Chronic stress induced tissue malondialdehyde level in amygdala nucleus lesioned wistar rat.

Abstract

Chronic Stress has significant adverse effects on health and is a risk factor for many illnesses. The idea
that the brain categorizes stressors and uses response pathways that vary according to the category has
gained significant support in the recent years. The present study was designed to elucidate the possible role
of amygdala nucleus on chronic physical and chronic psychological stress induced tissue lipid
peroxidation level. Adult Wistar albino rats were divided into three major groups normal, sham lesioned
and amygdala lesioned. Each group was further subdivided into control groups and stress groups. In each
group ten animals were taken. The amygdala lesion was performed according to the stereotaxic
coordinates by Paxinos and Watson. The stressed group of rats were subjected to chronic swimming
(Physical) and chronic immobilization (Psychological) stress for 60 days. Tissue MDA levels (nanomoles
of MDA/g wet tissue) was estimated by Kartha and Krishnamurthy method. MDA levels of heart, liver and
kidneys were estimated. The stress induced increase in MDA level in amygdala lesioned rats was
significantly more (P<0.001) during immobilization stress than swimming stress. The data of the present
study support the hypothesis that the brain recognizes at least two major categories of stressor, which has
been referred to as physical and psychological. Further, our study also provides the supportive evidence
that the response of a psychological stress to a greater extent is regulated by amygdala nucleus.

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European Journal of Biotechnology and Bioscience 2014; 2 (1): 57-60
ISSN: 2321-9122
www.biosciencejournals.com
EJBB 2014; 2 (1): 57-60
Received: 31-7-2014
Accepted: 12-8-2014
Nayanatara AK
Departments of Physiology, Center for
Basic Sciences, Kasturba Medical
College, Bejai, Mangalore, Manipal
University, Karnataka, India.
Tripathi Y
Department of Physiology, Santhosh
Medical College, Ghaziabad
Nagaraja HS
Discipline of Physiology and
Pharmacology Faculty of Medicine,
Health and Molecular Sciences, James
Cook University, Australia.
Jeganathan PS
Departments of Physiology,
A.J. Institute of Medical Sciences,
Mangalore, Karnataka, India.
Ganaraja B Sheila R Pai
Departments of Physiology, Center for
Basic Sciences, Kasturba Medical
College, Bejai, Mangalore, Manipal
University, Karnataka, India
Arun Kumar B
Departments of Chemistry, Mahatma
Ghandhi Memorial College Udupi,
Karnataka, India.
Nayanatara Arun Kumar
Associate Professor, Department of
Physiology, Kasturba Medical
College (Manipal University), Bejai,
Mangalore- 575004, Karnataka,
India
Chronic stress induced tissue malondialdehyde level in
amygdala nucleus lesioned wistar rat
Nayanatara AK, Tripathi Y, Nagaraja HS, Jeganathan PS,
Ganaraja B, Sheila R Pai, Arun Kumar B
Abstract
Chronic Stress has significant adverse effects on health and is a risk factor for many illnesses. The idea
that the brain categorizes stressors and uses response pathways that vary according to the category has
gained significant support in the recent years. The present study was designed to elucidate the possible role
of amygdala nucleus on chronic physical and chronic psychological stress induced tissue lipid
peroxidation level. Adult Wistar albino rats were divided into three major groups normal, sham lesioned
and amygdala lesioned. Each group was further subdivided into control groups and stress groups. In each
group ten animals were taken. The amygdala lesion was performed according to the stereotaxic
coordinates by Paxinos and Watson. The stressed group of rats were subjected to chronic swimming
(Physical) and chronic immobilization (Psychological) stress for 60 days. Tissue MDA levels (nanomoles
of MDA/g wet tissue) was estimated by Kartha and Krishnamurthy method. MDA levels of heart, liver and
kidneys were estimated. The stress induced increase in MDA level in amygdala lesioned rats was
significantly more (P<0.001) during immobilization stress than swimming stress. The data of the present
study support the hypothesis that the brain recognizes at least two major categories of stressor, which has
been referred to as physical and psychological. Further, our study also provides the supportive evidence
that the response of a psychological stress to a greater extent is regulated by amygdala nucleus.
Keywords: Amygdala, Lesion, MDA, Immobilization, Swimming, stress.
1. Introduction
In the modern era, stress has undoubtedly become an integral part of human life. Literature survey
shows that stressful conditions alter physiological homeostasis leading of variety diseases.
Experimental models are required to better understand the role of brain nucleus in stress and
elaborate new therapy. Oxidative stress due to physical as well as psychological forms of stress
has become the focus of interest due to its deleterious effects on physiological systems.
Forebrain structures are known to influence stress responses [1]. Among them, the amygdala has
attracted continued interest because of its central role in processing emotional information [2].
Literature survey shows the involvement of amygdala in autonomic responses to stress [3, 4]. The
role of specific brain nucleus in response to the type of stressors has remained unanswered to this
date. It is still a matter of debate that whether the brain recognizes two major categories of
stressor, which were referred as ‘physical’ and ‘psychological’ based on the type of nucleus
which is activated. Elevated levels of malondialdehyde (MDA) one of the by-products of lipid
peroxidation has been reported in the cardiovascular, neurological and in other diseases [5]. There
is a lack of information available in literature on the precise influence of the amygdala on chronic
stress-induced formation of lipid peroxidation level in different tissues .The present study was an
attempt to elucidate the comparative analysis of regulatory role of amygdala nuclei in regulating
physical and psychological stress.
2. Materials and methods [6]: All procedures in this present study were performed in accordance
with the guidelines established by the Institutional Animal Ethics Committee and of the Society
for Neuroscience Policy on the Use of Animals in Research. Adult albino rats (150 to 250 g) of
Wistar strain were used in the present study. The rats were procured from the central animal
breeding center at our university. Animals were housed individually in polypropylene cages (29
cms x 22 cms x 14 cms) during the experimental period at 28±2 0C temperature and 50±5%
humidity. The rats were maintained under standard laboratory conditions with 12 h light: 12 h
dark cycle. Rats were anesthetized (Pentobarbital sodium, 40 mg/kg, i. p.) and then sacrificed by
giving the lethal dose of Pentobarbital sodium. The amygdala lesion was performed according to
the stereotaxic coordinates prescribed in the Paxinos and Watson rat stereotaxic atlas [7]. The

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procedure was repeated on either side to produce bilateral
lesions. The surgical procedures performed in the sham lesioned
control group were the same, except for the passing of the DC.
The animals were divided into three major group. Each group
was further subdivided into three subgroups as control group
and two experimental stress groups. Each subgroup contained
ten animals. The stressed groups of rats were subjected to
swimming stress and immobilization stress. All the stress
experiments were done between 10 AM to 12 Noon to minimize
circadian variability.
2.1 Chronic Stress Procedure
Chronic Immobilization stress [8, 9]: The immobilization
chambers used in this study were plastic tubes of varying sizes
to accommodate all sizes of rats (15 cms long and 4 cms
diameter, 16 cms long and 5cms diameter, 17 cms long with 6
cms diameter). The tubes had a conical head at one end. The
conical head area contained numerous perforations which
served as breathing holes. The rat was placed inside the tube
with head in the conical end. The rats were totally restrained by
packing the rear end of the tube and closing it firmly with a
stopper. Rats were exposed to chronic stress in the form of
immobilization for 2 hour per day for a period of 60 days.
Chronic swimming stress [10]: The rats were allowed to swim in
the plastic tubs containing tap water maintained at room
temperature. The water level in the plastic tub was always kept
at 30 cms from the bottom. Rats were forced to swim in this tub
until exhaustion. The point at which the animals became unable
to stay at surface and showed signs of sinking was considered to
be the point of exhaustion. After the stress session, the rats were
towel dried and then placed back in their respective cages where
water and food were available ad libitum. Animals were
subjected to force swimming daily for 60 days.
2.2. Grouping of Animals
Normal Control (NC) - This subgroup of normal rats was not
subjected to any kind of stress
Normal chronic Swimming stress- (N-CS)- This subgroup of
normal rats was subjected to chronic swimming stress daily for
sixty days.
Normal chronic Immobilization stress- (N-CI) - This
subgroup of normal rats was subjected to chronic
Immobilization stress daily for sixty days.
Amygdala sham lesioned control (ASL-C) - This group of rats
received the same surgical procedure for lesioning of amygdala
as mentioned above except for passing of the DC (direct)
current and these animals were not subjected to any kind of
stress.
Amygdala sham-lesioned chronic swimming stress (ASL-SS)
- This group of rats was subjected to sham lesion at amygdaloid
nucleus and underwent chronic swimming stress daily for sixty
days.
Amygdala sham-lesioned chronic immobilization stress
(ASL-IS)- This group of rats was sham lesioned at amygdaloid
nucleus and underwent chronic immobilization stress daily for
sixty days.
Amygdala lesioned control (AL-C)- This subgroup of
amygdala lesioned rats was not subjected to experimental stress.
Amygdala lesioned chronic swimming stress (AL-SS)- This
subgroup of rats was lesioned at amygdala nucleus and
subjected to chronic swimming stress for sixty days.
Amygdala lesioned chronic immobilization stress (AL-IS) -
This subgroup of rats were lesioned at amygdala nucleus were
subjected to chronic immobilization stress one hour per day for
sixty days.
2.3. Preparation of the tissue homogenate: At the end of
experiment, all rats were anaesthetized with sodium
pentobarbital (40 mg/kg body weight). heart, liver, kidney, were
quickly removed. Each of these tissues was separately
transferred to a glass homogenizer containing 10 ml of 10 mM
cold phosphate buffer saline (PBS - pH 7.4). The tissues were
homogenized using an electrical homogenizer (Remi 8000
RPM). The unbroken cells and cell debris were removed by
centrifugation at 3000 RPM for 10 minutes by using Remi C 24
refrigerated centrifuge (-4 °C). The obtained supernatant was
used for estimation of Lipid Peroxidation:
2.4. Estimation of Lipid Peroxidation: Lipid peroxidation was
estimated according to the method of Kartha and Krishnamurthy
[11]. This assay is based upon the reaction of TBA with
malondialdehyde (MDA) which is one of the aldehyde products
of lipid peroxidation. Five ml of the homogenate (freshly
prepared) was incubated in 50 ml conical flask at 38-39 °C for
30 minutes along with “the blank” in a water bath in separate
conical flasks. After incubation, 1ml of aliquots was added to
the tube containing 1.5 ml of 20% cold trichloroacetic acid
(TCA) and then centrifuged for 10 minutes. After
centrifugation, 2 ml of supernatant fluid was taken in a test tube
and to it was added 2 ml of 0.7% thiobarbituric acid (TBA) and
kept in the boiling water bath for 10 minutes. The development
of pink color was measured at 535 nm by using Spectronic D-20
Spectrophotometer. TBA reactive material was expressed in
terms of nanomoles of malondialdehyde (MDA)/g wet tissue,
taking molar extinction coefficient of malondialdehyde (MDA)
as 1.56 X 105.
2.5. Histological Analysis: On completion of the experimental
procedures, all the rats were sacrificed and their brains, after
transcardial formol saline injection, were dissected and
processed for histological study. Serial sections 5 µ thickness
were taken and stained with Haematoxylin and eosin (H&E) [12]
(Fig. 1). These sections were examined under dissection
microscope to confirm the lesion and the lesion sites were again
magnified under power 10X for a detailed view.
2.6. Statistical Analysis: The datas were summarized using
mean ± SEM or median and interquartile range depending on
the skewness. For normally distributed data one way ANOVA
was used and Kruskal Wallis test was used for skewed data.
This was followed by multiple comparison tests for significant
F value in ANOVA. The data of pre and post lesion was
analyzed using two ways ANOVA followed by post hoc tests in
case of significant F value of ANOVA. P<0.05 were considered
as statistically significant.
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European Journal of Biotechnology and Bioscience
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Figure 1.
Table 1: Swimming stress and Immobilization stress induced changes on tissue MDA following swimming stress and immobilization
stress. Values are expressed as meanSEM; nanomoles of MDA/g wet tissue.
Groups Heart Liver Kidney
NC 9.5860.788 20.187

0.514 14.2910.439
N-SS 20.220.86** 39.26

0.20** 20.120.88**
N-IS 18.800.22** 40.22

1.20** 22.910.54**
ASL-C 9.200.30NS 21.01

0.20 NS 14.120.22 NS
ASL-SS 21.4381.61 ‡ ‡ 41.425

0.703 ‡ ‡ 21.2910.633 ‡ ‡
ASL-IS 18.8650.49 ‡ ‡ 40.789

1.30 ‡ ‡ 21.210.95 ‡ ‡
AL-C 10.2440.36 19.70

2.27 18.1340.84
AL-SS 19.702.27§§ 40.88

0.55§§ 20.1340.84§§
AL-IS 23.6200.487§§# # 49.290

0.98§§# # 30.0100.56§§# #
n= number of rats
NC versus ASL-C – Non significant (NS)
** P < 0.001; compared between NC versus N-SS and N-IS
‡ ‡ P < 0.001; compared between ASL-C verus ASL-SS and ASL-IS
§§ P < 0.001; compared between AL-C verus AL-SS and AL-IS
# # P< 0.001; AL-SS versus AL-IS
3. Results: It is evident from the table I that the MDA level of
the heart, liver and kidney did not so any significant changes in
between the NC and ASL-C groups. Exposure to chronic
stressors significantly (P<0.001) increased the tissue MDA level
in the normal group, sham lesioned groups and lesioned groups
when compared to their respective control group. Further,
Exposure to immobilization stress in amygdala lesioned groups
showed a significant increase (P<0.001) in the MDA level of
heart, liver and kidneys when compared to amygdala lesioned
swimming stress groups.
4. Discussion: The biochemical manipulations in the body are
good markers that can be of great use to understand, target and
manage stress induced etiologies. The available literature
suggests that the stress regulatory circuits are activated by a
particular stressor which is crucially dependent on stimulus
attributes [13]. The central component of stress responses have
been recently redefined and presumed that various categories of
stressors have been handled to some extent in different ways [14].
Recent reviews have suggested the existence of two generalized
stress pathways namely limbic sensitive and limbic insensitive
[15, 16]. Although the stressors trigger a core set of endocrine,
autonomic and behavioral responses, the brain pathways that
drive these responses might differ according to the stressor
category. The nature of the stressor itself is important in the
pattern of expression of stress responses. The core element of
the physiological responses is broadly similar and it appears that
they are generated by the neural pathways that may vary
according to the stressor category. A number of pathological
conditions have been implicated due to free radical formation
[17]. Therefore, lipid peroxidation was estimated in different
brain tissues in order to evaluate their relative role in stress
induced regulatory mechanisms. Limbic and hypothalamic
brain structures integrate emotional, neuroendocrine, and
autonomic inputs that determine the magnitude, neural and
hormonal response to stressful experiences [18]. Studies have
also showed that amygdala nucleus is the “window of limbic
system” through which exteriorization of emotional
manifestation takes place, plays an important role in the
organization of stress response [19, 20]. In the present study, no
significant difference was observed in any of the parameters in
between the sham lesioned groups and the normal groups. This

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finding confirms that surgical procedure did not affect the
studied parameters of the study. Exposure to stress situations
can stimulate numerous pathways leading to increased
production of free radicals [21]. It is well known that free radicals
generate a cascade producing lipid peroxidation, protein
oxidation, and DNA damage and cell death [22]. Stress induced
oxidative metabolism on the body depends upon the
involvement of various tissues. Hence, the estimation of these
oxidative metabolic products on certain tissues was also
included in this study. In the present study, MDA level of the
tissues were estimated by TBARS (thiobarbituric acid reactive
species) assay to evaluate lipid peroxidation level in heart, liver
and kidneys, following exposure to two different models of
acute and chronic stress. This assay is the easiest method used
to study the effects of different treatments on lipid peroxidation
and can be applied to crude biological extracts [23, 24, 25]. The
results of the present study showed that swimming stress
significantly increased the MDA level in the heart, liver and
kidneys in normal groups and sham lesioned groups and
lesioned groups. Swimming stress elevates oxygen consumption
and metabolic activity due to increased muscle contraction [26].
Elevated oxygen consumption during increased metabolic
activity increases the electron leakage from the mitochondrial
transport system and causes an increase in oxidative stress, lipid
peroxidation and generation of free radicals, which can be
harmful to the cell [27]. The results of the present study is in
agreement with the previous reported studies [28, 29] as swimming
stress significantly increased oxidative free radical formation.
Although the effect of acute physical stress on free radical
production and antioxidant system in rats have been intensively
investigated, the precise effect of repeated physical stress on the
different organs is yet to be confirmed as the reported studies
have shown contradictory results by the investigators [29, 30].
In recent years it has been shown that psychological stress may
change the balance between pro-oxidants and antioxidant
factors inducing oxidative damage30. Immobilization stress
which is widely employed to provoke psychological stress has
also been reported to be associated with oxidative damage in
rats [30]. In the present study, repeated immobilization stress
increased the tissue lipid peroxidation level as observed in the
previous studies [30, 31]. Repeated psychological stress on daily
basis might lead to oxidative damage by changing the balance
between oxidant and antioxidants factors. The results of the
present study suggest the involvement of amygdala nucleus in
minimizing the stress induced formation of free radicals.
Exposure of stress, increased the tissue MDA level. Literature
survey show that the AVP (Arginine –Vasopressin) released
within the amygdala seems to be involved in the generation of
passive copying strategies in stressful situation during stress [32].
Due to the loss of stress coping strategies, stress might have
caused increased formation of free radicals. The present study
supports the definite role amygdala in stress tolerance. This
shows that amygdala nucleus appears to play a potent role in
minimizing the stress induced formation of lipid peroxidation
during an exposure to the psychological type of stress rather
than a physical type of stress like swimming.
The present study provides the supportive evidence that the
response of a psychological stressor to a larger extent is
regulated by the amygdala. There might be the presence of an
intricate web sf reciprocal independent connections of amygdala
nucleus to the brain areas regulating these responses designed
for the psychological stress. However, the precise role of these
nuclei and their interaction among themselves and other brain
areas in regulating homeostasis warrants further study.
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