Effect of 1800-2100 MHz Electromagnetic Radiation on Learning-Memory and Hippocampal Morphology in Swiss Albino Mice

Abstract

Introduction: With advancing technology the mobile phone with multiple features is used as a multipurpose device and attract people of all age groups. Increased usage of mobile phone raises the question of possible adverse effects on health. Aim: To assess the 1800-2100 MHz radiation effect on learning-memory and microscopic anatomy of hippocampal Cornu Ammonis (CA3) neurons in mice. Materials and Methods: A total of 18 albino mice were divided into 3 groups (6 Mice per group). Group-I: Control Group, Group-II: Exposed to Radio frequency-Electromagnetic radiation (RF-EMR) for 30 minutes/day for 3 months, Group-III: Exposed to RF-EMR for 60 minutes/day for 3 months. Followed by the exposure, learning memory was assessed by using HebbWilliams maze in all the groups. The mice were then sacrificed, brains were dissected out and sections were taken at the level of hippocampus and then stained with Haematoxylin and Eosin for microscopy. The results were expressed in Mean±SD and analysed by using one-way (analysis of variance) ANOVA followed by LSD (Least Square Difference) test for paired wise data. The p-value

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Journal of Clinical and Diagnostic Research. 2019 Feb, Vol-13(2): AC14-AC17
1414
DOI: 10.7860/JCDR/2019/39681.12630
Anatomy Section
Effect of 1800-2100 MHz Electromagnetic
Radiation on Learning-Memory and
Hippocampal Morphology in
Swiss Albino Mice
Original Article
INTRODUCTION
The extensive use of Global System for Mobile communication
(GSM) mobile phones throughout the world raises the possible
adverse effects on human health especially on the Central Nervous
System (CNS), the brain. In many countries more than half of the
population relies/depend on mobiles for wireless communication
and internet data [1]. In 2015, more than 7 billion people were using
mobiles in the world, estimating to 62.9% of the world’s population.
Rapid increase of mobile users in general and specifically upto
80% of youngsters owning a mobile has made communication and
technology easier [2].
In this concern there is a growing interest in scientific community for
the potential deleterious effects of Radio Frequency Electro Magnetic
Radiation (RF EMR) on the public health, especially much focus on
the effects of RF EMR on structural and functional integrity of the
brain because the radiation exposure is directly to the head region
[3]. In 2006 and 2010 World Health Organisation (WHO) issued a
research agenda for high priority research on effects of RF exposure
on ageing and neurodegenerative diseases in animals and effects of
pre and post natal RF exposure on development and behaviour in
animals [4,5]. The mobile phone releases non-ionising radiation which
has low frequency and considered to be safe, but recent studies
evidenced that it has an impact on the living tissues especially on the
brain which can cause headache, memory loss, heat over the ear,
decreased concentration and other cognitive effects [6].
The hippocampus is a part of brain which belongs to the limbic
system and is involved in cognitive functions like spatial learning
and working memory. It plays a crucial role in the formation of new
memories and it is considered as a sensitive region and is affected
by mobile phone radiation. The hippocampus is a “S”-shaped
folded structure located on the floor of the lateral ventricle on both
the cerebral hemispheres. Hippocampal formation consists of
hippocampus proper, dentate gyrus and subiculum. Hippocampus
proper is also known as Cornu Ammonis (CA), which consists of
CA1, CA2, CA3 and CA4 sub regions [7].
Studies have found that damage to the hippocampal neurons may
lead to impairment of memory and learning, behavioural disturbances
and impact on Hypothalamo-Pituitary-Adrenal (HPA) axis [3,8,9]. The
present study was undertaken to evaluate the long term exposure
effect of mobile phone radiofrequency electromagnetic radiation-4G
(1800-2100 MHz) on cognitive functions like spatial learning, working
memory and hippocampal morphology in adult swiss albino mice.
MATERIALS AND METHODS
The Experimental study was carried out after the approval
of Institutional Animal Ethical Committee (IAEC/PHARMA/
SDUMC/2017-18/04). The study was conducted at central animal
house Sri Devaraj Urs Medical College, Kolar from November 2017-
January 2018, the duration of the study was 3 months.
Animals
Six weeks old healthy male Swiss-Albino Mice were used in this
study, the animals were procured from committee for the purpose
of Control and Supervision of Experiments on Animals (CPCSEA)
registered brooders-Invivo Biosciences, Bengaluru.
KRISHNA KISHORE G1, VENKATESHU KV2, SRIDEVI NS3
Keywords: Cornu ammonis, Hebb-Williams maze, Radio frequency
ABSTRACT
Introduction: With advancing technology the mobile phone with
multiple features is used as a multipurpose device and attract
people of all age groups. Increased usage of mobile phone
raises the question of possible adverse effects on health.
Aim: To assess the 1800-2100 MHz radiation effect on
learning-memory and microscopic anatomy of hippocampal
Cornu Ammonis (CA3) neurons in mice.
Materials and Methods: A total of 18 albino mice were
divided into 3 groups (6 Mice per group). Group-I: Control
Group, Group-II: Exposed to Radio frequency-Electromagnetic
radiation (RF-EMR) for 30 minutes/day for 3 months, Group-III:
Exposed to RF-EMR for 60 minutes/day for 3 months. Followed
by the exposure, learning memory was assessed by using Hebb-
Williams maze in all the groups. The mice were then sacrificed,
brains were dissected out and sections were taken at the level
of hippocampus and then stained with Haematoxylin and Eosin
for microscopy.
The results were expressed in Mean±SD and analysed by using
one-way (analysis of variance) ANOVA followed by LSD (Least
Square Difference) test for paired wise data. The p-value<0.05
was considered as statistically significant.
Results: The time taken by the animal to reach the target
chamber was significantly increased in Group-III (exposed
60 minutes/day for 3 months), whereas group-II (exposed
30 minutes/day for 3 months) showed no significant changes
when compared to Group-I (control group). Microscopic
anatomy of hippocampal CA3 neurons in exposed group shows
less number of pyramidal cells with darkened nuclei, cytoplasm
was vacuolated and cells were scattered.
Conclusion: Exposure to 1800-2100 MHz radiation leads to
damage and decrease of neurons in hippocampal region, which
alters the learning and memory.

www.jcdr.net Krishna Kishore G et al., Effect of 1800-2100 MHz Electromagnetic Radiation on Learning-Memory and Hippocampal Morphology
Journal of Clinical and Diagnostic Research. 2019 Feb, Vol-13(2): AC14-AC17 1515
to prevent back entry. The time taken by the animal to reach The
Reward Chamber (TRC) from the start chamber was recorded. The
animals were trained for 3 days (3 trials/day) and the readings were
taken at the 4th day. Low scores indicates better memory, while the
high scores indicates poor memory in animals [Table/Fig-2] [10,11].
The Swiss-Albino Mice were kept in polypropylene cages with a
temperature of 23±2°C, humidity 55±5% and 10 hours light,
14 hours dark cycle and free access to standard pellet food and
water ad libitum. The experimental animal care was taken as per
the Committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA) guidelines.
Inclusion and Exclusion Criterion
Male healthy active Swiss-Albino mice with average weight of
20 grams when procured were included in this study. Female swiss
albino mice and lesser weight mice were excluded from this study.
Experiment Design
A total of 18 Male Swiss-Albino Mice were taken and they were
divided into three groups.
Group I: Control group-consists of 6 mice (non-exposed group).
Group II: 30 minutes exposure group-consists of 6 mice which
were exposed to Mobile Phones (MP) RF-EMR for 30 minutes/day
for 3 months.
Group III: 60 minutes exposure group-consists of 6 mice which
were exposed to MP RF-EMR for 60 minutes/day for 3 months.
Mobile phone: 4G android mobile phones (Micromax Bharat-2
with a Specific Absorption Rate (SAR) of 1.6 Watt/Kg) with same
specification and with same mobile network were used in this
study, keeping a GSM (2100 MHz) mobile phone in silent with auto
answer mode. The mobiles were hung down from the roof of the
mice cage and the radiation which they emitted during the exposure
was quantified by radiation frequency meter (Electrosmog Meter-ED
178 S) which was kept at the periphery, 1950 MHz of RF-EMR was
emitting till the periphery of the mice cage during the exposure, so
the similar amount of radiation may affect/enters the mice brain.
Exposure technique: Three Mice were kept in each cage during the
exposure. Animals of group II and III were exposed to 30 minutes
and 60 minutes/day for 3 months respectively. The mobile phones
were hung down in the center of the cages during the exposure
period for the uniformity of the radiation through out the cage
[Table/Fig-1].
[Table/Fig-1]: Image shows the cage with mice and mobile phone during radiation
exposure with Radiation Frequency Meter (Electrosmog Meter-ED 178 S) to quantify
the mobile radiation.
Hebb-Williams Maze: Hebb-Williams Maze is used to test the spatial
learning and working memory of the mice. The principle behind the
Hebb-Williams Maze test is “The faster the mice navigates the maze,
the better its spatial memory”. The Hebb-Williams maze is a square
shaped box which measures 60 cm (L)×60 cm (W)×10 cm (H) walls.
It consists of start chamber-A (Animal Chamber) which is attached to
the exploratory area-B (Middle Chamber) and a goal box-C, located
at the opposite end of the start chamber and contains a small food
reward. All three chambers were provided with removable doors to
allow the animal to move from one chamber to the next.
After 12 hours of fasting, the mice was placed in the start chamber-A
and allowed to enter into the exploratory area-B (middle chamber),
once the animal enters into middle chamber the door was closed
[Table/Fig-2]: Hebb-Williams Maze instrument to assess the spatial learning and
working memory in Swiss-Albino mice.
Tissue Processing
After the behavioural analysis the mice were euthanized, perfused
transcardially with normal saline and the brains were extracted out,
fixed in 10% buffered formalin, dehydrated in ascending grades of
ethyl based alcohol like 60%, 70%, 80%, 90% and absolute alcohol,
cleared in xylene, impregnated in paraffin wax at 60˚C, embedded
with the help of L-moulds and then 6 µm paraffin sections were
taken using rotary microtome at the level of the dorsal hippocampus
to assess the hippocampal CA3 cellular architecture with the help
of H and E staining. To prevent the bias every 5th section was taken
and the slides were decoded after the histological assessment.
Viable neuronal quantification was assessed with the help of ocular
micrometer fixed to light microscope (40X).
STATISTICAL ANALYSIS
The results were expressed in Mean±SD and analysed by using
one-way ANOVA followed by Least Square Difference (LSD) test
for paired wise data. The p<0.05 was considered as statistically
significant.
RESULTS
Body Weights of the Mice
The mean body weight of the control group mice was 32.3 grams,
30 min/day radiation exposed mice for 3 months had 31.8 grams
and 60 min/day radiation exposed mice for 3 months had
32.7 grams, the mean weight between the three groups didn’t
show any significant difference.
Effect of Radiation on Learning Memory in Hebb-
Williams Maze
The time taken by the mice to reach the target chamber from the
starting chamber was significantly increased in group II (30 min
exposed/day) and group III (60 min exposed/day) compared to
group I (non-exposed group).
The time taken by the animal to reach The Reward Chamber (TRC)
scores in Group I vs Group II (31±15.48 vs 49±17.62 seconds),
was not significant (p>0.05); Group I vs Group III (31±15.48 vs
64±22.99 seconds), was statistically significant (p<0.05) [Table/Fig-3].
Microscopic Anatomy of Hippocampal Cornu
Ammonis (CA3) Neurons
Histological sections of haematoxylin and eosin stained hippocampal
CA3 pyramidal neurons showed marked difference between control
group and RF-EMR exposed groups (group II and III). Sections of
control group showed 5-6 layers of compactly arranged pyramidal
cells which were healthy with clear nucleus [Table/Fig-4]. Group II
(30 min exposure for 3 months) showed less number of pyramidal
neurons With darkened nuclei (non-viable neurons) which was

Krishna Kishore G et al., Effect of 1800-2100 MHz Electromagnetic Radiation on Learning-Memory and Hippocampal Morphology www.jcdr.net
Journal of Clinical and Diagnostic Research. 2019 Feb, Vol-13(2): AC14-AC17
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scattered when compared to control group [Table/Fig-5]. Group
III (60 min exposure for 3 months) showed very less number of
pyramidal neurons with more number of darkened nuclei (more
non viable neurons) with vacuolation in cytoplasm and scattered
arrangement of pyramidal neurons when compared to group I and
group II [Table/Fig-6].
effects on the brain especially on the hippocampus, which is a sensitive
region on the temporal lobe of the brain responsible for spatial learning
and working memory, an important cognitive function [7].
In this study, Hebb-Williams maze analysis was used to assess
the learning and memory in albino mice exposed to mobile phone
radiation frequency and control group [10,11]. In the present study,
MP RF-EMR exposed mice took significantly increased time to reach
the target chamber in Hebb-Williams maze when compared to the
control group, which shows memory retention and memory retrieval
is being affected and leads to memory impairment in the mice.
Studies have shown that RF EMR exposure will impair the learning
and memory, which may be due to neurodegenerative changes and
alterations in the morphology of the hippocampus [7,8].
On histological examination, radiation exposed hippocampal CA3
neurons showed less number of pyramidal cells with darkened
nuclei (Non viable), vacuolated cytoplasm and cells were scattered
in arrangement. The altered structural integrity in the hippocampus
might be the cause for impairment of learning memory. Decrease
in pyramidal cell count may be due to inhibition of neurogenesis
and this was supported by Odaci E et al., [12]. Bolla SR reported
that exposure to 800 MHz mobile radiation for 30 days leads to
increased neuronal damage and decreased viable neurons in
hippocampal CA3 region [9].
Nittby H et al., reported that exposure to 900 GSM radiation will
reduce memory functions in rat, which is similar to our study [1].
MP RF exposure to 900-1800 MHz radiation leads to decrease in
nuclear diameter and reduce neuronal density in the hippocampus
[13]. Findings on exposure to 50-217 Hz low frequency radiation
with television and mobile phone have impact on learning and
memory [14]. Fragopoulou AF et al., reported that consolidation
and retrieval memory deficits were observed in mice exposed to
9 hr 30 mins for 4 days with 900 MHz non ionising radiation [15].
Heat shock proteins-HSP 27 and HSP 70 related stress levels are
elevated in rat hippocampus exposed to 2450 MHz radiation [16].
A 2.14 GHz Radiation frequency exposure at 4 Watt/kg specific
absorption rate increases the body temperature to 1.5°C compared
to baseline and upregulates some stress markers like HSP and Heat
Shock Transcription Factor (HSF) gene expressions in cerebellum
and cerebral cortex [17].
LIMITATION
The outcome of the present rodent study may not be extrapolate
with human population due to many reasons like Thickness of the
skull bone, Weight/Volume of the brain, Specific Absorption Rate
(SAR), Duration of exposure, Frequency of radiation and Lifespan of
the human population.
CONCLUSION
In this present study, we evaluated the chronic exposure effect
of MP RF-EMR- 4G (1800-2100 MHz) on cognitive functions like
spatial learning, working memory and hippocampal morphology in
adult swiss albino mice. We observed that MP RF-EMR exposed
mice took significantly increased time to reach the target chamber
in Hebb-Williams maze when compared to the control group.
Radiation exposed hippocampal CA3 neurons showed less number
of pyramidal cells with darkened nuclei (Non viable), vacuolation in
cytoplasm and cells were scattered in arrangement. The altered
structural integrity in the hippocampus may alter the spatial learning
and memory.
ACKNOWLEDGEMENTS
We thank Dr. Sarala N Prof., Department of Pharmacology and
Incharge-Animal house facility, Sri Devaraj Urs Medical College,
Kolar for providing animal house to carry out this doctoral study, we
thank Dr. Muninarayana Veterinarian for his suggestions during this
study and we also thank Mr. Ravi Shankar, Statistician for his help.
[Table/Fig-3]: Effect of Mobile phone radiofrequency-electro-magnetic radiation (MP
RF-EMR) on learning and memory by using Hebb-Williams maze- group I, II and III.
[Table/Fig-4]: Group I (non-exposed)-Control group H and E stained Hippocampal
pyramidal normal neurons (Arrow) in high power (40X)
[Table/Fig-5]: Group-II (30 min exposed for 3 months) H and E stained Hippocampal
pyramidal neurons (Arrow) in high power (40X) showed less in number, Non-Viable and
scattered with vacuolation in cytoplasm.
DISCUSSION
With advancement of technology like 2G to 3G, 3G to 4G in the
telecommunication field, the mobile phones are being used for
communication, internet data and as multipurpose device. However
over usage of mobiles with advance multiple features has adverse
[Table/Fig-6]: Group- III (60 min exposed for 3 months) H and E stained Hippocampal
pyramidal neurons (Arrow) in high power (40X) showed very less in number, Non-Viable
and scattered with vacuolation in cytoplasm

www.jcdr.net Krishna Kishore G et al., Effect of 1800-2100 MHz Electromagnetic Radiation on Learning-Memory and Hippocampal Morphology
Journal of Clinical and Diagnostic Research. 2019 Feb, Vol-13(2): AC14-AC17 1717
PARTICULARS OF CONTRIBUTORS:
1. Ph.D Scholar, Department of Anatomy, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka, India.
2. Professor, Department of Anatomy, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka, India.
3. Professor and Head, Department of Anatomy, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka, India.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR:
G Krishna Kishore,
Department of Anatomy, Sri Devaraj Urs Medical College, Tamaka, Kolar, Karnataka, India.
E-mail: krishnakishore.dev@gmail.com
FINANCIAL OR OTHER COMPETING INTERESTS: None.
Date of Submission: Sep 18, 2018
Date of Peer Review: Nov 23, 2018
Date of Acceptance: Dec 31, 2018
Date of Publishing: Feb 01, 2019
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