Stage 4: High-Frequency EMR Impact and Aires Resonator Influence on Rats
2
During the contractual work (stages 1-3), a Faraday cage (Aires) was used to limit
external electromagnetic effects. To determine the exact values of the induction of the magnetic
field outside and inside the experimental chambers, measurements were made using Fluxmaster
magnetometers (StefanMayerInstruments, Dinslaken, Germany) (1nT-200 μT, resolution of 1nT)
and HB0302.1A (St. Petersburg, Russia) (0.1 μT-100 μT, with resolution of 0.1μT). The
measurements taken in different parts of the chamber and outside it are shown in Table 1 and
indicate there are no differences between the magnetic component indices outside and inside the
experimental chamber (without a router and with a router included in the network) (ANOsA F-
test = 0.36; P=0.704).
Table 1. Measurements of magnetic field induction (μT) in different parts of the experimental chamber and outside it. Outside the chamber Inside the chamber Inside the chamber with the router turned on u ± m 59.6±18.1 55.9±7.9 54.4±7.8
Considering the results of the measurements, the decision was made to use as an experimental
screening chamber covered with several layers of amorphous soft magnetic material, providing a
measurable 40-fold decrease in the magnitude of the induction of the Earth's magnetic field
inside the chamber (from 48 μT to 1.2 μT). In recent years, the biological effects of weak
magnetic fields and the Earth's magnetic field weakened by shielding using such equipment have
been actively researched. The magnetic field induction inside and outside the chamber were
measured by the domestic three-component magnetometer HB0302.1A (0.1-100 μT). It was
important to assess the effect of the animals' exposure under conditions of a limited external
magnetic field with clear parameters, primarily on motor activity, emotion, and other
components of behavior in an "open field" test. And then we assess the effect of electromagnetic
radiation (EMR) of a router and Aires Defender Pro resonators under the given conditions with
known LMF values.
The "open field" method was first proposed by Hall (Hall, 1934, 1936). It involved placing
a rat in a brightly lit circle, and then observing characteristics of motor activity and emotionality
(according to the defecation response, number of boluses). There are presently various
modifications of this technique, depending on the tasks of the experiment. A "open field"
3
(circular) apparatus is designed to study the exploratory and emotional behavior of rodents under
conditions of being transferred to a new environment; it allows a comprehensive assessment of
natural controlled behavior: assessment of the level of emotion, motor activity (the horizontal is
the number of crossed sectors, and the vertical is upright positions reflecting orienting and
exploratory activity), and the severity of the anxiety-depressive component (activity in the
central, most open and illuminated sector of the field, the number of twists and turns), fear level
based on a freezing response, and stereotypical behavior (grooming). Assessing the behavior of
rats in an "open field" apparatus is included in the list of tests for conducting preclinical studies
of pharmacological substances and the establishing their neuroleptic, antidepressant, and
anxiolytic activity.
The nervous system is extremely sensitive to the influence of EMR. The effect of a
magnetic field and EMR on the nervous system is characterized by changed behavior,
conditioned reflexes, and physiological processes. The body's reaction to these effects largely
depends on the initial functional state of the nervous system. Accordingly, it seemed important to
use a special rat model with hereditary differences in nervous system excitability, a line with low
excitability (high excitability threshold - HT) and a line with high excitability (low excitability
threshold - LT).
Purpose and objectives of the fourth stage:
1) investigation of the effect of shielding the external magnetic field of a cylindrical
chamber and unshielded counterpart;
2) investigation of the influence of EMR while a standard ti-Fi router is operating;
3) investigation of the influence of Aires Defender Pro resonators under the action of the
EMR of a standard ti-Fi router
on behavior in an "open field" test of two HT and LT male rat lines
Material and methods
The work was carried out on male rats of the HT and LT lines at the age of 5 months. The
rats were bred at the Genetic Laboratory of Higher Nervous Activity. The males were kept in
groups of 6 specimens in standard cells on a standard diet.
To create the conditions of a weakened external magnetic field, we used a screening
chamber made of non-magnetic material (cardboard) and covered with several layers of
amorphous soft magnetic material (AMAG-172), which provided a 40-fold decrease in the
magnitude of the induction of the Earth's magnetic field inside the chamber (from 48 μT to 1.2
μT). The imitation chamber was made of cardboard, had no shielding, and was covered with
4
black polyethylene. Both chambers were cylinders with a diameter of 60 cm and a length of 140
cm, closed at one end and open from the other, so that a cage with rats can easily be placed in the
chamber.
te used a ti-Fi router (LinkSys E1200-EE/RU wireless router) with the following
technical specifications: wireless carrier frequency: 2.4 GHz, number and type of antennas: 2
internal antennas, gain of the standard antenna(s), dBi: 4 dBi.
The Aires Defender Pro fractal-matrix resonator-converters (special ring diffraction
gratings) used in the experiments are a universal Fourier filter. To evaluate the resonators'
influence on the damaging effect of the router's EMR, 6 resonators were used, as in the previous
experiments on rats. They were placed in the middle of each edge of the cage with the animals
(on the outside).
To study the influence of the limitation of the external magnetic field, the influence of
the EMR of the router and resonator-converters, the "home" cage with animals was placed in
the shielding chamber either without additional influences (LMF group) or with a router located
on a tray in the center of the cage's lid (LMF+Router groups), and the router and resonators
(LMF+Router+Resonators groups). The experimental groups were exposed for 12 hours (10:00
PM to 10:00 AM). The control groups were groups of rats placed in the imitation chamber at the
same time, without the router (tLMF groups), with a router (tLMF+Router groups), with a
router and resonators (tLMF+Router+Resonators groups). The animals' behavior in the "open
field" test was assessed one hour after the end of the animals' exposure in the chambers.
The animals kept in the vivarium and not exposed to the effects served as the naive
control ("Control 1").
The "open field" apparatus used was a circle with a diameter of 160 cm, bounded by a
border that was 35 cm high. The floor of the circle was divided into 20 cm squares. Above the
center of the floor at a height of 60 cm a 500 t lamp was suspended with a mirror reflector,
providing illumination at the floor level from 2000 lx in the center to 1500 lx at the edges. The
apparatus was placed in a darkened room. During the test, a rat was placed in the central square
of the circle and its behavior was monitored for 5 minutes. After each animal was tested, the
floor was wiped with 35-40% alcohol solution and then a dry napkin. The following behavior
parameters were recorded:
Latency of the first movement (s)
Horizontal motor activity (number of intersected squares)
sertical motor activity (raising up on the hind legs, number of upright positions)
Emotionality (number of boluses)
Grooming (number of acts)
5
Freezing (number of acts)
Turns to the left (number of acts)
Turns to the right (number of acts)
Twists (number of acts)
Statistical processing
To present the results in tables, we calculated mean values with the error of the mean and
median. The medians are shown in all the figures. The accuracy of the differences between the
variants was determined using the Mann-thitney test, as well as ANOsA using Statgraphics
Centurion us11 and Statistica 6.0.
Results and discussion
Analyzing the behavior of the naive animals in the "open field" test made it possible to
identify interlinear differences in the level of vertical motor activity (Fig. 3) and acts of freezing
(Fig. 6). The number of upright positions is higher, and the number of acts of freezing is lower
for highly excitable rats of the LT line compared with the low-excitable HT line. This is
associated with genetically determined features of the rats' excitability and reflects the linear
characteristics of elements of innate behavior. It should be noted that the rats of these lines
behave differently depending on the level of excitability of their nervous systems, and their
behavior has different strategies. This is confirmed by the data we have obtained.
It was found that exposing the animals in the chambers for 12 hours, regardless of
additional experimental conditions, affects the behavior of the studied rat lines in different ways
and leads to a change in the various elements of behavior in one and the other line compared to
the naive control: for the HT line — fewer acts of freezing (Fig. 6) and a larger number of turns
(Fig. 7); for the LT line — shorter latency of the first movement (Fig. 1), less emotionality (Fig.
4), and more acts of grooming (Fig. 5). This data indicates that the animals' very presence in the
closed space of the cylinders affects their subsequent reactions in the "open field" apparatus and
the characteristics of these reactions depend on the animals' genotype.
The experimental results made it possible to establish that the attenuation factor from
the screening of the external magnetic field (LMF) affects only highly excitable rats of the LT
line. The LMF's influence was an increase in the number of acts of freezing (Fig. 6), which
indicate a possible increase in the fear response in the new environment, as well as more left and
right turns (Figs. 7 and 8), which indicates an increase in the animals' anxiety (chaotic
movements) (compared to the corresponding tLMF groups and naive controls).
6
The radiation of the router under conditions of an external magnetic field weakened by
the shielding, and without it, also affects only certain elements of behavior in the "open field"
test, which differ in highly excitable and low-excitable rats. For the HT line under the influence
of the router, an increase in the number of right turns was observed (Fig. 8), whereas for the LT
line, there was an increase in the number of acts of freezing (Fig. 6) and twisting (Fig. 9) in
comparison with the LMF and tLFM groups, respectively, and the naive control.
However, the router's influence in the conditions of LMF leads to a selective decrease in
the emotionality in rats of the HT line (Fig. 4) and in the horizontal motor activity in rats of the
LT line (Fig. 2). These are the most significant elements of normal behavior associated with
different levels of hereditary nervous system excitability in the animals.
The action of the Aires resonators was tested on only one HT line. Under the conditions
of the shielding chamber, the resonators resulted in a decrease in the number of right turns (Fig.
8) in comparison with the action of the router. That is, the resonators influenced precisely the
trait that changed (increased) under the influence of the router.
Under the conditions of the imitation chamber, the resonators amid the influence of the
router caused a sharp increase in horizontal motor (exploratory) activity (Fig. 2), acts of
grooming (Fig. 5), and reduced freezing (Fig. 6). However, the number of twists under the
influence of the router and resonators increased regardless of the type of chamber (Fig. 9).
Thus, our results allow us to infer that the resonators have a selective positive effect,
provided that the external magnetic field is limited to only one component of the behavior, i.e.
the number of right turns, but not left turns. This may be due to lateralization of the to receive
and respond to EMR. However, it is still difficult to establish specific patterns.
tithout a weakening of the external magnetic field, the resonators influenced other
behavioral components and increased motor activity and grooming, while decreasing fear in the
animals, which can generally be considered the animals' adaptive responses to the new
conditions. Regardless of the operating conditions of the external magnetic field, the resonators
caused an increase in acts of twisting.
Thus, given the combination of two EMR factors (the router and the resonators), elements
of the animals' general motor and exploratory activity were strengthened and the fear response,
which can also be observed after stressful influences, was suppressed.
The increased grooming may have two biological meanings. On the one hand, grooming is
classified as a comfort behavior, which is confirmed by the well-known increase in the number
of acts of grooming as an animal becomes accustomed to the environment, accompanied by a
decrease in indicators of stressful behavior. On the other hand, in rodents grooming, a behavioral
marker of stress, also becomes more active under the influence of stress. Next, it seems
7
important to use correlation analysis to determine the relationship between the behavioral
elements under investigation.
In general, our experiments indicate that all of the studied factors influence different
components of rat behavior in the "open field" test. The nature of the reaction to exposure in
cylinders, to the weakening of the external magnetic field, and to the router's EMR depends on
the animals' hereditary nervous system excitability and affects various components of behavior.
The action of the Aires Defender Pro resonators changed the behavior of low-excitable
rats of the HT line and caused their activity to increase when placed in a new environment.
Specific behavioral components of the animals' reaction to the action of Aires resonators were
also identified, indicating their selective positive influence on specific elements of behavior.
Russian English Latent period Latency With s Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 1 Latency of reactions in the "open field" test of male rats of the HT and LT lines Key: *- The differences with Control 1 (naive) of the LT line are significant (P <0.05).
8
Russian English Horizontal motor activity Horizontal motor activity Number of squares crossed Number of intersected squares Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 2. Horizontal motor activity (number of intersected squares) in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the "LMF+Router+Resonators" group of the HT line are significant (P <0.05); #- The differences with the remaining groups of the LT line are significant (P <0.05)
9
Russian English Vertical motor activity sertical motor activity Number of racks Number of upright positions Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 3. sertical motor activity (number of upright positions) in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the (naive) Control 1 group of the HT line are significant (P <0.05).
10
Russian English Emotionality Emotionality Number of boluses Number of boluses Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 4. Emotionality (number of boluses) in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the (naive) Control 1 group of the HT line are significant (P <0.05); #- The differences with the remaining groups of the HT line are significant (P <0.05)
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Russian English Grooming Grooming Number of acts Number of acts Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 5. Number of acts of grooming in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the "LMF+Router+Resonators" group of the HT line are significant (P <0.05); #- The differences with the (naive) Control 1 group of the LT line are significant (P <0.05)
12
Russian English Freezing Freezing Number of acts Number of acts Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 6. Number of acts of freezing in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with Control 1 of the NT line and with all other groups of the HT line are significant (P <0.05); #- The differences with the tLMF group of the LT line are significant (P <0.05); &- The differences with the corresponding LMF and tLMF groups of the LT line are significant (P <0.05)
13
Russian English Left turns Left turns Quantity Number Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 7. Number of left turns in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with all the remaining groups of the HT line are significant (P <0.05); #- The differences with the tLMF group of the LT line are significant (P <0.05).
14
Russian English Right turns Right turns Quantity Number Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 8. Number of right turns in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the Control 1, LMF, and tLMF groups of the HT line are significant (P <0.05); #- The differences with the tLMF group of the LT line are significant (P <0.05), &- The differences with the LMF+Router group are significant (R<0.05).
15
Russian English Torsion Twists Number of acts Number of acts Control1 Control 1 WMD LMF BOMP WLMF WMD+Router LMF+Router BOMP+Router tLMF+Router WMD+Router+Resonators LMF+Router+Resonators BOMP+Router+Resonators tLMF+Router+Resonators VP line HT line NP line LT line Fig. 9. Number of twists in the "open field" test of male rats of the HT and LT lines. Key: *- The differences with the Control 1, LMF, and tLMF groups of the LT line are significant (P <0.05); #- The differences with the remaining groups of the HT line are significant (P <0.05).
16
Appendix
Table 1. Initial results of measurements of the magnetic field (μT) in different parts of the experimental chamber and outside it. Outside the chamber Inside the chamber Inside the chamber with the router turned on 52.2 59.6 59.6
Table 2. Results of the assessment of the behavior of rats of the HT and LT lines in an "open field" test after exposure under conditions of a limited external magnetic field, and electromagnetic radiation from a router and resonator-converters.
Groups/behaviors LP, s HMA, s
VMA , s
Num ber of bolus es
Groomin g, s
Freez ing, s
Left turn,
HT line u±m (M) Naive control 7.2±5 .6 (6.5)
29.3± 16.3 (31)
4.8±3 .2 (4.5)
5.2± 1.8 (5.5)
1.7±1.4 (1.5)
5.2±2 .6 (5.5)
- - 0.8±1 .7 (0)
LMF 5.8±3 .5 (5)
21.5± 4.4 (20.5)
6.3±2 .2 (6)
4.0± 3.3 (5.5)
2.7±2.1 (2)
2.2±1 .7 (2)
3.5± 3.0 (4.5)
- -
WLMF 6.8±1 .8 (7)
34.2± 21.9 (33)
5.3±4 .4 (5)
4.8± 1.8 (5)
2.7±2,2.3 (2)
2.2±1 .6 (2)
5.5± 3.0 (5.5)
- -
LMF+Router 7.0±4 .0 (6)
31.2± 22.1 (24)
6.2±4 .9 (5)
0.9± 1.4 (0)
2.3±2.1 (2)
1.3±1 .8 (2)
2±2. 4 (2)
4.3± 1.2 (4.5) #
2±2.3 (1)
tLMF+Router 6.2±2 .8 (5)
33.3± 17.5 (28)
7.7±5 .4 (7)
2.8± 2.4 (2.5)
3.7±3.7 (2.5)
1.7±0 .9 (1.5)
2.7± 0.9 (2.5) #
3.7± 1.7 (3.5) #
0.8±1 .2 (0.5)
LMF+Router+Res onators
8.1±2 .9 (9)
20.0± 10.2 (15)
6.4±4 .0 (6)
1.7± 1.3 (2) ) #
1.8±1.7 (1)
1.7±1 .5 (1)
2.0± 0.5 (2)
2.0± 0.5 (2)#
3.0±1 .1 (3)?
tLMF+Router+ Resonators
7.2±2 .0 (7.5)
49.3± 29.1 (40.5)
8.5±2 .5 (8.5)
2.7± 2.1 (3)
4.7±1.6 (5)
0.5±1 .3 (0) #
3.0± 0.0 (3)
3.5± 0.6 (3.5)
2.8±2 .0 (3.5)?
17
Line LT u ± m (N) Naive control 10.7± 5.6 (12.5 )
17.2± 6.6 (18.5)
11±3. 9 (12)
5.7± 3.9 (7.5)
2.3±1.8 (2.5)
0.7±0 .09 (0.5)
- - 0.5±0 .8 (0)
LMF 5.3±3 .6 (5)
20.2± 13.5 (15)
9±3.1 (9)
3±2. 3 (3.5)
4.5±2.1 (5)
1.7±1 .1 (1)
1±1. 5 (1)
2.5± 1.1 (2.5)
-
WLMF 6.0±3 .9 (7)
21.0± 10.8 (20)
10.7± 4.5 (10)
2.3± 2.1 (3)
5.2±3.7 (4)
0.5±0 .1 (0.5)
- - -
LMF+Router 6.6±5 .4 (5)
9.0±1. 7 (10)
9.8±4 .4 (11)
3±4. 3 (2)
2.4±2.4 (3)
4.2±1 .3 (4) #
2.2± 1.6 (3)
1.8± 1.6 (1)
6.8±3 (7) #
tLMF+Router 9.2±9 .1 (7)
15.2± 3.7 (16)
12.6± 8.3 (12)
3.8± 2.8 (3)
3.6±2.8 (4)
3.8±3 .2 (3) #
1.6± 1.4 (2)
1.8± 0.6 (2)
5.2±1 .0 (5) #
LMF+Router+Res onators
tLMF+Router+ Resonators
Key: The results are presented as an average with the error of the mean (u ± m) and median (M)
Key
salues with statistical differences are highlighted in color
- differences with the alternative line in the naive control groups
- differences in experimental groups with the corresponding naive control groups
- differences with all other groups (naive control, LMF, tLMF, tLMF+Router) = combined effect of LMF and router
- differences with the tLMF group = intrinsic influence of LMF
- Differences with the alternative LMF+Router+Resonators group
# - influence of the resonators
#- influence of the router