Description of the apparatus
Experiments on the effects of the ELF (Extremely Low Frequency) weak electromagnetic fields have been performed by using the electronic equipment QUEC-PHISIS QPS1™, provided by PROMETEO S.r.l. (an Italian firm which has developed a device able to generate ELF field on a coil aimed at stimulating the motion of selected ionic species through the ionoresonance effect).
The electronic device being used is able to generate waves in the range where the cyclotron frequencies of the ions involved in metabolic processes lie. Moreover, it is well known from clinical impedencymetry [15
] that e.m. fields belonging to a specific frequency range, peaked on 50 KHz, are able to cross the cell membrane with the maximum efficiency. Therefore, even if the information directed to the ionic flux control travels on low (less than 100 Hz) frequencies signals, higher frequency signals have to be carried on in order to affect the intra cellular environment and the ionic exchanges. To this aim, the device generates programmable patterns of signals, carrying the basic information with a superimposed amplitude modulation at 50 KHz. The signal feeds a coil or a stack of coils which, in turn, generates the magnetic field.
The magnetic field produced inside the cage was measured during the exposure with a gauss meter F.W Bell (Sypris Q3 Test and Measurements) Mod. 7010 equipped with a low field Hall sensor MOX 71-2506-05, with the resolution of 0.1 nT.
In order to study the influence of a prolonged exposure to these fields, initially one coil of 75 cm of diameter was used, which allowed the exposure of two rats in one cage positioned just in the middle of the coil Such a dimension let us to dwell easily a cage for long time, thus avoiding disturbing the animal's daily life. The field produced had a frequency of f = 31.6 Hz and an amplitude of about 40 μT, in order to stimulate the Ca2+ dynamics. The amplitude was set equal to the amplitude of B0 in order to accomplish to the range of validity of the Edmonds model.
In a subsequent experiment, a set of identical Helmoltz coils arranged on a rack (Fig. ) was assembled thus allowing the simultaneous exposure of up to 16 animals housed in 5 cages. Each cage has been put on a shelf of the rack, however, it must be remembered that the Helmoltz coils set up allows to have a uniform magnetic field inside the coils provided that they are spaced of a length equal to their radius. The limitations of the maximum output power of the device (16 Watt) sets a limit to the maximum diameter of the coil. The exposure frequency was again chosen f = 31.6 Hz to stimulate the Ca2+ ion dynamics. In fact the q/m ratio of Ca2+ ion is 4.81 × 10-6 Coul/kg, the corresponding fc/B0 is 0.76 (Hz/μT) and the resonant frequency has been calculated being the measured geomagnetic field of 41.5 μT.
Set of exposure. Each coil has a diameter of 75 cm and the distance between cages is 37 cm.
However, the peculiar geometry and the materials of the rack and of the grids of the cages (stainless steel) did not allow a uniform static magnetic field level to be reached neither along the ax of the rack nor on the plane of the five cages. Thus, the B0 magnetic field amplitude, accurately measured during the exposure, ranged between 35.5 and 51.6 μT, as it can be seen in details in Fig. . In such a circumstance the cyclotron frequency varies along the plane of each cage accordingly to the variation of the static magnetic field. Also other ions as Co3+ and Mn3+ having an fc/B0 ratio quite similar to the Ca2+ might have been activated.
Measured static magnetic field (geo magnetic field) B0 along the plane of each of the 5 cages.
Animal exposure and sample collection
This Research Project was communicated on July the 7th 2005. to the Ministry of Public Health as stated by Laws (D.lgs 116/92).
The animals were kept in stabulary controlled conditions all over the duration of the experiments : temperature 21°C ± 1; hygrometry between 40% and 70%; air change per hour 8 to 12 volumes; nyctohemeral cycle 12 h/12 h; lightning 300 to 600 lux at 1 meter over the floor; noise < 60 db. The cages bases were made out of polypropylene while the ceilings were in stainless steel. In each cage up to 4 adult animals, or 3 aging animals, could be housed. A complete maintenance diet for rats (Standard Diet GLP – 4 RF 21; Charles River-Italy), administered dry ad libitum with water at disposal, was utilised.
In a preliminary experiment two WISTAR SPF(Specific Pathogen Free)/VAF(Virus Pathogen Free) male rats (age at the beginning 50 weeks) were exposed to a field specifically designed to modify calcium dependent enzymatic activities as indicated in the previous paragraph, for 35 days continuously. The animals were then observed without any other intervention for 182 days after the exposure. The limited number of animals used was due to the necessity of a constant exposure and the availability of a small set up. Furthermore, two animals of the same strain, used as controls, were kept in the same conditions without exposure.
In the main protocol, 13 male OFA (Oncins France Strain) SPF/VAF rats, from Charles River Laboratories Italia (Calco-LC), were exposed for a total time of 50 days.
Eight animals were 8–12 weeks old, and Eight animals were 68–72 weeks old. They were hosted into the cages according to the scheme: 4 adult rats in cage 1; 4 adult rats in cage 2; 2 aging rat in cage 3; 3 aging rats in cage 4; 3 aging rats in cage 5. After 15 days of adaptation, all rats were subjected to 1st blood drawing (1st day of the trial – field OFF). After this first drawing 2 adult rats and 1 aging rat died. From 2nd to 29th day, all rats were subjected to continuous irradiation by low intensity and frequency electromagnetic fields (ELF) (field ON); on 15th and 29th day respectively, 2nd and 3rd blood drawing was carried out. From 30th to 36th day ELF were deactivated (field OFF); on 36th day 4th blood drawing was carried out. From 37th to 50th day of trial ELF were reactivated (field ON); on 50th day 5th blood drawing was carried out. After the third and fourth drawing two other aging rat were lost. On 50th day of trial all rats were sacrificed by intracardiac inoculation of embutramide + mebezonium iodide + tetracaine hydrochloride.
After each blood drawing the rats returned to the same cage. During the experiment, it was decided to submit to euthanasia subject 3 of cage 5 before the others for its health status, while rat 2 of cage 4 died twenty days before the end of the trial.
The individual body weight was controlled on 1st, 15th, 29th, 36th, and 50th day together with blood drawing, and the amount of food consumed by each group was noted during the trial.
The data shown in the Figures are the averages on the animals which survived along the whole duration of the experiment, i.e. 6 adult and 5 aging rats.
A complete post-mortem examination of all the animals was performed after euthanasia and lungs, heart, spleen, liver, pancreas, kidneys, skeletal muscle and brain samples were fixed in buffered 10% formalin. Half of the brain, ocular tract and eyes were frozen and stored at -80°. Tissues were then processed in routine manner for histological evaluation, cut at 5 μm thickness, and stained with H&E. Liver sections were also stained with periodic acid-Schiff (PAS)/diastase techniques to assess hepatic glycogen.
Blood sample analysis
All blood samples were obtained by intracardiac drawing after anaesthesia with tyletamine hydrochloride and zolazepan hydrochloride combined with xilazine hydrochloride. Blood samples added to K2-EDTA were utilized to determine several haematology and biochemical parameters (see below). Blood sampling was performed in animals with no restriction to food availability. In order to reduce variability due to recent food uptake, care was taken to obtain samples at the same time of the day, namely between 3 and 4 pm, i.e. 7–8 hours after light start.
Determinations of haematological and biochemical parameters
Haematological parameters (WBC, RBC, haemoglobin, hematocrit, MCV, MCH, MCHC, platelets, and differential leukocyte count: neutrophils, lymphocytes, monocytes, eosinophils, basophils) were measured in blood samples, added up with K2-EDTA, by ADVIA 120 HEMATOLOGY SYSTEM (BAYER Corp. Diagnostic Division, Tarrytown, NY, USA) provided with specific veterinary software for rats.
Biochemical parameters (Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Creatine kinase (CK), creatinine, cholesterol, glucose, total protein, tryglicerides, urea) were measured in plasma samples, obtained after centrifugation of blood samples added with K2-EDTA, by automatic analyser ROCHE HITACHI 912 PLUS (ROCHE Diagnostic Corp., Indianapolis, USA).
Fatty acids (palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1ω
9), linoleic acid (C18:2ω
6), linolenic acid (C18:3ω
3), eicosatrienoic acid (C20:3ω
9), arachidonic acid (C20: 4ω
6), docosaesanoic acid (C22: 6ω
3), and nervonic acid (C24: 1ω
9)), derived as methyl esters, were measured in plasma samples according to Carnielli [16