Validation of an original incubator set-up for the exposure of human astrocyte cells to X-band microwaves in a GTEM-chamber

A current concern about the biological effects of electromagnetic fields (EMF) is increasing with the wide spread use of X-band microwaves (MW, 8-10 GHz range). Gigahertz transverse electromagnetic (GTEM) field flat transmission lines are currently being used for experimental exposure of biological samples to high frequency EMF. Experiments carried out on human cells in culture require optimal growing temperature conditions, i.e. 37°C, 5% CO2 in a humidified atmosphere. The aim of our work has been: i) to built up an original incubator set-up, the so called GTEM-incubator, for exposure of human cells in culture to MW inside a GTEM-chamber, under optimal growing physical conditions; ii) to make the validation of the GTEM-incubator by growing cell samples inside the non-energized GTEM-chamber (test sample) comparing the results with the ones obtained from cell samples grown inside a standard incubator (control samples). The features for comparison were: cell morphology, expression and distribution of cytoskeleton proteins, genotoxicity, viability and cell cycle progression. Any variation in any of the studied parameters would allow for detecting any possible failure or misconception in our GTEM-incubator working test. The results obtained in control and test incubators showed non-significant differences in the development of both cell populations for any of the studied parameters. Thereby our GTEM-incubator is considered valid for our purposes of human cell exposures to X-band MW

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltagegated- delayed-rectifier-K+ channel; SK2-smallconductance- Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxinsensitive voltage-dependent potassium channel; BKCamaxi- conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gatedcalcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of colocalization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity

Exposure to ELF-pulse modulated X band microwaves increases in vitro human astrocytoma cell proliferation

Common concern about the biological effects of electromagnetic fields (EMF) is increasing with the expansion of X-band microwaves (MW). The purpose of our work was to determine whether exposure to MW pulses in this range can induce toxic effects on human astrocytoma cells. Cultured astrocytoma cells (Clonetics line 1321N1) were submitted to 9.6 GHz carrier, 90% amplitude modulated by extremely low frequency (ELF)-EMF pulses inside a Gigahertz Transversal Electromagnetic Mode cell (GTEM-cell). Astrocytoma cultures were maintained inside a GTEMincubator in standard culture conditions at 37±0.1°C, 5% CO2, in a humidified atmosphere. Two experimental conditions were applied with field parameters respectively of: PW 100-120 ns; PRF 100-800 Hz; PRI 10-1.25 ms; power 0.34-0.60 mW; electric field strength 1.25-1.64 V/m; magnetic field peak amplitude 41.4-54.6 μ Oe. SAR was calculated to be 4.0x10-4 W/Kg. Astrocytoma samples were grown in a standard incubator. Reaching 70-80% confluence, cells were transferred to a GTEM-incubator. Experimental procedure included exposed human astrocytoma cells to MW for 15, 30, 60 min and 24 h and unexposed shamcontrol samples. Double blind method was applied. Our results showed that cytoskeleton proteins, cell morphology and viability were not modified. Statistically significant results showed increased cell proliferation rate under 24h MW exposure. Hsp-70 and Bcl-2 antiapoptotic proteins were observed in control and treated samples, while an increased expression of connexin 43 proteins was found in exposed samples. The implication of these results on increased proliferation is the subject of our current research

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 μm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 μm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 μm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields