Stress, 2012; Early Online: 111

q Informa Healthcare USA, Inc.

ISSN 1025-3890 print/ISSN 1607-8888 online
DOI: 10.3109/10253890.2012.719052

Chronic stress-induced oxidative damage and hyperlipidemia are

accompanied by atherosclerotic development in rats

M. DEVAKI, R. NIRUPAMA, & H. N. YAJURVEDI

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Department of Zoology, University of Mysore, Manasagangotri, Mysore, India

(Received 12 January 2012; revised 1 August 2012; accepted 3 August 2012)

Abstract
Although stress-induced hyperlipidemia and increased oxidative stress have been reported and implicated in etiology of
atherosclerosis, experimental evidence for stress-induced atherosclerotic development concomitant with these alterations is
lacking. In this study, exposure of adult male albino Wistar rats (Rattus norvegicus) to restraint for 1 h and after a gap of 4 h to
forced swimming for 15 min every day for 2, 4, or 24 weeks resulted in a duration of exposure-dependent hyperlipidemia as
shown by significant increases in concentrations of blood cholesterol, low-density lipoproteins, and triglycerides and decrease
in high-density lipoprotein concomitant with increased oxidative stress as indicated by decrease in hepatic antioxidant enzyme
activities and increase in lipid peroxidation in the liver, kidney, and heart. These alterations were accompanied by
development of fibrous layer, formation of foam cells, reduction in elastic fibers, and accumulation of Oil-Red-O-positive lipid
droplets in the intima of thoracic aorta following 24 weeks of stress exposure, but not after 4 weeks. The study demonstrates
for the first time that (i) chronic stress-induced hyperlipidemia and oxidative damage are coupled with atherosclerotic
development in rats fed with normal diet and (ii) chronic stress effects prevail even after the cessation of stress exposure as
indicated by high concentration of blood cholesterol and reduced hepatic superoxide dismutase activity 20 weeks after 2 or
4 weeks of stress. This study exemplifies long-term allostatic regulation leading to a pathological state, with long-term
hyperlipidemia and oxidative damage from chronic stress resulting in atherosclerosis.

Keywords: Aorta, atherosclerosis, foam cells, glucocorticoid, hyperlipidemia, oxidative stress

Introduction
Stress is a state of altered homeostasis, and the ability
to cope with stress is a crucial determinant of health
and disease. Chronic stress is known to alter the blood
lipid profile. A significant elevation in circulating
concentrations of cholesterol, low-density lipoproteins
(LDLs), very-low-density lipoproteins (VLDL), and
triglycerides (TGs) following immobilization in
rabbits (Lata et al. 2004) and apoE2/2 mice (Gu
et al. 2009) has been reported. Decreased highdensity lipoprotein (HDL) concentration following
exposure to immobilization (Ruiz de Gordoa et al.
1994), chronic unpredictable stressors (Nayanatara
et al. 2009) and restraint (Akhtar et al. 2008) in rats
has been found. Similarly, emotional stressors such
as workload problems, depression, anger, and social
isolation cause alterations in the blood lipid profile in

humans (Richards et al. 2000). In addition, preferential expression of genes related to lipid metabolism
following immobilization in rats has also been
reported (Sato et al. 2006). Furthermore, stress also
causes leakage of cytoplasmic enzymes, glutamate
pyruvate transaminase (GPT), and glutamate oxaloacetate transaminase (GOT) into the blood in humans
(Kayashima et al. 1995) and laboratory animals
(Sen et al. 1992; DSouza et al. 2004), indicating
tissue damage. Significant increase in the weight of the
heart and adrenal glands, heart rate, and activities of
serum glutamate pyruvate transaminase (SGPT)
and serum glutamate oxaloacetate transaminase
(SGOT) was observed following restraint (Sen et al.
1992) and isolation or immobilization (DSouza et al.
2004) in rats.
Moreover, stress also causes an increase in the production of free oxygen radicals (Lakshmi et al. 2009).

Correspondence: H. N. Yajurvedi, Department of Zoology, University of Mysore, Manasagangotri, Mysore 570 006, India. Tel: 91 0821
2419778. Fax: 91 0821 2419363/2419301. E-mail: hnyajurvedi@rediffmail.com

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M. Devaki et al.

An imbalance between oxidants and antioxidants has

been implicated in various disorders including
neurodegenerative diseases, gastric ulcerogenesis,
and impaired cognitive function (Nadeem et al.
2006). Several studies have shown altered antioxidant
status due to stress. For instance, restraint, forced
swimming, exposure to flashing light, isolation
(Manoli et al. 2000), and repeated restraint
(Nayanatara et al. 2005; Bhat et al. 2007; Zafir and
Banu 2009) increase lipid peroxidation and decrease
activities of antioxidant enzymes in rat brain.
However, Bhattacharya et al. (2001) reported
increased superoxide dismutase (SOD) activity
though there was a decrease in the activities of
catalase and glutathione peroxidase (GPx) following
foot shock in the brain of rats. In addition, an increase
in the concentration of thiobarbituric acid reactive
substances in the cerebral cortex (Fontella et al. 2005)
and levels of plasma peroxides (Nadeem et al. 2006) in
rats has been observed following restraint and
immobilization.
Adaptive responses of the body to stressors have
been termed allostasis (McEwen 2002), which
involves compensatory alterations in physiological
variables, overriding local feedback (Sterling 2004).
Allostatic regulation is through mediators produced
by the hypothalamic pitutary adrenal (HPA) axis,
autonomic nervous system, and immune system. With
prolonged stress, the mediators of allostasis are
considered to produce wear and tear of the body, or
allostatic load (McEwen 2002). This includes
stress-induced alterations in blood lipid profile and
oxidative damage. However, whether long-term
alterations in lipid profile concomitant with altered
antioxidant status lead to severe consequence such as
atherosclerosis is not clear, although hyperlipidemia
(Rainey et al. 1992) and oxidative stress (Lakshmi
et al. 2009) induced by conditions other than stress
can cause atherosclerosis. In earlier studies, stressors
such as isolation in cynomolgus monkeys (Macaca
fascicularis; Shively et al. 1989) and social deprivation
in male Apo2/2 mice for 20 weeks (Bernberg et al.
2008) resulted in atherosclerotic development. However, stress alone was not implicated in atherosclerotic
development in these studies because the monkeys
were fed an atherogenic diet coupled with isolation,
and the mice were of a mutant strain. Kaplan et al.
(1983) reported development of coronary arterial
atherosclerosis, without alterations in serum lipids
following social stress in monkeys and suggested that
psychosocial factors may influence atherogenesis in
the absence of hyperlipidemia. However, whether
oxidative stress was also evident in chronically stressed
monkeys was not investigated (Kaplan et al. 1983).
Furthermore, rats subjected to chronic unpredictable
stress for 15 days showed hypertrophy of the intima
and tunica media of thoracic aorta, increasing serum
lipid concentration and atherogenic index without

changes in HDL concentration (Neves et al. 2009),

although there was no evidence for atherosclerotic
plaques and oxidative damage was not investigated.
This study investigated the hypothesis that chronic
intermittent stress can induce alterations in blood lipid
profile and antioxidant status that are dependent on
duration of stressor exposure and accompanied by
atherosclerotic development in the thoracic aorta. The
study tested whether stress-induced changes in lipid
profile and antioxidant status are reversible.
Materials and methods
Animals
Adult male Wistar rats weighing 180 200 g obtained
from the central animal facility, University of Mysore,
were housed two or three rats per cage in a 12:12-h
lightdark cycle (lights on 07:00 19:00 h), temperature 27 ^ 28C. The rats had free access to standard rat
chow and water ad libitum. All experiments were
conducted in accordance with standard ethical
guidelines and were approved by the institutional
animal ethics committee.
Stressors
Two kinds of stressors were used (Grissom et al.
2008): (i) restraint in a Plexiglas cylindrical restrainer
of 22.3 cm long, 6.7 cm diameter, closed with a
perforated lid, for 1 h per episode and (ii) forced
swimming for 15 min per episode in a glass jar of
45.7 cm high, 22.2 cm outer diameter, water depth of
30 cm, and temperature of 27 ^ 28C.
Experimental plan and procedures
At the commencement of the experiment, 37 adult
male rats were randomly chosen from the colony and
weighed (Figure 1). Five randomly chosen rats were
killed by exposing to ether (anesthetic ether, I.P.
Hydroquinone 0.002% w/v) to obtain baseline data
(initial controls). The thorax and abdomen were
opened, a blood sample was collected with a syringe
from the vena cava, and the liver, kidney, heart, and

Randomly selected
adult male rats, n=37

Stress groups

Control groups
Initial controls
killed day 0, n=5

2 weeks stress,
killed, n=5

4 weeks stress,
killed, n=5

No stress, killed
week 24, n=5

2 weeks stress
+recovery, killed
+22 weeks, n=5

4 weeks stress
+recovery, killed
+20 weeks, n=5

24 weeks stress,
killed, n=7

Figure 1. Experimental groups showing period of stress exposure

or recovery. Blood and tissue samples were collected at autopsy.

Biochemical analyses
Blood samples were centrifuged at 604 g for 15 min,
and plasma was separated and stored at 2 208C.
Plasma concentrations of cholesterol (assay sensitivity
0.14 mmol/l), HDL (sensitivity 2.5 mg/dl) and TGs
(sensitivity 0.4 mmol/l) and activities of plasma GOT
(sensitivity 350 U/l) and lactic dehydrogenase
(LDH; sensitivity 2400 U/l) were determined using
kits (Agappe diagnostics, Kerala, India). Activity of
plasma glutamate pyruvate transaminase was determined following the method of Segal et al. (1962;
sensitivity 0.30 U/l). Plasma concentrations of LDL
and VLDL were determined by the method of
Friedewald et al. (1972). Adrenal glands were
homogenized to estimate the activity of 3b-hydroxysteroid dehydrogenase (3b-HSDH) by measuring the
rate of conversion of pregnenolone into progesterone
(Shivanandappa and Venkatesh 1997; sensitivity
0.04 nmol/mg of protein). Liver homogenate (10%)
was used to determine the activities of g-glutamyl
transferase (GGT; Rosalki and Tarlow 1974; sensitivity 9 U/l), SOD (Marklund and Marklund
1974; sensitivity 16 unit/mg protein), glutathioneS-transferase (GST; Habig et al. 1974; sensitivity
11 U/mg of protein), glutathione reductase (GR;
Carlberg and Mannervik 1985; sensitivity 250 U/mg
of protein), catalase (Aebi 1984; sensitivity 0.25
4 nmol/min/ml), and GPx (Tappel 1978; sensitivity
0.5 mU/ml). The concentration of malondialdehyde
in the liver, heart, and kidney was determined
(Ohkawa et al. 1979; sensitivity 4 nmol/mg protein).

Intra- and interassay variability tests were conducted

for all the assays, and variability was in the range of
3 5%.
Histology of aorta
A portion of the thoracic aorta (aortic root) was
removed at autopsy and fixed in 10% buffered
formalin. The fixed aorta was stained with Sudan IV
to reveal sudanophilic plaques (Holman et al. 1958).
After Sudan IV staining the aorta was embedded in
paraffin wax, 5-mm-thick transverse sections were cut
and stained with hematoxylin eosin. Lipid deposition
and elastic fibers and collagen in the blood vessel
were detected using Oil Red O (Preece 1972) and
van Gieson staining (Culling 1963), respectively.
The measurements of intima thickness (IT), media
thickness (MT), total intima media thickness (IMT),
and wall thickness were made in hematoxylin eosin
stained sections of thoracic aorta under a microscope
(100 magnification; Olympus BX41, Tokyo, Japan)
using ProgResw CapturePro 2.7 software. The intima
limit was the zone between the luminal surface and the
internal elastic lamina, and a lesion comprised the
entire intima including lipid cores and fibrotic
components. The distance between the lumen intima
interface and the media adventitia interface was the
IMT. The total thickness of the aortic wall was the
distance between the endothelium lining and the
adventitia. Thickness of the different layers was
measured in five randomly chosen areas per section

(A)

70
24 weeks no stress
60

Mean % change in body weight SEM

adrenal glands were removed. Blood was transferred

to an eppendorf tube containing 0.1% ethylenediaminetetraacetic acid, and plasma was separated by
centrifugation. Biochemical analyses were carried
out to obtain baseline data. Remaining rats were
randomly divided into two groups, the no stress
group (controls; n 05) and the stress group (n 25).
Rats in the stress group were restrained for 1 h, and 4 h
later they were subjected to forced swimming for
15 min at random times between 07:00 and 19:00 h,
every day for 24 weeks. To minimize habituation,
the sequence of stressors and timing of stress exposure
were randomly changed every day. Five rats after
2 weeks exposure, and five rats after 4 weeks exposure
in the stress group were killed, as above. The
remaining rats were killed after 24 weeks exposure to
the stressors, and the no stress controls were killed
at the same time. Five rats exposed to the stressors
for 2 weeks and five rats exposed for 4 weeks were
then maintained without exposure to stressors
(recovery groups) until the 24th week when they
were killed. Blood samples and tissues were collected
as above. Postmortem body weight and adrenal gland
weight were recorded. Rats were killed 16 h after the
last exposure to a stressor.

2 weeks stress

50

4 weeks stress

40

24 weeks stress

30
*

20

10

Recovery after
2 weeks stress
Recovery after
4 weeks stress

0
10
20
30

40
(B)
Mean relative weight (mg/100g body
weight) of the adrenal gland SEM

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Chronic stress and atherosclerosis

40
35

30
25
20
15
10
5

Initial controls
*

2 weeks stress
4 weeks stress

24 weeks stress
24 weeks no stress
Recovery after
2 weeks stress
Recovery after
4 weeks stress

Figure 2. Vertical bars are mean ^ SEM. Percentage change in

body weight compared to initial body weight (A) and relative adrenal
weight (B) in control, stressed, and recovery groups, n 5 rats per
group. *p , 0.05 versus controls, ANOVA, and Duncans test.

M. Devaki et al.
Table I. Stress and adrenal 3b-HSDH activity and plasma lipid profile.
Concentration (U/l)
Activity of 3b-HSDH
(mmol/mg/min)

Groups (n 5)
Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
4 weeks stress recovery, week 24
24 weeks no stress
F values (df 6,34)
p value

0.15 ^ 0.009
0.28 ^ 0.009b
0.39 ^ 0.01c
0.33 ^ 0.05bc
0.12 ^ 0.17a
0.11 ^ 0.01a
0.13 ^ 0.002a
17.02
p , 0.05

Cholesterol

LDL
a

43.1 ^ 1.7
60.9 ^ 2.5b
69.5 ^ 1.6c
82.2 ^ 2.6d
57.7 ^ 6.8b
58.4 ^ 1.2b
39.8 ^ 1.3a
32.84
p , 0.05

TG
a

31.3 ^ 1.8
42.3 ^ 2.2bc
50.3 ^ 0.96c
60.1 ^ 3.13d
42.8 ^ 7.4bc
40.4 ^ 1.9b
24.7 ^ 1.34a
18.84
p , 0.05

VLDL
a

58.6 ^ 2.1
92.7 ^ 2.1bc
97.6 ^ 3.3c
110.2 ^ 14c
73.5 ^ 6.7ab
64.1 ^ 2.4a
61.9 ^ 5.1a
7.61
p , 0.05

HDL
a

11.7 ^ 0.4
18.5 ^ 0.4bc
19.5 ^ 0.6c
22.0 ^ 2.8c
14.6 ^ 1.3ab
12.8 ^ 0.4a
12.3 ^ 1a
8.710
p , 0.05

33.4 ^ 1.8ab
28.9 ^ 1.3ac
24.0 ^ 1.2cd
20.5 ^ 0.7d
30.0 ^ 1.2ac
26.9 ^ 4.3ac
36.7 ^ 2.9b
7.861
p , 0.05

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Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. LDL, low-density lipoproteins; TG, triglycerides;
VLDL, very-low-density lipoproteins; HDL, high-density lipoproteins.

of the blood vessel (i.e. five readings/section/layer) and

from 10 randomly selected sections per rat.

Statistical analysis
Each parameter was expressed as the group mean ^
SEM. One-way analysis of variance (ANOVA)
followed by Duncans multiple test was used to detect
significant differences among groups; level for
significance was set at p , 0.05.

p , 0.05, Figure 2B) at 2, 4, and 24 weeks of stress

exposure compared to unstressed controls. The actual
weight of the adrenal gland showed a significant
increase in 4 and 24 weeks stressed rats, but not in rats
stressed for 2 weeks or in the recovery groups,
compared with controls (adrenal weight, mean ^
SEM, mg, were initial controls, 25.6 ^ 1.2, 2 weeks
stress, 36.2 ^ 1.3, 4 weeks stress, 52.4 ^ 3.6, 24 weeks
stress, 57.2 ^ 6.3, recovery after 2 weeks stress,
32.3 ^ 1.4, recovery after 4 weeks stress, 36.6 ^ 2.5,
no stress, 33.0 ^ 4.7, F value 10.65, df 6, 34,
p , 0.05).

Results
Body and adrenal weights

Adrenal 3b-HSDH activity

Gain in the body weight of rats exposed to stress for 4

or 24 weeks was significantly lower than controls, and
there was a loss in body weight after 2 weeks stress
(F 11.12, df 6, 34, p , 0.05, Figure 2A). Rats in
the recovery group gained body weight, but significantly less than controls (Figure 2A). There was a
significant increase in relative adrenal gland weight
(weight/100 g body weight) (F 17.75, df 6, 34,

Compared to controls (basal or no stress for 24 weeks),

there was a significant (ANOVA, p , 0.05; Table I)
increase in adrenal 3b-HSDH activity in rats exposed
to stress for 2 weeks, and further significant increases
after 4 or 24 weeks of stressor exposure; in the recovery
groups (no stress after 2 or 4 weeks exposure) adrenal
3b-HSDH activity at 24 weeks did not differ from that
in the unstressed controls at 24 weeks.

Table II. Stress and activities of hepatic antioxidant enzymes and GGT.
Activity
(mmol/mg/min)
Groups (n 5)

CAT activity
(nmol/mg/min)

Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
24 weeks stress recovery, week 24
24 weeks no stress
F values (df 6, 34)
p value

24.3 ^ 1.3a
13.4 ^ 1.5b
0.6 ^ 0.004c
0.4 ^ 0.008c
18.0 ^ 1.1bd
23.3 ^ 3.0a
20 ^ 1.8ad
62.5
p , 0.05

GPx
0.15 ^ 0.006a
0.13 ^ 0.006ab
0.094 ^ 0.003ab
0.027 ^ 0.02b
0.21 ^ 0.04a
0.19 ^ 0.13a
0.14 ^ 0.02ab
2.566
p , 0.05

GST
68.1 ^ 2.2a
43.3 ^ 3b
29.8 ^ 3.1c
8.95 ^ 1d
68.3 ^ 11a
74.7 ^ 5.1a
64.1 ^ 3.5a
39.46
p , 0.05

GR activity
(U/ml)

SOD activity
GGT activity
(U/mg protein)
(U/l)

46.9 ^ 0.66a
14.8 ^ 0.1b
15.8 ^ 4.2b
14.2 ^ 0.9b
39.6 ^ 4.9a
61.5 ^ 8.0c
43.2 ^ 2.0a
34.12
p , 0.05

20.3 ^ 0.2a
18.1 ^ 1.08ab
14.9 ^ 0.4bc
10.9 ^ 0.4d
12.6 ^ 1.2cd
14.7 ^ 0.7bc
20.8 ^ 2.7a
10.92
p , 0.05

47.6 ^ 9.31a
60.9 ^ 6.7b
65.5 ^ 7.7b
84.2 ^ 3.3c
59.6 ^ 5.08b
48.0 ^ 3.1a
46.7 ^ 6.1a
5.811
p , 0.05

Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. CAT, catalase; GPx, glutathione peroxidase; GST,
glutathione-S-transferase; GR, glutathione reductase; SOD, superoxide dismutase, GGT, g-glutamyl transferase.

Chronic stress and atherosclerosis

Table III. Stress and aminotransferases and LDH activities.

Activities (U/l)
Groups (n 5)
Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
4 weeks stress recovery, week 24
24 weeks no stress
F values (df 6, 34)
p value

Plasma LDH

Plasma GOT

226 ^ 11.5
240 ^ 27.9a
380 ^ 20.1b
401 ^ 12.1b
244 ^ 24.08a
281 ^ 37.2a
225 ^ 12.5a
14.06
p , 0.05

105 ^ 63.8
184 ^ 18.4a
549 ^ 37.01b
623 ^ 42.3b
249 ^ 22.7a
236 ^ 76.5a
240 ^ 18.2a
21.862
p , 0.05

Plasma GPT
8.56 ^ 0.6a
35.3 ^ 0.33c
40.6 ^ 0.6c
58.2 ^ 5.4d
27.1 ^ 1.5b
24.2 ^ 3.1b
9.4 ^ 0.8a
48.813
p , 0.05

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Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. LDH, lactate dehydrogenase; GOT, plasma
glutamate oxaloacetate transaminase; GPT, plasma glutamate pyruvate transaminase.

Blood lipid profile

HDL concentration was significantly decreased after
stress exposure for 4 or 24 weeks compared to the
24 weeks no stress or recovery groups (ANOVA,
Duncans, p , 0.05) (Table I). There was a significant
increase in total cholesterol and LDL (Table I)
concentrations after 2 weeks stressor exposure, with
further significant increases after 4 or 24 weeks
exposure (ANOVA, Duncans, p , 0.05). In the
recovery groups, total cholesterol and LDL concentrations were significantly less than in the
group stressed for 24 weeks but were greater than in
the unstressed controls (ANOVA, Duncans,
p , 0.05). TGs and VLDL concentrations were
significantly increased after stress exposure for 2, 4,
and 24 weeks compared to the initial control, no
stress, and recovery groups, with no significant
differences among groups stressed for 2, 4, or 24
weeks (ANOVA, Duncans, p , 0.05).
Activities of hepatic antioxidant enzymes and GGT
Hepatic CAT activity was significantly decreased after
stress exposure for 2 weeks and was further reduced

after 4 or 24 weeks treatment compared to initial

controls or the group unstressed for 24 weeks
(Table II); in the recovery groups, CAT activity was
not different from the group not exposed to stress
(ANOVA, Duncans, p , 0.05). Hepatic GPx activity
was significantly reduced after 24 weeks of stress
exposure compared to initial controls or the group not
exposed to stress for 24 weeks (ANOVA, Duncans,
p , 0.05), but was not changed after stress for 2 or
4 weeks, and was similar to controls in the recovery
groups. GST activity decreased progressively with
duration of stress exposure (ANOVA, Duncans,
p , 0.05) and returned to normal in the recovery
groups. GR activity was significantly and similarly
reduced in the groups stressed for 2, 4, or 24 weeks, as
compared with initial controls and the group not
exposed to stress for 24 weeks (ANOVA, Duncans,
p , 0.05); GR activity in the groups recovering from
stress increased to or above the control levels. SOD
activity decreased progressively with the duration of
stress exposure and did not return to normal in the
recovery groups (ANOVA, Duncans, p , 0.05).
GGT activity was significantly increased after stress
exposure for 2 or 4 weeks compared to controls and

Table IV. Stress and lipid peroxidation.

Concentration of malondialdehyde (nmol/mg protein)
Groups (n 5)
Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
4 weeks stress recovery, week 24
24 weeks no stress
F values (df 6, 34)
p value

Liver
23 ^ 2.1a
61.1 ^ 1.7b
74.2 ^ 3.4c
101 ^ 7.09d
31.1 ^ 0.72a
27.4 ^ 2.5a
23.1 ^ 5a
55.94
p , 0.05

Kidney
11.1 ^ 0.9a
29.2 ^ 2.7c
61.5 ^ 3.09d
81.6 ^ 3.05e
21.8 ^ 1.6bc
27.2 ^ 4.4c
14.3 ^ 1.8ab
104.3
p , 0.05

Heart
11.4 ^ 0.95a
36 ^ 2.2c
64.8 ^ 3.8d
85.4 ^ 6.3e
28.7 ^ 1.8c
25.3 ^ 2.08bc
14.6 ^ 0.95ab
63.83
p , 0.05

Notes: All values are mean ^ SEM. Values with same superscript letters in the given column are not significantly different; values with
different superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test.

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Figure 3. Transverse sections of similar areas of thoracic aorta of a control rat (24 weeks no stress; A and B), and a rat stressed for 24 weeks
(C and D) and a part of the aorta of a stressed rat under high magnification to show a portion of an atherosclerotic plaque (E) and foam cells
(arrow). Note the thickening of wall of the aorta and a fibrous layer in the vessel of the stressed rat. B and D are higher magnification images of
A and C, respectively. Hematoxylineosin stain; AD, adventitia; EN, endothelium; FB, fibrous layer; FC, foam cells; ME, media.

was further increased after 24 weeks (ANOVA,

Duncans, p , 0.05). GGT activity was normalized
at 24 weeks after cessation of exposure at 4 weeks, but
not at 2 weeks.

compared to the controls (ANOVA, Duncans,

p , 0.05). In the recovery groups, plasma GPT
activity was significantly reduced compared with the
stress groups but remained greater than in the
controls (ANOVA, Duncans, p , 0.05).

Plasma aminotransferase and LDH activities

Plasma LDH and plasma GOT activities were
significantly increased after exposure to stress for 4
weeks or 24 weeks compared to initial controls and
the group unstressed for 24 weeks (ANOVA,
Duncans, p , 0.05) (Table III); activities were
normalized at 24 weeks after cessation of exposure
at 2 or 4 weeks. Plasma GPT activity was
significantly increased after stress exposure for 2 or
4 weeks, and further increased after 24 weeks,

Malondialdehyde concentrations
Malondialdehyde (MDA) concentrations in the liver,
heart, and kidneys progressively increased in stressed
rats compared to the control groups (ANOVA,
Duncans, p , 0.05) (Table IV). In the recovery
groups, MDA concentrations were decreased compared with the stress groups (ANOVA, Duncans,
p , 0.05) and normalized in liver, but in heart and
kidney remained greater than in the controls.

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Chronic stress and atherosclerosis

Figure 4. Transverse sections of the thoracic aorta of a control rat (24 weeks no stress; A), and a rat stressed for 24 weeks (B) stained with Oil
Red O for localization of lipids. Note the Oil-Red-O-positive lipid deposition (arrow heads) in the section from a stressed rat (B). Transverse
sections of the thoracic aorta of a control rat (24 weeks no stress; C), and a rat stressed for 24 weeks (D) stained with van Gieson technique to
demonstrate elastic fibers (arrow head) and collagen (solid arrow). Note the presence of abundant elastic fibers in the control rat aorta (C) and
depletion of elastic fibers and abundant collagen deposition in the stressed rat aorta (D).

Histology of aorta
There were no marked histological changes in the
thoracic aorta of rats exposed to stress for 4 weeks, but
at 24 weeks histopathological changes were evident
(Figure 3). The aorta of controls (24 weeks not
exposed to stress) did not show prominent lipid
deposition following Sudan IV staining, and the
sections revealed endothelial lining and intima with
little collagen (Figure 3A,B). The stressed rats
(24 weeks) showed dark red spots in the aortic vessel
following Sudan IV staining, and the sections revealed
thickening of the vessel wall and the appearance of
fibrous layer in the intima (Figure 3C,D). Strikingly,

foam cells were evident in the intima of the aorta of

stressed rats (Figure 3E). The foam cells were large
and irregular in shape with eccentric nuclei containing
prominent nucleoli and contained minute pink
granules in the cytoplasm (Figure 3E). Oil Red O
staining showed a higher lipid content in the stressed
rats compared to the control group (Figure 4A,B).
van Gieson staining showed higher levels of elastin in
the aorta of the control group than that of the stressed
rats, as revealed by the intense blackish-brown colored
staining (Figure 4C) than in the aorta of stressed rats
(Figure 4D). In addition, the elastic structure was
disrupted and pinkish-stained collagen fibers were

Table V. Stress and thickness of layers of thoracic aorta wall.

Mean thickness (mm) ^ SEM
Groups (n 5)
Initial control
4 weeks stress
24 weeks stress
4 weeks stress recovery, week 24
24 weeks no stress
F values (df 4, 24)
p value

Intima
11.9 ^ 1.1a
12.0 ^ 0.8a
60.4 ^ 4.6b
13.4 ^ 0.8a
14.6 ^ 0.9a
80.85
p , 0.05

Media
67.3 ^ 3.2 a
72.6 ^ 3.1a
202 ^ 19.8b
78.3 ^ 5.5a
69.6 ^ 2.3a
34.65
p , 0.05

Total intimamedia

Total aortic wall

71.3 ^ 4.2 a
81.6 ^ 2.4a
252 ^ 18.8 b
90.3 ^ 7.01a
73.03 ^ 3.3a
66.6
p , 0.05

153 ^ 3.09a
160 ^ 0.6a
322 ^ 41.5b
186 ^ 15.8a
152 ^ 2.1a
11.9
p , 0.05

Notes: Values with same superscript letters in a given column are not significantly different; values with different superscript letters are
significantly ( p , 0.05) different by Duncans multiple post hoc test.

M. Devaki et al.

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present in the aorta of the stressed rats (Figure 4D).

These changes lead to the formation of atherosclerotic
plaques, which consisted of a fibrous layer (Figure 3D,
FB) beneath the endothelium (Figure 3D, EN)
followed by a fatty streak containing foam cells
(Figure 3D, FC) and extracellular lipids. There was a
significant increase in mean thickness of the intima,
media, total intima media, and total aortic wall in rats
stressed for 24 weeks, but not 4 weeks, compared to
the control groups (Table V). Of seven rats exposed to
stressors for 24 weeks, two died in the 22nd week, four
rats showed formation of atherosclerotic plaques
(thickening of intima media and foam cells), and
the remaining rat showed slight thickening of intima
without foam cells.
Discussion
In this study, exposing rats to restraint followed by
forced swimming every day for 2, 4, or 24 weeks
increased adrenal 3b-HSDH activity, a key enzyme in
adrenal steroidogenesis, and the weight of the adrenal
gland indicating stress-induced activation, with
expected increased corticosterone secretion, driven
by the hypothalamus and adrenocorticotrophic hormone (ACTH) (Tsigos and Chrousos 2002). Hence,
the alterations in the blood lipid profile and antioxidant
enzyme activities and lipid peroxidation that were
found were associated with adrenocortical stress
responses. In contrast to earlier studies, in which
alterations in lipid profile (Ruiz de Gordoa et al. 1994;
Richards et al. 2000; Lata et al. 2004; Akhtar et al.
2008; Gu et al. 2009; Nayanatara et al. 2009) and
antioxidant status (Manoli et al. 2000; Fontella et al.
2005; Nayanatara et al. 2005; Nadeem et al. 2006;
Bhat et al. 2007; Zafir and Banu 2009) were studied
after a relatively shorter duration of stress exposure
(maximum 30 days), this study clearly indicates that
the HPA axis as well as other physiological mechanisms
continued to respond to the stressors for a long period
despite repeated daily exposure. As prolonged stress
induces decreased food intake and loss in body weight
(Zardooz et al. 2006), the reduced gain in body weight
in rats after 4 or 24 weeks of stress exposure in this
study further supports a long-term stress response.
This study clearly demonstrates that stress due to
repeated restraint and forced swimming increased
blood concentrations of cholesterol, TG, LDL, and
VLDL and decreased HDL. Decreased activities of
hepatic antioxidant enzymes coupled with increased
MDA concentrations in heart, liver, and kidney at all
the stress durations studied indicate hyperlipidemia
and compromised antioxidant status. While forced
swimming can be considered an exercise model, it is
considered as a stressor because of emotional factors
associated with forced exercise (Nagaraj and Jeganathan 1999). Hence, the metabolic alterations might
be a combined effect of physical exercise and stress.

Exposure of laboratory animals to a variety of

stressors (Lata et al. 2004; Sato et al. 2006; Nayanatara
et al. 2009) and emotional stress in humans (Richards
et al. 2000) cause an increase in serum cholesterol, TG,
LDL, VLDL, and decrease in HDL concentrations.
Though results of this study are similar to earlier
reports, they clearly demonstrate that stress-induced
increases in blood cholesterol and LDL and decrease in
HDL are duration dependent, hence the alterations
became more severe with increase in exposure period,
which was not reported in earlier studies. It was also
observed that all the components of the lipid profile did
not show the same response to stress. Although TG
and VLDL concentrations showed significant
increases with repeated stress, the levels did not vary
according to the duration of exposure. The findings
revealed that the changes in TG and VLDL induced by
stress are evident within a short duration of exposure to
stress for 2 weeks and remain at the same level
throughout the exposure period of 24 weeks. An
increase in the blood concentration of cholesterol with
stress might reflect release of cholesterol from adipose
tissue or increased synthesis in the liver or decreased
excretion; similarly, increased circulating TG levels
with chronic stress might reflect lipolysis in adipose
tissue, with the released fatty acids serving as substrate
for TG synthesis (Luoma 2010).
There was a reduction in the antioxidant status with
the chronic stress paradigm used here, as indicated
by reduced activities of hepatic primary (CAT, SOD,
GPx) and secondary (GR, GST, G6PDH) antioxidant
enzymes, coupled with increased lipid peroxidation in
the liver, heart, and kidney, in accordance with the
earlier studies (Manoli et al. 2000; Fontella et al.
2005; Nayanatara et al. 2005; Nadeem et al. 2006;
Zafir and Banu 2009). However, the present data
indicate that antioxidant potential remains compromised even after a long exposure period of 24 weeks,
which was not evident in earlier studies. Reduction in
activities of primary antioxidants might be due to
cross-linking or exhaustion of enzymes (Salo et al.
1990). In addition, increase in hepatic GGT activity
following chronic stress in this experiment also
indicates increased oxidative stress as GGT is involved
in superoxide anion generation (Pompella et al. 2004),
and high-GGT activity is found in human populations
with increased risk for cardiovascular diseases (Emdin
et al. 2005). Tissue injuries are expected due to high
level of oxidants under stressful conditions. Indeed, in
this study there was an increase in the activity of
plasma GOT, plasma GPT, and LDH which leak into
blood following tissue injury (Arakawa et al. 1997)
and thereby indicate organ damage, including heart.
The unwanted effect of increased secretion
of glucocorticoids (GCs) due to stress includes
overproduction of reactive oxygen species (Bjelakovic
et al. 2007), dysregulation of physiological processes,
and increased lipolysis (Brindley 1995). GCs decrease

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Chronic stress and atherosclerosis

concentration of low density lipoprotein cholesterol
(LDL-C) receptors on hepatocytes (Rainey et al.
1992), leading to higher LDL-C concentration and
increased production and secretion of VLDL in the
liver (Brindley 1995). As effects of GCs on lipid
metabolism persist for a long period (Eisenstein
1973), it is possible that alterations persist even after
the challenge is past, resulting in chronic hyperlipidemia. In addition, GCs increase reactive oxygen radical
generation in cells (McIntosh and Sapolsky 1996;
Bjelakovic et al. 2007), reduce expression of primary
antioxidant, SOD, on chronic exposure (McIntosh
et al. 1998), and deplete glutathione (GSH) (Slater
et al. 1995). Here, adrenal enlargement and increased
activity of a GC synthesizing enzyme (3b-HSDH)
provide evidence for increased GC production with
the present chronic stress model. Such increased GC
production likely provides a common pathway in this
model that alters both lipid and antioxidant status, as
hyperlipidemia and reduced antioxidant potential were
observed in chronically stressed rats in this study, in
contrast to earlier studies which either focused on lipid
profile changes (Ruiz de Gordoa et al. 1994; Richards
et al. 2000; Lata et al. 2004; Akhtar et al. 2008; Gu
et al. 2009; Nayanatara et al. 2009) or oxidative
damage (Manoli et al. 2000; Fontella et al. 2005;
Nayanatara et al. 2005; Nadeem et al. 2006; Zafir
and Banu 2009) following chronic stress exposure.
Several risk factors are implicated in the development of atherosclerosis including increased oxidized
LDL-C and endothelial dysfunction induced by
reactive oxygen species (Ross 1999) and induced by
endothelial dysfunction reactive oxygen species (Ross
1999). HDL is a protective factor for atherosclerotic
development as it reduces oxidation of LDL and helps
in reverse transport of cholesterol. Hence, any factor
that simultaneously causes oxidative stress and
hyperlipidemia might induce atherosclerosis. Stress is
most likely a factor as it results in high levels of GCs
which cause dyslipidemia as well as oxidative damage
as discussed above. However, though stress is
implicated in pathogenesis of atherosclerosis, thus far
there is no experimental evidence to link stress alone to
the development of atheroma because earlier studies
have used other factors along with stress to induce
atherosclerosis. For instance, though monkeys showed
atherosclerosis following stress due to isolation, they
were fed an atherogenic diet during experimentation
(Shively et al. 1989). Likewise, mice that developed
atherosclerosis due to isolation were deficient in
apolipoprotein (Bernberg et al. 2008). In contrast, in
this study chronic exposure to stressors for different
durations resulted in a significant elevation in circulating cholesterol, LDL, and TG and decrease in HDL,
concomitant with increased oxidative stress (lipid
peroxidation). Hence, the stress protocol used in this
study was effective in simultaneously inducing both
risk factors, i.e. lipid profile and oxidative damage for

the development of atherosclerosis. Indeed, there was

an atherosclerotic development in the aorta after
24 weeks stress exposure. A typical atherosclerotic
plaque consists of a fibrous cap, accumulation of lipid
droplets, a large number of foam cells in the intima and
depletion of smooth muscle (Libby 2006). The
macrophages that enter the intima region during
pathogenesis of atherosclerosis accumulate oxidized
LDL to become foam cells and are the hallmark of
atherosclerotic lesions (Witztum and Steinberg 1991),
whereas the normal blood vessel wall does not contain
these cells. In rats stressed for 24 weeks, the aorta
showed the development of a fibrous layer beneath the
endothelium and a fatty streak, consisting of lipid
droplets and foam cells, coupled with reduction in
elastic fibers and smooth muscle and appearance of
collagen fibers. Hence, our observations correspond
with the description of a typical atherosclerotic plaque.
Significant increases in IT, MT, and IMTand thickness
of the aortic wall in rats exposed to stress for 24 weeks
in this study further support atherosclerotic development as IMT is also a characteristic of atherosclerosis
(Bots et al. 2007). Thus, this study is the first to provide
experimental evidence, of a link between stress,
compromised antioxidant status, and hyperlipidemia
in the rat for the development of atherosclerosis. In
addition, the stress protocol developed provides an
animal model for studying pathogenesis and prevention of atherosclerosis. Although rats are generally
considered an atherosclerosis-resistant species due to
their hypo-responsiveness to dietary cholesterol,
combination of a cholesterol-rich diet with other
factors can induce atherosclerosis (Moghadasian
2002). For instance, hyperlipidemia and atherogenesis
are induced by high-cholesterol/high-fat diet containing cholic acid and thiouracil (Joris et al. 1983;
Moghadasian 2002; Jawien et al. 2004). Hence, the
combined effect of prolonged hyperlipidemia due to
long-term stress exposure and oxidative damage might
have contributed to atherosclerotic development in
rats in this study.
Normalization of stress-induced changes especially
dyslipidemia, reduced antioxidant status after the
cessation of the exposure, and the duration required
for normalization are the important factors to be
considered because of their involvement in atherosclerosis. In this study, in the recovery group rats
studied 20 22 weeks after the cessation of 4 or
2 weeks of stress exposure, respectively, the blood
lipid levels, i.e. HDL, TG, and VLDL, did not differ
from controls, whereas cholesterol and LDL concentrations remained higher than in controls. Likewise,
activities of hepatic catalase, GPx, GR, GST, and
GGT and hepatic MDA concentration in the recovery
groups did not differ from controls. However, lipid
peroxidation (MDA concentration) in heart and
kidney and hepatic SOD and plasma GOT activities
remained increased compared with controls in the

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10

M. Devaki et al.

recovery groups. Thus, the present experimental

results importantly reveal that though the majority of
the parameters of lipid metabolism and the antioxidant system are restored to normalcy after
cessation of stress exposure, some (cholesterol,
LDL, lipid peroxidation) remain elevated even after
a long withdrawal period (20 weeks). This observation
gains importance because of pathological consequences of high concentrations of LDL-C and
oxidative stress.
This study provides an example of long-term
allostatic regulation leading to a pathological state.
Lack of adaptation to repeated stress is a feature of
allostasis (McEwen 2002), and many diseases are
related to long-term chronic stress responses, when
allostasis shifts from a healthy condition to a
pathological state (Goldstein and McEwen 2002;
McEwen 2002). The stress paradigm used here
evidently did not result in habituation, as the stressed
rats continued to show elevated adrenocortical
activity, hyperlipidemia, and compromised antioxidant status, even after 24 weeks, and these changes
were accompanied by atherosclerotic development
(allostatic load) in thoracic aorta. These alterations
may be mediated by GCs as they act as allostatic
mediators (McEwen 2002). Another feature of
allostasis is the inability to shut off allostatic responses
after the challenge (stressor) is past (McEwen 2002).
The finding here that the blood concentrations of
LDL and cholesterol and hepatic MDA level
remained significantly greater than in controls, even
20 weeks after cessation of exposure to the stressors,
not only confirms a model of allostasis but also
provides new evidence for the persistence of stress
effects for a long period. With a similar regime of stress
exposure in our earlier study (Nirupama et al. 2012),
hyperglycemia remained for several weeks in rats after
the cessation of stress exposure. Together, these
findings support the validity of our animal model for
studying allostasis and allostatic load.
Declaration of interest: The work was supported by
a grant from Ministry of Human Resource Development, Government of India, through the University
Grants Commission to University of Mysore, under
the Institution of Excellence scheme. The authors
report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.

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