENCES [1] [2]

[3] [4]

[5] Fig. 1 Management circuit of probe i-th

In addition, it is worth highlighting that another DAC module, integrated within the microcontroller, allows to finely control each working voltage (OverVoltage) of the selected SiPM. Furthermore, a -27.5 V voltage reference is used in order to keep all the unselected SiPMs at a voltage level just below their threshold, thus obtaining a faster response than the one obtained when starting the SiPM power supply from 0 V. III. EXPERIMENTAL RESULTS A first test has been carried out on a volunteer subject: on his forehead a probe board has been fixed by using a black elastic band. During a time window of 200 s, a trial was recorded. During such test, the subject under investigation was normally breathing with the exception of the time interval between 70 and 100 s, during which a breath holding phase was set up. Data collected from a SiPM positioned at a distance of about 2.6 cm from the LED source are shown in Fig. 2. The region enclosed by the two green vertical lines points out the breath holding phase. Raw data have been first elaborated by following the modified Beer-Lambert’s law and then they have been filtered through a 300 mHz low-pass filter so to reject the unwanted cardiac pulse (located at about 1.1 Hz). The oxygenated (red curves, HBO2), deoxygenated (blue curves, HB) and total (yellow curves, blood volume, BV) haemoglobin concentration levels are depicted in Fig. 2. It could be noticed that a significant variation of the total haemoglobin appears during the breath holding phase. This should clarify that the grey matter of the brain has been reached. In particular, such experimental results are in accordance with other similar tests reported so far, but with a completely different equipment [13].

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A. Bakker, B. Smith, P. Ainslie and K. Smith, “Near-Infrared Spectroscopy”, Applied Aspects of Ultrasonography in Humans, Prof. Philip Ainslie (Ed.), pp. 65-89, 2012 B. Chance, E. Anday, S. Nioka, S. Zhou, L. Hong, K. Worden, C. Li, T. Murray, Y. Ovetsky, D. Pidikiti and R. Thomas, “A novel method for fast imaging of brain function, non-invasively, with light”, Optical Express, vol. 2, pp. 411-423, 1998 P. Rolfe, “In Vivo Near Infra-Red Spectrophotometry”, Annual Reviews in Biomedical Engineering, vol. 2, pp. 315-354, 2000 G. Adamo, D. Agrò, S. Stivala, A. Parisi, C. Giaconia, A.C. Busacca and G. Fallica, “SNR measurements of silicon photomultipliers in the continuous wave regime”, Photonics West 2014 - Silicon Photonics IX, 2014, paper no. 8990-43 G. Adamo, D. Agrò, S. Stivala, A. Parisi, G. C. Giaconia, A. C. Busacca, M. Mazzillo, D. Sanfilippo and G. Fallica, “Measurements of Silicon Photomultipliers Responsivity in Continuous Wave Regime”, IEEE Transactions on Electron Devices, vol. 60, n. 11, pp. 3718-3725, 2013 G. Adamo, D. Agrò, S. Stivala, A. Parisi, L. Curcio, A. Ando’, A. Tomasino, C. Giaconia, A.C. Busacca, M. C. Mazzillo, D. Sanfilippo and G. Fallica, “Responsivity measurements of 4H-SiC Schottky photodiodes for UV light monitoring”, Photonics West 2014 - Silicon Photonics IX, 2014 , paper no. 8990-41 R. Pernice, G. Adamo, S. Stivala, A. Parisi, A.C. Busacca, D. Spigolon, M.A. Sabatino, L. D’Acquisto and C. Dispenza, “Opals infiltrated with a stimuli-responsive hydrogel for ethanol vapor sensing”, Optical Materials Express, vol. 3, n. 11, pp. 1820-1833, 2013 G. Adamo, D. Agrò, S. Stivala, A. Parisi, G. C. Giaconia, A. C. Busacca, M. Mazzillo, D. Sanfilippo and G. Fallica, “Responsivity measurements of N-on-P and P-on-N silicon photomultipliers in the continuous wave regime”, Proc. SPIE 8629, Photonics West 2013, Silicon Photonics VIII, 86291, pp. 86291A-1–86291A-9, 2013 D. Sanfilippo, G. Valvo, M. Mazzillo, A. Piana, B. Carbone, L. Renna, P. G. Fallica, D. Agrò, G. Morsellino, M. Pinto, R. Canicattì, N. Galioto, G. Adamo, S. Stivala, A. Parisi, L. Curcio, C. Giaconia, A. C. Busacca, R. Pagano, S. Libertino and S. Lombardo, “Design and development of a fNIRS system prototype based on SiPM detectors”, Photonics West 2014 - Silicon Photonics IX, 2014, paper no. 8990-40 H. Xue, M. Bestonzo, U. R. Acharya and F. Molinari, “Design and Implementation of a Continuous Wave Near Infrared Spectroscopy System for Bedside and Home Monitoring”, Journal of Medical Imaging and Health Informatics, vol. 1, pp. 317–324, 2011 A. Bozkurt, A. Rosen, H. Rosen and B. Onaral, “A portable near infrared spectroscopy system for bedside monitoring of newborn brain”, BioMedical Engineering OnLine, vol. 4, pp. 1-11, 2005 R. Zimmermann, F. Braun, T. Achtnich, O. Lambercy, R. Gassert and M. Wolf, “Silicon photomultipliers for improved detection of low light levels in miniature near-infrared spectroscopy instruments”, Biomedical Optics Express, vol. 4, pp. 659-666, 2013 S. Vikrant, “Near Infrared Spectroscopy: a study of celebral hemodynamics during breathholding and development of a system for hotflash measurement”, M.S.Thesis, University of Arlington Texas, 2005

A versatile, nanosecond electric pulse generation system for in vitro bioelectric research studies Stefania Romeo*, Olga Zeni*, Anna Sannino*, Luigi Zeni*,#, Maria Rosaria Scarfì * *

Institute for Electromagnetic Sensing of the Environment (IREA), National Research Council (CNR), via Diocelziano 328, 80124, Napoli, Italy # Dept. of Industrial and Information Engineering, Second University of Naples, via Roma 29, 81031 Aversa, Italy

Abstract— Two prototypes of a double switch, Blumlein pulse generator were designed and realized to expose cell suspensions to ns pulsed electric fields by means of either microscope-slide based electrodes or electroporation cuvettes. Both systems provide pulses with variable duration, amplitude, polarity and repetition rate, and are suitable for either real time or post-pulse biological investigations.

I. INTRODUCTION The electroporation (EP) phenomenon lies in a nonlethal alteration of the plasma membrane permeability induced by the application of intense pulsed electric fields (PEF), and allows the entry of genetic material and pharmaceutical agents into living cells. Intense electric pulses with durations in the millisecond to microsecond time scale are currently used in both medical and industrial applications [1, 2], while nanosecond electric pulses have been more recently demonstrated to interact with both the plasma and the intracellular membranes, inducing morphological (membrane permeabilization) and functional (enhanced calcium release and gene expression, apoptosis) effects [3], and showing promising applications for cancer treatment [4]. In all cases, the characteristics of applied electric pulses (amplitude, duration, repetition rate) are the parameters that mainly affect the EP induction and the dynamics of the phenomenon. As a consequence, versatile pulse generation systems, providing well defined pulses with variable characteristics, are strongly required in the framework of the studies on biological effects of pulsed electric fields. Moreover, such pulse generators have to be compatible, from the physical and electrical point of view, with the different sample holders chosen on the base of the biological technique to be employed (electroporation cuvettes, microscope slide-based applicators, needle arrays of electrodes or tweezers). In this work, the design and realization of a nanosecond pulse generator, based on the double-switch Blumlein topology, will be reported, which provides pulses with variable amplitude, duration, polarity and repetition rate. Two prototypes have been realized for exposing cell suspensions by means of either microscope-slide based electrodes or electroporation cuvettes, in order to perform different biological assays either in real time or delayed with respect to pulse exposure.

II. MATERIALS AND METHODS The Blumlein pulse forming network (PFN) is a well established circuit topology in pulsed power engineering, and is currently one of the main architectures adopted to generate nanosecond, high voltage electric pulses for electroporation

[5]. A Blumlein PFN is usually configured with a high voltage source charging paired transmission lines, series connected to the load, which is required to have an impedance as large as twice the characteristic impedance of each transmission line. The generator operates in two phases, charge and discharge, driven by the activation of a single switch, and provides rectangular pulses with fixed length. In our previous works, it was demonstrated that pulses with variable duration and polarity can be generated by adopting a double-switch Blumlein configuration. The pulse duration depends on the time delay between the switches activation, while the polarity is determined by the activation order [6]. Both the prototypes of double-switch Blumlein PFN here presented were designed and realized according to the scheme depicted in Figure 1.

Fig. 1 Diagram of the double-switch Blumlein prototypes The system is composed by a command station, a fiber-optic link and the Blumlein PFN. The command station comprises a PC and a couple of low voltage, digital pulse generators (USBpulse100, Elan Digital System, Fareham, UK) which provide the control signals for the switches activation. These signals are propagated to the switches by means of a fiberoptic link, consisting of two transmitters, two receivers, and a fiber-optic patch-cord. This solution has been set up to separate the control side of the system from the high voltage side, thus avoiding interferences that could be conducted through common grounding connections and cause undesired commutation of the switches. Different solutions for the high voltage switches and for the transmission lines were implemented in the two prototypes, in order to optimize the pulse generation system for exposing

cell suspensions in either microscope slide-based electrodes or electroporation cuvettes, as reported in the results section. III. RESULTS Microscope slide-based pulse applicators were realized by mounting a couple of stainless-steel electrodes on a glass microscope slide, as shown in Figure 2. By adopting 1 cm long, 100 μm thick electrodes, with 100 μm gap among each other, and by considering that resistivity of the medium in which cells are suspended is 100 Ωcm, a load impedance of about 100 Ω was obtained. Therefore, since it is required the load impedance to be twice that of each transmission lines, 50 Ω coaxial cables were used in this first prototype. Moreover, due to the very thin gap among the electrodes, relatively low voltages are required to gain very intense electric fields in the gap, and therefore, two fast RF power MOSFET switches (DE275-102N06A; Ixys, Fort Collins, CO) were employed, that can hold a maximum voltage of 1 kV. The final prototype is able to provide pulses with variable duration (30 to 200 ns), polarity, and electric fields as large as 10 MV/m can be generated for cells exposure under confocal microscopy [7].

values are required to generate sufficiently intense PEFs, and hence, two solid state switches of the HTS-UF (ultra fast) series by Behlke company (Germany) were employed, that can hold a maximum voltage of 8 kV. The final prototype provides pulses with durations between 10 and 60 ns, and with variable polarity and repetition rate [8].

Fig. 3 Microstrip transmission line realized for the doubleswitch Blumlein prototype Overall, the double-switch Blumlein prototypes here presented can be adopted in the framework of in vitro studies on the evaluation of biological effects of nsPEFs, with pulse characteristics variable over wide ranges, and involving both qualitative biological assays in real time with the exposure (e.g. confocal microscopy analysis) and post-pulse quantitative investigations (e.g. , flow-cytometry). ACKNOWLEDGMENTS The work was partially supported by the Regione Campania (L.R. N. 5, 28/3/2002). REFERENCES [1]

Fig. 2 Microscope slide-based electrodes for nsPEF applications under confocal microscopy [2]

The second Blumlein PFN prototype was realized for exposing cell suspensions in electroporation cuvettes which are commercially available with different gaps among the electrodes (1, 2, 4 mm) and allow the exposure of relatively large volume samples, suitable for post-pulse investigations. According to the type of cuvette adopted, and to the electromagnetic characteristics of the pulsing buffer, the PFN has to be matched to different load impedances. As a consequence, microstrip transmission lines were chosen in this case as transmission line, since they can be easily designed in such a way to exhibit a particular characteristic impedance, and can be realized with a low cost and fast procedure (Figure 3). Three microstrip-lines have been realized, to be matched to 1) a low impedance load (10 Ω) made by a biological medium (σ = 1 S/m) placed inside a 0.1 or 0.2 cm electroporation cuvette; 2) an intermediate impedance load (50 Ω); 3) a high impedance load (133 Ω) made by a low conductivity medium (σ = 0.15 S/m) placed inside a 0.4 cm electroporation cuvette. The strips were realized in meander-shape in order to gain more compact structures, that are interchangeable within the system. Due to the larger electrode gap, larger high voltage

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M. Marty, G. Sersa, J. R. Garbay, J. Gehl, C. G. Collins, M. Snoj, V. Billard, P. F. Geertsen, J. O. Larkin, D. Miklavcic, I. Pavlovic, S. M. Paulin-Kosir, M. Cemazar, N. Morsli, Z. Rudolf, C. Robert, G. C. O'Sullivan and L. M. Mir, “Electrochemotherapy - An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study,” Europ. J. Cancer Suppl., Vol. 4, pp 313, 2006. H. Akiyama, T. Sakugawa, T. Namihira, K. Takaki, Y. Minamitani, and N. Shimomura, “Industrial applications of pulsed power technology,” IEEE Trans. Dielectr. Elect. Insul., Vol. 14, No. 5, pp. 1051-1064, 2007. R. P. Joshi and K. H. Schoenbach, “Bioelectric effects of intense ultrashort pulses,” Critical Reviews in Biomedical Eng., Vol. 38, pp 255-304, 2010. M. Breton and L. M. Mir, “Microsecond and nanosecond electric pulses in cancer treatment,” Bioelectromagnetics, Vol. 33, No. 2, pp. 106-123, 2012. J. F: Kolb, S. Kono and K. H. Schoenbach, “Nanosecond pulsed electric field generators for the study of subcellular effects,” Bioelectromagnetics, Vol. 27, pp. 172-187, 2006. A. de Angelis, J. F. Kolb, L. Zeni, and K. H. Schoenbach, “Kilovolt Blumein pulse generator with variable pulse duration and polarity,” Rev. Sci. Instr., Vol. 79, No. 4, pp. 1-4, 2008. S. Romeo, M. Sarti, M. R. Scarfì, and L. Zeni, “Modified Blumlein pulse forming networks for bioelectrical applications,” J. Memb. Biol., Vol. 236, No. 1, pp. 55-60, 2010 S. Romeo, C. D’Avino, O. Zeni, and L. Zeni, “A Blumlein-type, nanosecond pulse generator with interchangeable transmission lines for bioelectrical applications,” IEEE Trans. Dielectr. Elect. Insul., Vol. 20, No. 4, pp. 1224-1230, 2013.

Evaluation of the occupational risk for workers exposed to the magnetic field of an induction heater Luigi Cerra* #

SOLVECONSULTING, Strada Ex Alifana - 81030 San Marcellino (CE), [email protected]

Abstract — This study aimed to provide minimum health and safety requirements regarding the exposure of workers to the risks arising from electromagnetic fields of an induction heater. The measurement results were compared with the action values of European 2004/40/EC (repealed) and 2013/35/UE Directives. For completion of this study have provided some information to limit worker exposure.

I. INTRODUCTION The goal of this study is to be aware of the EMF sources in order to limit the emissions and to select the best counter measures to be adopted in case their EMF emissions exceed acceptable levels. The more the biological effects of electromagnetic fields on human body are investigated, the more the subject is becoming important, since the metal industries have to take care of workers’ safety. As part of the metal industries is very frequent use of induction heating for materials processing. The heat necessary to work the metal and related products is developed to effect the flow of eddy currents that are made on the circular metal piece to be machined. In turn, the eddy currents are generated by the action of a magnetic field by means of the circulation of electric currents of variable frequency within a coil, commonly known as the induction coil. The following figure illustrates the diagram of operation

definition of an index that we define, heare, as ICs calculated taking into account of the corresponding indices of the cartesian components ICsx, ICsy, ICsz. The index ICs must be less than 1 to consider the effects of simultaneous exposure and action values at various frequencies. For the calculation of index ICs are used the following formulas

where: Bx (f ), By (f ), Bz (f ), are respectively the Cartesian rms i rms i rms i components of the magnetic field measured as root mean square (rms) at the i-th frequency. BL (fi) is the corresponding action value (rms) defined by European Directives at the i-th frequency. Formulas 1 and 2 are valid also for the electric field . Spot measurements of the electric field showed that the emissions are much lower than the action values, and therefore, in this study has been paid attention only to the emission of the magnetic field. The restriction in equation (2) (standard method or spectral method) is based on the assumption that the spectral components are in phase. This approach is reasonable when the number of spectral components is limited and their phases vary randomly. Therefore it was decided to analyze exposures by using the spectral method (standard method). For the measurements was used in the Narda EHP50C analyzer field, the datalogger PMM8053B NardaProbe and software for remote control from a PC, via a fiber optic cable. The probe detects signals of sinusoidal electric and magnetic in the range of frequencies (5 Hz, 100 Khz).

Fig. 1 induction heating

In reference to the induction heater object of this study, the waveform of the magnetic field generated is a the sum of three sinusoidal dominant signals, the first at 50Hz, the second at 300Hz and the third at 2.2 kHz frequency. To support the fusion of the metal alloy in a short time, the plant develops a power of about 50 kW. II. MATERIALS AND METHOD In the case of complex signals, when the number of spectral components of the signal is limited the statement of the 2003 ICNIRP proposes to apply for these waveforms weighted sum approach already codified in the guidelines of 1998 and reconfirmed in the 2010 guidelines and alternatively to the method of the weighted peak. This leads to the

III. RESULTS As shown in the figure below for the the measurements were selected workstations typical of the operator A1, A2, A3 and A4:

Fig. 2 plant layout.

The tables below show the results of measurements at the points A1, A2, A3, A4 and related processing for the

calculation of ICs, respectively, in the cases that you apply the action values of 2004/40/CE Directive and LA action levels of new European 2013/35/UE Directive. TABELLA I A1 (melting metal room)

f Bx (Hz) (µT) 50 1,1 300 1,8 2225 5,3 ICs (40/CE) ICs (35/UE)

By (µT) 2,6 3,5 1,2

Bz (µT) 0,8 1,1 1,5

B LA (µT) (40/CE) 2,9 500 4,1 83 5,6 30,7 0,22 0,05

LA inf (35/UE) 1000 1000 135

Fig. 3 A4 point

TABELLA III A2 (ingot metal room- front area)


By (µT) 3,1 10,7 2,2

Bz (µT) 1,1 2,4 3,1

B LA (µT) (40/CE) 3,7 500 11,4 83 20,8 30,7 0,75 0,16

LA inf (35/UE) 1000 1000 135

TABELLA IIIII A2 (ingot metal room- rear area)


By (µT) 5,3 13,2 3,1

Bz (µT) 2,2 3,2 5,5

B LA (µT) (40/CE) 6,4 500 14,2 83 29,4 30,7 1,05 0,23

LA inf (35/UE) 1000 1000 135

TABELLA IVV A3 (75cm from the current generator)


By (µT) 7,3 18,7 20,2

Bz (µT) 2,9 4,5 6,3

B LA (µT) (40/CE) 8,7 500 20,2 83 89,9 30,7 3,07 0,68

TABELLA V A3 (50cm from the current generator)

f B (Hz) (µT) 50 13,8 300 33,4 2225 144 ICs (40/CE) ICs (35/UE)

LA (40/CE) 500 83 30,7

LA inf (35/UE) 1000 1000 135 4,6 >1

TABELLA VI A4 (25cm from the power cables)

f B (Hz) (µT) 50 43,5 300 94,5 2225 1149 ICs (40/CE) ICs (35/UE)

LA (40/CE) 500 83 30,7

LA inf (35/UE) 1000 1000 135 >>1 >1

LA inf (35/UE) 1000 1000 135

IV. CONCLUSION The study showed that the condition ICNIRP for limiting the electrical stimulation relevant to 10Mhz effect of simultaneous exposure to multiple frequency is not observed for the workstations near to the current generator and the power cables of the heater. Also during the measurements in position A3, were recorded acute effects such as nausea and dizziness for the induced currents in the body by the strong magnetic field. The employer shall take the following minimum health and safety requirements: • Denial of access to workers electromagnetic risk-sensitive (workers with implanted pacemakers, implanted insulin pumps) • Health surveillance of workers occupationally exposed in agreement with the competent doctor • Reducing exposure through the maintenance on the induction heater • Administrative controls to limit access • It is recommended the use of visual signals and zoning area with the following information “strong magnetic field – area 2 rif. Standard EN-50499” BIBLIOGRAFIA [1] ICNIRP, Guidelines for limiting exposure to time-varying electric and magnetic fields (1 hz to 100 khz). Published in health physics 99(6):818-‐836; 2010

[2] ICNIRP, Guidance on determining compliance of exposure to pulsed and complex non-sinusoidal waveforms below 100 khz with icnirp guidelines- Copyright © 2003 Health Physics Society

[3] ICNIRP, Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz), Health Physics, Vol. 74, n. 4, pp. 494-522 (1998)

[4] Andreuccetti D., S. Priori, N. Zoppetti, population exposure to ELF sources with complex waveform

[5] Directive 2013/35/EU - electromagnetic fields of 26 June 2013 on the minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents (electromagnetic fields) (20th individual Directive within the meaning of Article 16(1) of Directive 89/391/EEC) and repealing Directive 2004/40/EC

[6] Directive 2004/40/EC of the European Parliament and of the Council of 29 April 2004 on the minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents (electromagnetic fields)

[7] CENELEC EN 50499:2008-12: "Procedure for the assessment of workers' exposure to electromagnetic fields."

Electromagnetic Radiation by the MUOS Satellite Earth Station in Sicily (Italy): Health Risk Assessment Alessandro Polichetti, Roberta Pozzi Technology and Health Department, National Institute of Health, Viale Regina Elena, 299, 00161 Rome (Italy), [email protected], [email protected] Abstract— Due to complaints by nearby residents, the installation of a U.S. military satellite earth station in the Italian territory was suspended awaiting a health risk assessment by the Italian National Institute of Health. In this relation, methodology and results of the study, as concerns electromagnetic field exposures, are shown and discussed.

I. INTRODUCTION The Mobile User Objective System (MUOS) is a military satellite communication system, composed of five geostationary satellites and four earth stations, which will provide simultaneous voice, video and data communication for U.S. troops in movement and which will replace the current U.S. military narrowband tactical communications system, known as the Ultra High Frequency Follow-On (UFO) system. The MUOS earth stations are composed of three 18.4 diameter circular parabolic Ka-band dish antennas (MUOS antennas) transmitting at 30-31 GHz, and two helical UHF antennas which transmit at 240-315 MHz. Two of these earth stations are located in the territory of the U.S.A. (in Virginia and in Hawaii), one in Australia, and one in Italy, in the Naval Radio Transmitter Facility (NRTF) of the U.S. Naval Air Station Sigonella, at few kilometres from the Sicilian town of Niscemi. Concerns about the possibility of health risks due to electromagnetic radiation emitted by the MUOS antennas have spread among the population of Niscemi during the construction of the earth station which was suspended due to the protests, pending a health risk assessment by the Italian National Institute of Health (ISS), involved in the issue by the Italian Ministry of Health. A multidisciplinary Working Group of the ISS has evaluated the possible health impact of the electromagnetic emissions by the new MUOS installation, which will add to those by the already active antennas of the NRTF transmission facility, along with the environmental impact of the emissions in air by a petrochemical plant in the near town of Gela; the health status of people residing in Niscemi was also analyzed. Methodology and results, relevant to electromagnetic fields, of the study carried out by the Working Group of the ISS will be here described and discussed. II. METHODS A. Risk Assessment Criteria In order to assess the health risks for people that will be exposed to electromagnetic fields by the antennas of the MUOS earth station located in the NRTF facility of Niscemi, it has been necessary to separately consider three kinds of health effects: short-term effects; indirect effects due to electromagnetic interferences with medical devices; long-term effects.

For the first two types of effects, clearly established by scientific research, quantitative risk assessments have been performed. The possibility of health risks linked to thermal short-term effects has been assessed by comparing the evaluated exposure levels with ICNIRP reference levels [1]. As concerns indirect effects on medical devices, the electromagnetic immunity requirements for medical electrical equipment and for active implantable medical devices like cardiac pacemakers [2,3] have been taken into account for the sake of comparison with exposure levels. As regards long-term effects of electromagnetic fields, they are not scientifically established, and exposure-response relations are not available, implying that a quantitative evaluation of the health risk corresponding to a specified exposure level is not possible: just qualitative considerations are possible based on actual scientific knowledge. However, considering that Italian regulation [4], in addition to exposure limits for protection against short-term effects (numerically different from the recommendations by ICNIRP [1]), foresees a precautionary action value of 0.1 W/m2 for homes and, more generally, locations used for prolonged stays, the conformity to the national law has also been taken in consideration. B. Exposure Evaluation Radio electrical and geometrical parameters of the UHF antennas used in this analysis are: transmission frequency range = 240 - 315 MHz; power P = 200 W; gain G = 16 dBi; lenght L = 4 m. Relevant positions for general population are always in the far-field region of these antennas (2L2/λ = 33.6 m at 315 MHz) where power density S can be calculated as

S=

G(θ, φ) × P 4πr 2

In the case of MUOS dish antennas, the fundamental parameters are: transmission frequency = 30 - 31 GHz; power P = 1600 W (200 W declared by U.S. Navy, but some contradictions in various reports led us to consider the highest value reported); gain G = 71.4 dBi; diameter d = 18.4 m. In this case the radiative near field (Fresnel) region extends up to a distance of several kilometres (0.6d2/λ = 21 km at 31 GHz). Far field evaluations are indeed useless, since antennas point upwards at a minimum elevation of 14.7 degrees. It was not possible, due to limitations in time and resources in this official duty assigned to ISS by the Ministry of Health, to perform numerical simulations for the near field, which are particularly needed for off-axis evaluations. However, a rough estimation is possible on the basis of information from technical literature [5-7], which can be sufficient for a health risk assessment if the evaluated field levels are very lower than exposure limits.

III. RESULTS AND DISCUSSION As regards UHF antennas, far field calculations allow to exclude established short-term effects, interferences on medical devices, and violations of the Italian precautionary attention values, already at a 230 m distance (data not shown). Results for MUOS dish antennas are shown in Table I. TABLE I ELECTROMAGNETIC FIELD LEVELS EMITTED BY MUOS DISH ANTENNAS Position In front of the antenna’s aperture Maximum value on-axis in Fresnel region

Power density 24 W/m2 9.3 W/m2

Maximum off-axis value in Fresnel region at a 18.4 m lateral distance from the beam

0.093 W/m2

Maximum off-axis value in Fresnel region at a 36.8 m lateral distance from the beam

10-3 W/m2

Notes Compliance to occupational ICNIRP limits (50 W/m2). Compliance to general population ICNIRP limits (10 W/m2). Compliance to the Italian precautionary attention value (0.1 W/m2).

As a consequence of the large size of the MUOS parabolic antennas, the near field region of the radiated electromagnetic fields extends for tens of kilometres, making difficult an accurate theoretical evaluation of the electromagnetic field levels in a very large area comprising not only the town of Niscemi but also other towns. However, most part of the radiated electromagnetic energy in the near-field area is confined inside cylinders coaxial to the parabolic reflectors, with a base of 74 m diameter and a height of about 20 km, outside of which the electromagnetic fields levels are at most two orders of magnitude lower than the precautionary action values set by Italian regulations. In the far field, where radiation beams begin to diverge, distances from antennas are so high that possible exposures of population at secondary lobes of radiation are negligible. Compliance to ICNIRP recommendations imply that risks to human health related to known short-term effects of electromagnetic fields are not foreseeable. Even in the case of accidental exposures of people to the main beam, involving at most exposures higher than exposure limits recommended for general population by ICNIRP, but lower than those recommended for workers, no damages for human beings are foreseeable, apart from possible exposures of cardiac pacemaker wearers. Generally, cardiac pacemakers have to be immune to electromagnetic fields below the general population ICNIRP limits [3], and electrical medical equipments have to be immune at least up to 3 V/m electric fields [2] (0.024 W/m2 in terms of power density); however, the electromagnetic interference (EMI) tests are foreseen just up to 3 GHz, below the frequencies of MUOS dish antennas. This upper frequency limit for EMI tests reflects various considerations, e.g. about the increased device protection afforded by the attenuation of the enclosure and body tissues at microwave frequencies, or about the reduced sensitivity of circuits at microwave frequencies: while it is reasonable that the low intensity 30 GHz microwaves outside the main beams of MUOS dish antennas do not interfere with pacemakers and electrical medical equipments, a particular caution would be

advisable in the case of workers wearers of pacemakers, if any, possibly exposed in front of the dish antennas’ apertures. In 2011, the International Agency for Research on Cancer (IARC) has evaluated the evidences of carcinogenicity of RF electromagnetic fields, which were classified as “possibly carcinogenic in humans” (Group 2B) [8]. This classification, essentially due to the results of epidemiological studies on cell phone users and head tumours, confirms the absence of a coherent evidence of carcinogenicity. IARC took into account also studies about environmental sources, but epidemiological evidence was considered insufficient for any conclusion. Current scientific knowledge, along with the very low estimated exposures, does not suggest risks related to longterm exposure of population to electromagnetic fields radiated by MUOS antennas. IV. CONCLUSIONS The large size of the MUOS dish antennas, which on one side is an important determinant of the very low expected exposures to electromagnetic radiation, on the other side may play an important role in the high perception of risk in the population resident in the area. A correct information about the possible health risks associated with exposure to electromagnetic fields, and in this particular case also, and above all, on the real field levels to which population will be exposed, is essential to avoid unjustified concerns that may be detrimental to the psychological well-being of people, and therefore to health, defined by the World Health Organization as “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity”. REFERENCES [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

International Commission on Non-Ionizing Radiation Protection (ICNIRP), “Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz)”, Health Phys., vol. 74, pp. 494-522, Apr. 1998. European Committee for Electrotechnical Standardization (CENELEC), Medical electrical equipment - Part 1-2: General requirements for basic safety and essential performance - Collateral standard: Electromagnetic compatibility - Requirements and tests, EN 60601-1-2: 2007-08. European Committee for Electrotechnical Standardization (CENELEC), Active implantable medical devices - Part 2-1: Particular requirements for active implantable medical devices intended to treat bradyarrhythmia (cardiac pacemakers), EN 45502-21: 2003-12. Decree of the President of the Council of Ministers 8 July 2003, “Setting of exposure limits, attention values, and quality goals to protect the population against electric, magnetic, and electromagnetic fields generated at frequencies between 100 kHz and 300 GHz”, Gazzetta Ufficiale della Repubblica Italiana, n.199, 28 Aug. 2003. N. Hankin, The Radiofrequency Radiation Environment: Environmental Exposure Levels and RF Radiation Emitting Sources. U.S. Environmental Protection Agency, Washington, D.C. 20460. Report No. EPA 520/1-85-014, July 1986. Federal Communications Commission Office of Engineering & Technology, Evaluating Compliance with FCC Guidelines for Human Exposure to Radiofrequency Electromagnetic Fields, OET Bulletin 65, edition 97-01, Aug. 1997. R.L. Lewis, A.C. Newell, “Efficient and accurate method for calculating and representing power density in the near zone of microwave antennas”, IEEE Trans. Antennas Propagat., vol. 36, pp. 890-901, June 1988. International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 102, Nonionizing radiation, Part II: Radiofrequency Electromagnetic Fields. Lyon, France: IARC, 2013.

Health Effects from Cell Phone Radiations. The Verdict of the Supreme Court of Cassation in Italy Bruno Bisceglia*, Vitulia Ivone+, Pietro Persico° *

Dept of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, Salerno, Italy, [email protected] + Dip. di Scienze Giuridiche - Scuola di Giurisprudenza, University of Salerno Via Giovanni Paolo II, 132, 84084 Fisciano, Salerno, Italy, [email protected] °

Pietro Persico, Magistrato Tribunale di Roma, Sezione Distaccata di Ostia, [email protected]

Abstract — The Court of Appeal of Brescia recognized the occupational origin of a trigeminal schwannoma in a user of mobile telephones: a case-study in the framework of the use (and misuse) of scientific evidences in toxic-tort litigation. The Supreme Court of Cassation of Italy has recently determined that the tribunal correctly followed the necessary procedures required under Italian law.

I. INTRODUCTION The exposure of a user to cell phone radiations depends on the technology of the phone, the distance between the phone’s antenna and the user, the extent and type of use, and the user’s distance from cell phone towers. Studies thus far have not shown a consistent link between cell phone use and cancers of the brain, nerves, or other tissues of the head or neck. More research is needed because cell phone technology and how people use cell phones have been changing rapidly. The effect of mobile phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in mobile phone usage throughout the world (as of June 2009, there were more than 4.3 billion subscriptions worldwide) [1,2]. The WHO (World Health Organization) has classified mobile phone radiation on the IARC (International Agency for Research on Cancer) scale into Group 2B - possibly carcinogenic to humans on May 31, 2011. «Could be some risk» of carcinogenicity, so additional research into the longterm, heavy use of mobile phones needs to be conducted [3,4]. II. THE VERDICTS. SUPREME COURT OF CASSATION, 2012. COURT OF APPEAL OF BRESCIA, 2009 The Supreme Court of Cassation of Italy has affirmed a ruling granting worker’s compensation to a businessman who developed a tumor after using a cell phone for 12 years. This is the first time that a high court —in any country— has ruled in favor a link between mobile phone radiation and tumor development. The Court of Cassation determined that the 2009 tribunal correctly followed the necessary procedures required under Italian law. [5, 6] On October 12, 2012, Italy’s highest civil court, the Supreme Court of Cassation, upheld a lower court ruling that an Italian man’s cranial nerve tumor was causally linked to his cell phone use. The plaintiff is 60-year-old Innocente Marcolini, who claimed that he developed a brain tumor near his left ear as a

consequence of speaking on his cellular and cordless telephones in connection with his work for up to six hours a day for twelve years. When the tumor was first detected, the plaintiff filed a claim for workers’ compensation from the Italian workers’ compensation authority (Istituto Nazionale Infortuni sul Lavoro, INAIL), which was denied on the grounds that there was no evidence that his tumor had been caused at work and that there is not enough evidence linking cell phone use to brain tumors. INAIL relied on conclusions from the Interphone study, based on research conducted by the World Health Organization’s IARC. The organization offers insight about the safety of mobile phone use, but states that “to date, no adverse health effects have been established as being caused by mobile phone use.” The plaintiff then filed a civil suit against INAIL’s decision in the Court of Appeal of Brescia, which found that there was a causal link between the use of cellular and cordless telephones and tumors. A further appeal from INAIL brought the case before the Supreme Court of Cassation. In its decision, the Labor Law section of the court held that the lower court’s decision was justified and that scientific evidence advanced in support of the claim was reliable. A. History Mr. Marcolini believed that his constant use of the telephone had caused him to have a serious health condition. As such, on the 17th of November 2003, he asked INAIL for compensation due to his health condition but was denied stating that there was no causal link between Mr. Marcolini’s occupation and his health problems as reported. INAIL opposed the application, always in terms of the lack of causation and the lack of oral evidence supporting his claim. INAIL produced various documentation to support their refusal. Having fulfilled the preliminary testimony, the sitting judge, after confirming the claimant’s intense use of cellular and cordless phones and involving technical experts for the Court, rejected the application for lack of causation, in accordance to the comments submitted by the Technical Consultant (CTU). During Mr. Marcolini’s appeal, the INAIL reminded the court that there was no reliable scientific studies regarding the harmfulness of electromagnetic waves. In 2004, after the diagnosis of an adrenal lesion, he underwent further surgery at the European Institute of Oncology. The histological diagnosis confirmed that he had a Pheochromocytoma, a rare tumor with possible secretion of catecholamines.

In June 2002 Mr. Marcolini began to be affected by a benign tumor which had affected his cranial nerves, in particular the acoustic nerve, and on November 8th 2002 Mr. Marcolini underwent brain surgery for the removal of ganglion of Gasser. A postoperative MRI [7] showed that there was still a residual tumor. In 2004, after the diagnosis of an adrenal lesion, he underwent further surgery at the European Institute of Oncology. The histological diagnosis confirmed that he had a Pheochromocytoma, a rare tumor with possible secretion of catecholamines. Mr. Marcolini applied to the Court of Brescia on the 7th of June 2007 citing his serious and complex brain disorder. In 2009 the Court of Appeal of Brescia, as an Occupational tribunal, issued a ruling recognizing the occupational nature of his disease and approved the awarding of 80% disability to Mr. Marcolini. Towards the end of 2009, the Court of Appeal of Brescia (as the authority invested to judge Occupational Health cases) awarded compensation to Mr. Innocent Marcolini, a company manager, in a decision against the INAIL judging that Mr. Marcolini’s disability of 80% was caused by his workplace and that the resulting onset of a neuroma of the ganglion of Gasser could be attributed to his professional environment, in particular, the constant exposure to electromagnetic waves when using the office cordless and mobile telephones. From 1981 until 1993 Mr. Marcolini had worked as a business manager, and in that capacity he claimed to have used the company’s land-line, cordless and mobile telephones for an average of 5-6 hours a day for the period of 12 years. Being right handed, he regularly held the mobile to his left ear while using his right hand either to answer the desk phone or to take notes.

C. Dosimetric Remarks The court appointed expert witness takes in account the use of mobile and cordless from Mr Marcolini. He has used such devices (800-1900 MHz) 5-6 hours in a day (Table II). «Nel periodo in cui ha lavorato c/o San Giacomo SPA per i primi 3 anni utilizzava telefono mobile – cordless (deposte 5-6 h al dì), dal 1993-4 al cordless fu associato l’uso di telefono cellulare fino al settembre 2003. I telefoni mobili (cordless) e i telefoni cellulari funzionano attraverso le onde elettromagnetiche» [7]. TABLE II COURT APPOINTED EXPERT WITNESS

Cordless (varie generazioni) Tel. cellulari (varie generazioni)

«Il quesito proposto può essere così svolto: l’esposizione a Radiofrequenze, anamnesticamente per un tempo efficace (>10 anni), ha molto verosimilmente avuto un ruolo concausale nell’evoluzione della neplasia patita dal sig. Marcolini. La menomazione dell’integrità fisica legata alla malattia ed ai suoi esiti si stima in misura dell’80%.»

The court appointed expert witness takes in account the use of mobile and cordless from Mr Marcolini. He has used such devices (800-1900 MHz) 5-6 hours in a day (Table II). III. CONCLUSION Is there some evidence from studies of cells, animals, or humans that radiofrequency energy can cause cancer? A limited number of studies have shown some evidence of statistical association of cell phone use and brain tumor risks, but most studies have found no association [8]. The verdict of the Court of Appeal of Brescia and the Verdict of the Supreme Court of Cassation are paradigmatic in the controversial debate about health effects of exposure of humans to cell phone radiations [9]. The judgment does not represent a scientific truth, but a judicial truth.

B. Symptoms According to the documents produced in court, in June 2002 Mr. Marcolini began to be affected by a benign tumor which had affected his cranial nerves, in particular the acoustic nerve, and on November 8th 2002 Mr. Marcolini underwent brain surgery for the removal of ganglion of Gasser. A postoperative MRI [7] showed that there was still a residual tumor. [1] The symptoms noted after the operation are collected in table I: TABLE I

[2]

SYMPTOMS AFTER SURGERY

Ulcer of the left cornea Syndrome of algo-dystrophic to the left hemiface with severe chronic pain persistent paraesthesia of the hemiface disturbance to the masticatory mechanism double vision partial epilepsy memory loss reduced attention span reduced adaptability temporal lobe syndrome with disturbance to smelling, hearing, taste, balance, vision, and psychiatric

Banda di Frequenza 800- 1900 MHz 800- 1900 MHz

[3] [4] [5] [6] [7] [8] [9]

REFERENCES Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR): Health Effects of Exposure to EMF. Opinion adopted at the 28th plenary on 19 January 2009. European Commission, Directorate General Health Consumers. S. Lagorio et al., “An Italian Court recognizes the occupational origin of a trigeminal neuroma in a mobile telephone user: a case-study of the complex relationships between science and law”, Med. Lav., 102(2):144-62, Mar-Apr 2011. “Electromagnetic fields and public health: mobile phones”, World Health Organization, Fact sheet N.193, June 2011. European Parlament Document, http://www.europarl.europa.eu/document/ Sentenza di Merito, Corte di Appello di Brescia, 22.12.2009, n. 614. Corte di Cassazione, sez. Lavoro, sentenza 3, 12.10.2012, n. 17438. Court appointed expert witness, CTU 2° grado. B. Bisceglia and V. Ivone, “Health effects from cell phone radiations: the verdict of the court of appeal of Brescia”, XIX Riunione Nazionale di Elettromagnetismo, Rome, 10-14 September 2012. B. Bisceglia, “Health effects from cell phone radiations. The verdict of the Court of Cassation of Italy”, BioEm 2013, Joint Meeting of the Bioelectromagnetics Society and the European BioElectromagnetics Association, Thessaloniki, Greece, June 10 - 14, 2013

What is the state of present Bioelectromagnetic Research? Lesson from the School of Bioelectromagnetism “A. Chiabrera” and from the activity in the Editorial Board of Bioelectromagnetics. F.Bersani Department of Physics and Astronomy, University of Bologna, Viale Berti Pichat 6/2, 40127, Bologna (Italy) [email protected]

Abstract - Starting from his long lasting experience of research in Bioelectromagnetics, his work as former Ass. Editor and currently member of the Editorial Board of Biolectromagnetics, and from the experience as Director of the School of Bioelectromagnetism “A. Chiabrera”, the author expresses his motivated opinion on the present situation of research on the biological effects of electromagnetic fields (EMF), underlining, on one side, the improvement of its quality in some areas during the last years, and, on the other side, stressing the weakness still present in this kind of research. Suggestions are given to the Italian researchers in the context of the ICEmB promoting activity.

I. PONDERING THE PRESENT SITUATION When I receive, as member of the Editorial Board of Bioelectromagnetics, a paper for review, I’m always in the hope to read something new, something that is potentially able to consistently improve the knowledge in this controversial area of research. Unfortunately, this is not the case for the most part of papers. After many years of this job together with my ‘first hand’ experience as Director of the School of Bioelectromagnetism “A. Chiabrera” at the Majorana Centre in Erice, I recently tried to start thinking about the recent improvement in the Bioelectromagnetic (Bioem) research, and on the other hand about the still present weaknesses of this area, also taking into account the recent report of the SCENIHR [1]. In the last decade, a large number of papers were published in this field, part in a dedicated journal like Bioelectromagnetics, part in other journals, both biological and biophysical. One general observation is that such papers are more carefully done than before, regarding the exposure conditions, including adequate field measurement, good dosimetry, and quality of the exposure systems, all this being also due to more stringent filters on behalf of the reviewers. Of course this is especially true for Bioelectromagnetics, much less for other journals not directly specialized in this area, and thus more prone to accept sound biology, but often more compliant regarding the technical aspects concerning the exposure conditions. This defect is obviously linked to the peculiar aspect of this kind of research, exquisitely interdisciplinary, thus requiring a high standard level in two completely different areas like technical and physical on one side and biological on the other. A gold standard can be only achieved if a research group is strictly interdisciplinary. The result is that, especially in not specifically dedicated journals, you can find good biology not accompanied by adequate and well defined exposure conditions. This is even more evident in papers coming from research groups which are newcomers in this area. Of course

it often happens the vice-versa, namely acceptable exposure conditions (all including), but mediocre biology. But the most important weaknesses are perceived not looking at the single works, but considering the problem from a wider perspective. To appreciate this point we should first consider that Bioelectromagnetics (Bioem) includes many different subjects, some of them based on sound interaction mechanisms, even if not totally known in the details; an example are the so called thermal effects, another one are the effects due to short or ultra-short high intensity electric pulses. The other studies, which represent the large majority, mainly regard the so called non thermal effects due to weak EMF, both in the range of ELF and RF. Obviously, their major presence in the current publications is mainly due to their interest regarding the public concern about the so called (improperly) environmental EM pollution, and in minor cases, due to their potential for therapeutic applications. For these category of fields not clear “primary” mechanisms are known, and the research is driven either by health protection motivations or, in some instances, medical perspectives. In the first case the EM signals used are those taken from the telecommunication standards for RF and for the electric distribution for ELF, while in the second case there are a proliferation of signals, from AC to pulsed, with a variety of frequencies, intensities and waveform, whose specific choice has often “rarefied” scientific justifications. Very often the choice of the biological targets are many, but without a coherent rationale. All this results in a proliferation of papers with different signals, exposure conditions, biological models etc., making difficult a clear comparison and a coherent pattern for any definite conclusion. This is a sort of little‘tragedy’, that makes difficult to raise the research from a phenomenological level to a higher level of understanding. II. CRITICAL REMARKS AND SUGGESTIONS FOR THE FUTURE It’s here worth stressing that the “core” problem for the comprehension of the mechanisms for weak field is, on one side, to better understand the biochemical pathways involved in the effects, and eventually, to link these effects to the primary biophysical interaction. Without this in mind we risk to maintain all the research at a purely phenomenological level. This state of affair is paradoxically worsened by the motivations leading to the source of funds. In the last decades the major motivation was the potential health risk associated with human exposure to EMF used in telecommunications and electric power distribution (besides specific situation relative to the workplace exposures); this leads to a sort of purely “toxicological” and “epidemiological” approaches, not based on a pure scientific endeavour to understand what really

happens in the biological systems when exposed to EMF. I don’t’ intend to subclass the society-driven research, which is important beyond any doubt, but It’s sometimes wise to remember that the ultimate goal of research is “understanding”, not only “to verify”. I don’t’ intend to subclass the often “fishing” attitude of many researchers either. In fact, in a still virgin forest like that of Bioem, and the still poor understanding of the interaction mechanisms, this is acceptable: when the first explorers tried to investigate the Amazonian forest, they proceeded, opening the way using machete, partially by chance, partially by a sort of inspiration. This is OK, provided that we take immediately the opportunity when found: this means to try to systematize the “chance discovery”. Out of metaphor, I sometimes read apparently good biological results, in some way potentially significant and far reaching, then I don’t’ see any consistent follow up of the first results. Sometimes the same authors extend a little bit the research changing a few parameters, without going far enough. What do I mean for “going far enough”? I means to link the two ends of the string: from the biological outcome to the possible primary targets through a detailed intermediate analysis of the biochemical pathways. The vice-versa was sometimes prospected, namely from primary mechanisms to some expected biological outcome (“hypothesis-driven” research). Unfortunately few works were done in this direction, and not much convincing, often resulting in an initial emphasis and excitation, eventually dropping down, running away like a mirage. Of course, I well know that this is a high and difficult task, also in consideration of the limited funds at our disposal, but at least “trying” is currently a duty. A wise attitude would be to reach a fruitful blending of the two approaches taking the opportunity of many new methods in the biological research (including advanced biophysical techniques and theories, high throughput data analysis, System Biology, in ‘silico’ experiments etc., allowing top-down and down-top approaches). In this perspective, the School of Bioelctromagnetism during this decade has played a peculiar and unique role in trying to focalize the hot topics in our field, trying to stimulate the young researchers to put their work on a critical and informed base. My constant attention was devoted to show how to proceed in this still controversial, though very stimulating, research, trying to take the opportunity of having strictly interrelated interdisciplinary skills. This was facilitated by a constant presence in our courses of a young audience coming from different disciplines, that stimulated the search for a common language. This is not a very exciting period, concerning the source of funds, especially in the non-leading areas of Science; but, at this point I would like to ‘make a virtue out of necessity’, and to take the opportunity of this apparently critical situation, to make a sort of fruitful pause, and to “think” before “to act”, to restart with a new and awareness of our present state of affairs.In Italy we can take the opportunity to have a unique concentration of researchers in this field, with the advantage of being potentially under the umbrella of a common patronage like ICEmB. I don’t’ have any “magic wand” to solve the hot problems, but I would like to give a few practical suggestions: -Let take the advantage to have here represented many different competencies, ranging from General Biology to

Biophysics, Neurobiology, Physics and Engineering, with some groups experts in mechanisms, exposure systems and dosimetry, others in environmental Biology, others in Biostatistics and System Biology and so on, to start a real collaboration and coordination. Just to make some examples: -Each biological group must have a technical reference for the exposure systems, the exposure conditions and dosimetry; our research is, as mentioned before, very interdisciplinary, and no one can be alone! (this suggestion is especially addressed to the newcomers in the field). - If one biological group claims to have found a relevant and consistent result it must try, first, to check the degree of reproducibility involving other groups, and then to make an effort to link the two ends of the string, asking other biophysical groups, interested in mechanisms and/or having other complementary skills to enlarge and better understand the results obtained. -For those interested in mechanisms: try to take advantage of a relevant result, if any, found by in the biological experimentation, and try to model the possible interaction mechanisms underlying the biological outcome, proposing further experiments to prove the hypothesis. In other words, do not working alone, but taking a complete advantage of a possible network of researchers (potentially improvable by ICEmB), to make a joint effort to make a real quality jump in the future Italian research. -It’s fundamental, though obvious, working together, since It’s today the only way to survive, and, much more important, to raise the level of research, and who doesn’t’ understand this point, jealous of his tiny flowerbed, is on the way to die, killing our discipline. I will be very glad to read in the near future papers signed by many authors coming from different and complementary skills. -Moreover, a strict large scale collaboration is not only important for a high quality research, but results also in clear advantages in crude periods of restricted funds. -Let me make an astounding example of the advantage of large (in this case huge!) collaborations: never would the High Energy Physics have advanced without bringing together a very high number of different people with complementary skills in one single experiment. In our field, we don’t’ need such huge enterprise to discover something interesting, but we need this kind of spirit of collaboration and integration. In conclusion I strongly believe that, one of the major role of the School of Bioelectromagnetism may presently to represent an excellent opportunity for researchers (mostly at the beginning of their career) coming from all over the world, to direct the future research in the right way, while in Italy, Institutions like ICEmB may be of great relevance in the near future, to improve the collaboration between the Italian researchers, and organizing (as done in the recent past) very special meetings on concrete possibilities to work together on some hot subjects, emerging from our research. REFERENCES [1] SCENIHR preliminary opinion on “Potential health effects of exposure to electromagnetic fields (EMF)” available at:http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_ 041.pdf.