Millimeter-wave pulsed heating in vitro: cell mortality and heat shock response.

Millimeter wave (MMW)-induced heating represents a promising alternative for non-invasive hyperthermia of superficial skin cancer, such as melanoma. Pulsed MMW-induced heating of tumors allows for reaching high peak temperatures without overheating surrounding tissues. Herein, for the first time, we evaluate apoptotic and heat shock responses of melanoma cells exposed in vitro to continuous (CW) or pulsed-wave (PW) amplitude-modulated MMW at 58.4 GHz with the same average temperature rise. Using an ad hoc exposure system, we generated 90 min pulse train with 1.5 s pulse duration, period of 20 s, amplitude of 10 °C, and steady-state temperature at the level of cells of 49.2 °C. The activation of Caspase-3 and phosphorylation of HSP27 were investigated using fluorescence microscopy to monitor the spatial variation of cellular response. Our results demonstrate that, under the considered exposure conditions, Caspase-3 activation was almost 5 times greater following PW exposure compared to CW. The relationship between the PW-induced cellular response and SAR-dependent temperature rise was non-linear. Phosphorylation of HSP27 was 58% stronger for PW compared to CW. It exhibits a plateau for the peak temperature ranging from 47.7 to 49.2 °C. Our results provide an insight into understanding of the cellular response to MMW-induced pulsed heating.

Millimeter-Wave Heating in In Vitro Studies: Effect of Convection in Continuous and Pulse-Modulated Regimes.

Shallow penetration of millimeter waves (MMW) and non-uniform illumination in in vitro experiments result in a non-uniform distribution of the specific absorption rate (SAR). These SAR gradients trigger convective currents in liquids affecting transient and steady-state temperature distributions. We analyzed the effect of convection on temperature dynamics during MMW exposure in continuous-wave (CW) and pulsed-wave (PW) amplitude-modulated regimes using micro-thermocouples. Temperature rise kinetics are characterized by the occurrence of a temperature peak that shifts to shorter times as the SAR of the MMW exposure increases and precedes initiation of convection in bulk. Furthermore, we demonstrate that the liquid volume impacts convection. Increasing the volume results in earlier triggering of convection and in a greater cooling rate after the end of the exposure. In PW regimes, convection strongly depends on the pulse duration that affects the heat pulse amplitude and cooling rate. The latter results in a change of the average temperature in PW regime. Bioelectromagnetics. 2019;40:553-568. © 2019 Bioelectromagnetics Society.

Tissue models for RF exposure evaluation at frequencies above 6 GHz.

Exposures to radiofrequency (RF) energy above 6 GHz are characterized by shallow energy penetration, typically limited to the skin, but the subsequent increase in skin temperature is largely determined by heat transport in subcutaneous layers. A detailed analysis of the energy reflection, absorption, and power density distribution requires a knowledge of the properties of the skin layers and their variations. We consider an anatomically detailed model consisting of 3 or 4 layers (stratum corneum, viable epidermis plus dermis, subcutaneous fat, and muscle). The distribution of absorbed power in the different tissue layers is estimated based on electrical properties of the tissue layers inferred from measurements of reflected millimeter wavelength energy from skin, and literature data for the electrical properties of fat and muscle. In addition, the thermal response of the model is obtained using Pennes bioheat equation as well as a modified version incorporating blood flow rate-dependent thermal conductivity that provides a good fit to experimentally-found temperature elevations. A greatly simplified 3-layer model (Dermis, Fat, and Muscle) that assumes surface heating in only the skin layer clarifies the contribution of different tissue layers to the increase in surface skin temperature. The model shows that the increase in surface temperature is, under many circumstances, determined by the thermal resistance of subcutaneous tissues even though the RF energy may be deposited almost entirely in the skin layer. The limits of validity of the models and their relevance to setting safety standards are briefly discussed. Bioelectromagnetics. 39:173-189, 2018. © 2018 Wiley Periodicals, Inc.

Microscale temperature and SAR measurements in cell monolayer models exposed to millimeter waves.

Due to shallow penetration of millimeter waves (MMW) and convection in liquid medium surrounding cells, the problem of accurate assessment of local MMW heating in in vitro experiments remains unsolved. Conventional dosimetric MMW techniques, such as infrared imaging or fiber optic (FO) sensors, face several inherent limits. Here we propose a methodology for accurate local temperature measurement and subsequent specific absorption rate (SAR) retrieval using microscale thermocouples (TC). SAR was retrieved by fitting the measured initial temperature rise to the numerical solution of an equivalent thermal model. It was found that the accuracy of temperature measurement depends on thermosensor size, that is, the smaller TC, the more accurate the temperature measurement. SAR determined using TC with lead diameters of 25 and 75 µm demonstrated 98.5% and 80.4% match with computed SAR, respectively. However, both TC provided the same temperature rises in long run (> 10 min). FO probe failed to measure adequately local heating both for short and long exposures due to the relatively large size of the probe sensor (400 µm) and time constant (0.6 s). Calculated SAR in the cell monolayer was almost two times lower than that in the surrounding liquid. It was shown that the impact of the cell monolayer on heating due to its small thickness (5 to 10 µm) can be considered as negligible. Moreover, we demonstrated the possibility of accurate measurement of MMW-induced thermal pulses (up to 10 °C) using 25 µm TC. Bioelectromagnetics. 38:11-21, 2017. © 2016 Wiley Periodicals, Inc.

Millimeter waves as a source of selective heating of skin.

This study demonstrates that 20-100 GHz range can be used for spatially-accurate focusing of heating inside the skin achieved by varying frequency and exposure beam size, as well as by enforcing air convection. The latter is also used to reduce overheating of skin surface. Heating at different skin depths depending on these parameters is investigated in detail using the hybrid bio-heat equation. In particular, it is shown that decreasing frequency and/or increasing exposure beam size at forced airflow result in elevation of heating of deeper layers of tissue and decrease of skin surface temperature. Changes of water content within 15%, which exceed those due to aging and presence of tumors, only slightly affect heating. Exposure intensity necessary to reach a target temperature significantly increases in different areas of body with elevated blood flow. Dependence on exposure intensity and hyperthermia treatment duration is also investigated and discussed. Results of this study suggest that the lower part of the millimeter-wave range is an attractive alternative for non-invasive thermal treatment of skin cancer with a high spatial resolution.

The role of the NF-κB, SAPK/JNK, and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves.

PURPOSE: To investigate the role of the toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), and stress activated protein kinases/Jun N-terminal kinase (SAPK/JNK) signalling pathways in the responses of RAW 264.7 macrophages to low-intensity microwaves (MW). MATERIALS AND METHODS: Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment. RESULTS: MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells. CONCLUSIONS: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.

Complex permittivity of representative biological solutions in the 2-67 GHz range.

The main purpose of this study is to provide experimental data on the complex permittivity of some biological solutions in the 2-67 GHz range at room and human body temperatures. The permittivity measurements are performed using an open-ended coaxial probe. Permittivity spectra of several representative monomolecular solutions of proteins, amino acids, nucleic acids, and carbohydrates are analyzed and compared. Furthermore, measurements have also been performed for complex biomolecular solutions, including bovine serum albumin (BSA)-DNA-glucose mixture, culture medium, and yeast extract solution. The results demonstrate that for concentrations below 1%, the permittivity spectra of the solutions do not substantially differ from that of distilled water. Measurements carried out for 4% and 20% BSA solutions show that the presence of proteins results in a decrease in permittivity. For highly concentrated RNA solutions (3%), a slight increase in the imaginary part of the permittivity is observed below 10 GHz. Experimental data show that free water permittivity can be used for modeling of the culture medium above 10 GHz. However, at lower frequencies a substantial increase in the imaginary part of the permittivity due to ionic conductivity should be carefully taken into account. A similar increase has also been observed for the yeast extract solution in the lower frequency region of the considered spectrum. Above 10 GHz, the high concentration of proteins and other low-permittivity components of the yeast extract solution results in a decrease in the complex permittivity compared to that of water. Obtained data are of utmost importance for millimeter-wave dosimetry studies.

Effect of millimeter waves and cyclophosphamide on cytokine regulation.

We have reported previously that millimeter waves (MMWs) protect T-cell functions from the toxic side effects of cyclophosphamide (CPA), an anticancer drug. Since the effect of MMWs has been reported to be mediated by endogenous opioids, the present study was undertaken to investigate the role of endogenous opioids in protection of T-cell functions by MMWs. The effect of MMWs (42.2 GHz, incident power density = 38 mW/cm²) was studied on CPA-induced suppression of cytokine release by T cells in the presence of selective opioid receptor antagonists (ORA). Production of cytokines was measured in CD4 T cells isolated from splenocytes. Treatment of mice with CPA suppressed the formation of Th1 cytokines (TNF-α, IFN-γ, and IL-2), shifting the overall balance toward Th2 (IL-4 and IL-5). MMW irradiation of CPA-treated groups up-regulated the production of Th1 cytokines suppressed by CPA. Treatment of the CPA+MMW group with selective kappa (κ) ORA further potentiated this effect of MMWs on Th1 cytokine production, whereas treatment with µ or δ ORA increased the imbalance of cytokine production in the Th2 direction. These results provide further evidence that endogenous opioids are involved in immunomodulation by MMWs.

Enhanced absorption of millimeter wave energy in murine subcutaneous blood vessels.

The aim of the present study was to determine millimeter wave (MMW) absorption by blood vessels traversing the subcutaneous fat layer of murine skin. Most calculations were performed using the finite-difference time-domain (FDTD) technique. We used two types of models: (1) a rectangular block of multilayer tissue with blood vessels traversing the fat layer and (2) cylindrical models with circular and elliptical cross-sections simulating the real geometry of murine limbs. We found that the specific absorption rate (SAR) in blood vessels normally traversing the fat layer achieved its maximal value at the parallel orientation of the E-field to the vessel axis. At 42 GHz exposure, the maximal SAR in small blood vessels could be more than 30 times greater than that in the skin. The SAR increased with decreasing the blood vessel diameter and increasing the fat thickness. The SAR decreased with increasing the exposure frequency. When the cylindrical or elliptical models of murine limbs were exposed to plane MMW, the greatest absorption of MMW energy occurred in blood vessels located on the lateral areas of the limb model. At these areas the maximal SAR values were comparable with or were greater than the maximal SAR on the front surface of the skin. Enhanced absorption of MMW energy by blood vessels traversing the fat layer may play a primary role in initiating MMW effects on blood cells and vasodilatation of cutaneous blood vessels.

Enhanced absorption of microwaves within cylindrical holes in Teflon film.

Earlier publications demonstrated that 0.9 GHz microwave exposure induced notable changes of the conductivity of modified bilayer lipid membranes (BLM) formed in holes in thin Teflon film (TF). The aims of this study were: 1) to perform detailed calculations of the microwave field distributions in holes formed in TF, using the finite-difference time-domain technique and 2) to model microwave heating of the hole under the conditions used in the BLM experiments but in the absence of BLM in the hole. We found that with the E-field oriented perpendicular to the TF plane the local-specific absorption rate in holes increased significantly. The increase became larger with increasing electrolyte concentration and with decreasing diameter of the hole and frequency. The calculated temperature elevations in the hole were in good agreement with those determined experimentally. These findings allowed us to conclude that the microwave effects on BLM conductivity reported previously resulted mostly from the enhanced absorption of microwave energy by the membrane-forming holes and subsequent local temperature elevation in the holes.

Millimeter wave effects on electrical responses of the sural nerve in vivo.

Millimeter wave (MMW, 42.25 GHz)-induced changes in electrical activity of the murine sural nerve were studied in vivo using external electrode recordings. MMW were applied to the receptive field of the sural nerve in the hind paw. We found two types of responses of the sural nerve to MMW exposure. First, MMW exposure at the incident power density >/=45 mW/cm(2) inhibited the spontaneous electrical activity. Exposure with lower intensities (10-30 mW/cm(2)) produced no detectable changes in the firing rate. Second, the nerve responded to the cessation of MMW exposure with a transient increase in the firing rate. The effect lasted 20-40 s. The threshold intensity for this effect was 160 mW/cm(2). Radiant heat exposure reproduced only the inhibitory effect of MMW but not the transient excitatory response. Depletion of mast cells by compound 48/80 eliminated the transient response of the nerve. It was suggested that the cold sensitive fibers were responsible for the inhibitory effect of MMW and radiant heat exposures. However, the receptors and mechanisms involved in inducing the transient response to MMW exposure are not clear. The hypothesis of mast cell involvement was discussed.

Millimeter-wave absorption by cutaneous blood vessels: a computational study.

The aims of the present study were to calculate the specific absorption rate (SAR) and E-field distributions inside cutaneous blood vessels and in surrounding tissues (dermis and fat) depending on the frequency of millimeter wave exposure. Most calculations were performed using the finite-difference time-domain (FDTD) technique. A rectangular block of homogeneous or multilayer tissue with blood vessels located in the center of the block was used as the basic geometry. We found that the SAR reached its maximal value in a long blood vessel oriented parallel to the E-field. It exceeded the SAR in the surrounding dermis by 40%-42% at 42.25 GHz. However, in the same blood vessel oriented perpendicularly to the E-field, the SAR was lower than that of the surrounding dermis. Absorption of millimeter waves in a cutaneous blood vessel was higher at 61.22 GHz than at 42.25 GHz. The SAR distribution in a blood vessel was nearly uniform. Because of the small sizes of cutaneous blood vessels relative to the wavelength, the SAR distributions in these blood vessels can be calculated by using quasi-static theory.

Millimeter wave induced reversible externalization of phosphatidylserine molecules in cells exposed in vitro.

In vitro exposure of refrigerated samples (4 degrees C) of anti-coagulated blood with millimeter waves (MMWs) at incident power densities (IPDs) between 0.55 and 1.23 W/cm2 has been found to induce clot formation. We found a small but statistically significant change in clot size with increasing IPD value. MMW exposure of blood samples starting at room temperature (22 degrees C) did not induce blood coagulation; neither did conventional heating at temperatures up to 40 degrees C. Since cell-free plasma did not clot upon MMW exposure, the role of blood cells was particularly analyzed. Experiments on various mixtures of blood cells with plasma revealed an important role of red blood cells (RBC) in the coagulation process. Plasma coagulation also developed within the MMW beam above dense keratinocyte (HaCaT) monolayers suggesting it lacked cell-type specificity. We hypothesized that alteration of the membrane surface in exposed cells might be responsible for the circumscribed coagulation. The thrombogenic role of externalized phosphatidylserine (PS) molecules is well known. Therefore, we carried out experiments for immunolabeling PS molecules with fluorescein isothiocyanate (FITC)-conjugated Annexin V on exposed cells. Fluorescence microscopy of the adherent human keratinocytes (HaCaT) and murine melanoma cells (B16F10) showed that MMW exposure at an IPD of 1.23 W/cm2 is capable of inducing reversible externalization of PS molecules in cells within the beam area without detectable membrane damage. Nonadherent Jurkat cells exposed to MMW at an IPD of 34.5 mW/cm2 also showed reversible PS externalization with flow cytometry, whether the cell temperature was held constant or permitted to rise. These results suggest that certain biological effects induced by MMWs could be initiated by membrane changes in exposed cells.