Chronic stress, but not hypercaloric diet, impaired vascular function in rats.

Full text

AUTHOR QUERY SHEET
Author(s): V. J. Neves, M. J. C. S. Moura, B. S. Almeida, R. Costa, A. Sanches, R. Ferreira, M. L. Tamascia,
E. A. O. Romani, P. D. Novaes & F. K. Marcondes
Article title: Chronic stress, but not hypercaloric diet, impairs vascular function in rats
Article no: 601369
Dear Author,
Please check these proofs carefully. It is the responsibility of the corresponding author to check against the origi-
nal manuscript and approve or amend these proofs. A second proof is not normally provided. Informa Healthcare
cannot be held responsible for uncorrected errors, even if introduced during the composition process. The journal
reserves the right to charge for excessive author alterations, or for changes requested after the proofing stage has
concluded.
The following queries have arisen during the editing of your manuscript and are marked in the margins of the
proofs. Unless advised otherwise, submit all corrections using the CATS online correction form. Once you have
added all your corrections, please ensure you press the “Submit All Corrections” button.
Q1 We have inserted a running head. Please approve or provide an alternative.
Q2 References Hsueh and Quin
˜ones (2003), Carvalho et al. (2006) and Miller et al. (1948) have been cited in
text but not provided in the list. Please supply reference details or delete the reference citations from the
text.
Q3 Please note that the reference citations Laute
´rio and Bond (1994), Mendis and Samarajeewa (2001) and
Soria et al. (2005) have been changed to Lauterio et al. (1994), Mendis et al. (2001) and Soria et al. (2001)
with respect to the reference list provided.
Q4 “Miristic” has been changed to “myristic”. Please check and confirm.
Q5 Please check the edit of the sentence.
Q6 A declaration of interest statement reporting no conflict of interest has been inserted. Please confirm whether
the statement is accurate.
Q7 Please provide the significance of “{” in Table II.

Chronic stress, but not hypercaloric diet, impairs vascular function
in rats
V. J. NEVES
1
, M. J. C. S. MOURA
2
, B. S. ALMEIDA
1
, R. COSTA
1
, A. SANCHES
1
, R. FERREIRA
1
,
M. L. TAMASCIA
1
, E. A. O. ROMANI
3
, P. D. NOVAES
3
, & F. K. MARCONDES
1
1
Laboratory of Stress, Department of Physiological Sciences, Piracicaba Dental School, University of Campinas, Piracicaba,
Sao Paulo, Brazil,
2
Life Sciences Center, Pontifical Catholic University of Campinas, Campinas, Sao Paulo, Brazil, and
3
Department of Morphology, Piracicaba Dental School, University of Campinas, Piracicaba, Sao Paulo, Brazil
(Received 25 September 2010; revised 28 April 2011; accepted 24 June 2011)
Abstract
The aim of this study was to evaluate vascular and metabolic effects of chronic mild unpredictable stress (CMS) and
hypercaloric diet (HD) without carbohydrate supplementation in rats. Male Sprague-Dawley rats were randomly assigned to
four groups: Control, HD, CMS, and HD plus CMS. CMS consisted of the application of different stressors for 3 weeks. The
rats were killed 15 days after CMS exposure. The HD group presented higher plasma lipid concentrations, without changes in
fasting glucose concentration, glucose tolerance test, and vascular function and morphology, in comparison with the control
group. Stressed rats presented higher fasting blood concentration of insulin, higher homeostasis model assessment index
values and area under the curve in an oral glucose tolerance test, in comparison with non-stressed rats. CMS increased the
plasma concentrations of corticosterone and lipids, and the atherogenic index values, without change in high-density
lipoprotein level. CMS increased intima-media thickness and induced endothelium-dependent supersensitivity to
phenylephrine, and lowered the relaxation response to acetylcholine in the thoracic aorta isolated from rats fed with control
or HD, in comparison with non-stressed groups. CMS effects were independent of diet. In non-stressed rats, the HD induced
dyslipidemia, but did not change glucose metabolism, vascular function, or morphology. The data from this study indicate
that CMS promotes a set of events which together can contribute to impair function of the thoracic aorta.
Keywords: Aorta, endothelium, insulin resistance, nitric oxide, lipids, stress
Introduction
In Western societies, regular consumption of hyper-
caloric diets (HDs) and obesity are often associated
with the occurrence of metabolic and cardiovascular
diseases. Atherogenic dyslipidemia is a major under-
lying cause for the development of atherosclerosis
(Sheril et al. 2009). However, a high-fat, high-sugar
diet alone cannot account for the current epidemic of
obesity (Ludwigh 2003) and coronary heart disease
(Gu et al. 2009). Moreover, despite the association
between atherosclerosis and an increase in serum lipid
concentration, many individuals develop severe ather-
osclerotic lesions while they have low serum lipid
concentration, and others develop far more severe
atherosclerosis than would be expected on the basis of
a modest elevation of serum lipids (Kaplan et al. 1983).
As exposure to chronic stress also correlates with an
increasing incidence of visceral obesity, insulin
resistance, hypertension, and atherosclerosis, stress
has been recognized as a risk factor for cardiovascular
and metabolic diseases (Kyrou and Tsigos 2007;
Shively et al. 2009). However, in response to stress,
some people lose weight while others gain weight
and the mechanisms involved in the relationships
among chronic stress, visceral obesity, dyslipidemia,
and cardiovascular diseases have not yet been
completely clarified.
Animal experimentation is a useful tool to
complement clinical research and epidemiological
Correspondence: F. K. Marcondes, Departamento de Cie
ˆncias Fisiolo
´gicas, Faculdade de Odontologia de Piracicaba, Universidade Estadual
de Campinas, Avenue Limeira, 901-Vila Areia
˜o, 13414-903 Piracicaba, SP, Brazil. Tel/Fax: 55 19 2106 5380/2106 5212. E-mail:
fklein@fop.unicamp.br
GSTR 601369—13/7/2011—SHANMUGAM—395854
Stress, 2011; 00(0): 1–11
qInforma Healthcare USA, Inc.
ISSN 1025-3890 print/ISSN 1607-8888 online
DOI: 10.3109/10253890.2011.601369
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110

data. Rodents generally adapt to repeated application
of stressors; however, this does not occur if the
presentation of stimuli is unpredictable, as it is in
chronic mild unpredictable stress (CMS). (Moreau
et al. 1994; Moreau 1997). In a previous study (Neves
et al. 2009), it was observed that CMS induced
proatherogenic changes in rats and that this effect
seems to be related to a decrease in the bioavailability
of endothelial nitric oxide (NO) induced by stress.
Here, we tested the hypothesis that CMS would
adversely alter vascular function independently of
high-fat diet.
Since insulin resistance and dyslipidemia are
involved in atherogenesis and endothelium dysfunc-
tion (Hsueh and Quin
˜ones 2003; Carvalho et al. 2006),
Q2
the aim of the present study was to evaluate the effects
of CMS and high-fat HD on the reactivity of thoracic
aorta to phenylephrine and acetylcholine, morphology
of the thoracic aorta, blood lipid concentrations, and
glucose metabolism.
Materials and methods
Animals and experimental design
Seventy-two male Sprague-Dawley rats (60 days old;
290 –340 g) were randomly assigned to four groups:
Control, HD, CMS, and HD plus CMS
(HD þCMS). Rats were housed one per cage at
22 ^28C with lights on from 06:00 to 18:00 h, and
received filtered water ad libitum. All procedures were
approved by the UNICAMP Committee on Animal
Research Ethics (Protocol number 900-1) and were in
accordance with the guidelines of the National
Council of Control of Animal Experimentation
(CONCEA).
For 7 weeks, the control and CMS groups were fed
a control diet based on AIN-93M, formulated for the
maintenance of adult rodents (Reeves et al. 1993),
containing 3.6 kcal/g (14.1% kcal protein, 75.9% kcal
carbohydrate, 10.0% kcal fat). The HD and
HD þCMS groups received a modified high-fat but
not high-sugar HD, based on a purified moderately
high fat (PMHF) diet (Lauterio et al. 1994) contain-
Q3
ing 4.41 kcal/g (16.8% kcal protein, 51.4% kcal
carbohydrate, 31.8% kcal fat). In this study, all the
butterfat (52 g) and cor n oil (118 g) in the PMHF diet
were replaced by coconut fat (44.2 g) and soybean oil
(118 g), respectively. The soybean oil was used to
replace corn oil because it was also used in the control
diet, and because the literature has postulated that it
may contribute to promoting insulin resistance in
rodents (Bueno et al. 2008). The coconut fat replaced
butterfat to increase the amount of saturated fatty
acids (SFA). Coconut fat is a highly saturated fat
Q4
(containing .76 g SFA/100g fat), rich mainly in
lauric, myristic and palmitic acids, which have been
shown to be capable of promoting dyslipidemia in rats
(Kamgang et al. 2005; Silva et al. 2006).
Stress protocol
The CMS protocol used in this study is based on the
model described by Moreau (1997) and modified
according to Neves et al. (2009). It consisted of the
application of different stressors for 7 days per week
for a period of three consecutive weeks (from the 3rd
to 5th week) as described in Table I. In this CMS
model, a different stressor is presented every day and it
does not allow the rodents to adapt to the stressors.
The absence of adaptation was confirmed in a pilot
study carried out in our laboratory; plasma corticos-
terone determinations showed that 1 and 15 days after
the end of CMS, stressed rats had higher corticoster-
one concentrations (32.6 ^2.4; 21.2 ^0.9 ng/ml,
respectively) in comparison with non-stressed rats
(2.4 ^0.4 ng/ml). As the aim of this study was to
evaluate long-term effects of CMS, the metabolic and
vascular evaluations were carried out 10 or 15 days
after CMS. Control rats were subjected only to the
procedures related to their normal care.
Oral glucose tolerance test and homeostasis model
assessment (HOMA index)
Ten days after stress, the rats were fasted for 6 h prior
to the collection of tail blood in heparin-coated tubes
for subsequent determination of fasting glucose and
insulin concentrations. Between 13:00 and 14:00 h,
the rats were anaesthetized with Halothane, the tail
was cut and blood samples were collected to
determine the glucose and insulin fasting concen-
trations. Immediately afterwards, an oral glucose load
(2 g/kg) solution was administered by oral gavage.
Additional blood samples were collected without
Table I. Chronic mild unpredictable stress procedure.
Morning Afternoon
Monday 08:00 h, 1 h immobilization 13:00 h, 1 h immobilization; 18:00h, overnight illumination
Tuesday 08:00 h, 1 h immobilization 14:00 h, 1 h immobilization followed by water and food deprivation for 18 h
Wednesday 08:00 h, access to food restricted for 2 h 13:00 h, 1 h immobilization followed by water deprivation for 18 h
Thursday 08:00 h, exposure to empty water bottle
for 2 h; 11:00 h, 1 h immobilization
14:00 h, 1 h immobilization; 15:00h, wet bedding for 17 h
Friday 08:00 h, 1 h immobilization 18:00 h, reversed light/dark cycle throughout the weekend
GSTR 601369—13/7/2011—SHANMUGAM—395854
V.J. Neves et al.2
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220

additional anesthesia at 30-, 60-, 90-, and 120-min
time intervals. The blood collected for all analysis was
obtained from the same site as the first bleeding,
which was halted by applying a tourniquet. In each rat,
only one tail cut was made. Plasma fasting insulin
concentration was measured with a commercially
available kit (LINCO Research
w
, St Charles, MO,
USA) with a sensitivity of 3.4 pmol/l. Blood glucose
was measured using the Prestige LX
w
glucosimeter.
The homeostasis model assessment (HOMA) index
was used to assess insulin resistance and it was
calculated by the formula: [fasting plasma glucose
(mmol/l) £fasting plasma insulin (mU/ml)]/22.5
(Søndergaard et al. 2006).
Analytic methods and tissue collection
All the rats were killed by decapitation 15 days after
the end of stress, without previous anesthesia to avoid
anesthetic-induced increase in plasma corticosterone
concentrations (Vahl et al. 2005). Trunk blood was
collected in heparin-coated tubes, and the plasma
was used to determine corticosterone and lipids by
enzymatic colorimetric assays. The thoracic aorta was
isolated. Three depots of visceral white adipose tissue,
which have been associated with insulin resistance and
cardiovascular diseases, were carefully removed and
weighed. The above-mentioned depots were the
epididymal fat pad (around the testis and ductus
deferens), retroperitoneal (located on the border
between the spine and the posterior abdominal wall)
and perirenal fat pad (along the posterior wall from the
kidney to the hip region) (Cinti 2005; Tran and Kahn
2010). The ratio of total fat pad weight to final body
weight was calculated (Levin et al. 2000).
Corticosterone was assayed using the Corticoster-
one Enzyme Immunoassay Kit (Assay Designs
w
,
Ann Arbor, MI, USA), with sensitivity of
0.027 ng/ml, the intra- and inter-assay coefficients of
variation being 7.7 and 9.7%, respectively. Total
cholesterol (TC) was assayed using a commercially
available kit (Laborlab
w
, Guarulhos, SP, Brazil), with
sensitivity of 0.03 mmol/l; the intra- and inter-assay
coefficients of variation were 3 and 2.7%, respectively.
Triglycerides (TGL) were also assayed using
the Laborlab
w
kit, with sensitivity of 0.02 mmol/l.
High-density lipoprotein (HDL) was determined
using the Laborlab
w
kit, with sensitivity of
0.01 mmol/l. The Friedewald’s formula: low-density
lipoprotein (LDL) ¼TC 2HDL 2(TGL £0.2)
was used to determine the plasma LDL concen-
trations (Friedewald et al. 1972). The atherogenic
index (AI) was determined using the formula: AI ¼
TC 2HDL/TC (Kamgang et al. 2005).
Concentration-effect curves
The middle portion of thoracic aorta was excised free
of fatty and connective tissue and then cut into two
3-mm-long to 5-mm-long rings. One ring was
Q1
manipulated carefully to avoid damaging the endo-
thelium, and the intimal surface of the second ring was
scraped gently with a scalpel blade to remove the
endothelial layer. The rings were suspended in a 20 ml
organ bath containing Krebs –Henseleit solution.
(Moura and Marcondes 2001). The aortic rings were
connected to an isometric force transducer model
7004 (Ugo Basile, Varese, Italy), which was in turn
connected to an Ugo Basile 2-channel Gemini
recorder model 7070 (Ugo Basile) for registration of
contractile responses. Concentration-effect curves
(CEC) were obtained for aortic rings with and without
endothelium to determine whether there were changes
in the contractile activity of the thoracic aorta and
whether the change was endothelium dependent, or
was mediated directly by changes in the vascular
smooth muscle (Cunha et al. 2005; Neves et al. 2009).
After 60 min of stabilization, the intactness of the
endothelium was assessed by determining the vasodi-
lating response to acetylcholine (1 mM) in rings pre-
contracted with phenylephrine (0.1 mM). Only aortic
rings that presented 100% relaxation by acetylcholine
were considered to have an intact endothelium.
The effectiveness of mechanical removal of the
endothelium was confirmed by the complete absence
of response to acetylcholine. The aortic rings
were rinsed and allowed to re-equilibrate for 60 min.
One cumulative CEC for phenylephrine (0.1 nM–
0.1 mM) was obtained in each ring and the maximum
response was evidenced when an increase in agonist
concentration did not produce any additional
response (Cunha et al. 2005; Neves et al. 2009).
Changes in thoracic aorta sensitivity to phenylephrine
were evaluated by determining the concentration
that produced 50% of the maximum response and
were expressed as the mean negative logarithm (pD
2
)
(Miller et al. 1948).
Q2
To evaluate the role of NO in modulating the
thoracic aorta sensitivity to phenylephrine, aortic rings
with intact endothelium, obtained from other rats
subjected to the same treatments, were isolated as
described above and were incubated with the NO
synthesis inhibitor, N
G
-nitro-L-arginine methyl ester
(L-NAME) (10 mM) for 40 min. A CEC for phenyl-
ephrine was obtained in the presence of L-NAME.
A CEC for acetylcholine was obtained in endo-
thelium-intact thoracic aorta rings isolated from other
rats subjected to the same treatments. In these
experiments, the aortic rings with intact endothelium
were pre-contracted with phenylephrine (1 mM). After
stabilizing the contractile response to phenylephrine,
acetylcholine (0.1 nM –0.1 mM) was cumulatively
added into the bath medium to induce relaxation.
GSTR 601369—13/7/2011—SHANMUGAM—395854
Chronic stress impairs vascular function in rats 3
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
320
325
330

The relaxation responses were expressed as percen-
tages of the response to phenylephrine (1 mM).
The stock solutions of phenylephrine and acetyl-
choline (Sigma Chemical
w
, St Louis, MO, USA) were
dissolved in 2% ascorbic acid and stored at 2108C for
1 week. The dilutions for the CECs were prepared
immediately before use and later discarded. For the
Krebs– Henseleit solution, standard salts (Merk
w
,
Darmstadt, Germany) were prepared in deionized
water. The quality of the water was assessed weekly by
measuring its conductivity and pH.
Morphometric study
To evaluate whether the functional changes in the
aorta observed in the CEC were associated with
morphological alterations, the inferior third of the
thoracic aor ta beginning 5 mm above the diaphragm
muscle was placed in Karnovsky’s fixative (Karnovsky
1965). In a previous analysis, we observed that the
supersensitivity to phenylephrine observed in CEC
obtained in the middle portion of thoracic aorta was
also observed in its inferior third. Therefore, in order
to decrease the number of rats used in the
experiments, the morphometric study was carried
out in the inferior third of the aorta isolated from the
same rats from which aortas were used for obtaining
the CEC to phenylephrine in the presence of L-
NAME. The aorta fragments were washed in 1%
osmium tetroxide in 0.1 M phosphate buffer, pH 7.3,
for 2 h at room temperature and dehydrated with 50,
60, 70, 90, and 100% acetone. The fragments were
embedded in Araldite-502 resin (Luft 1961), and
polymerized for 48 h at 608C.
Semi-thin (1 mm) transverse sections of the aorta
were cut on a SORVALL
w
Porter-Blum MT2-B
ultramicrotome with a glass knife, and collected at
10 mm intervals. The sections were stained with 0.5%
toluidine blue in 1% sodium borate for 50 s followed
by 1% basic fuchsin for 30 s. Five sections of each
aorta in five rats per group were obtained, totaling 25
sections in each studied group for analysis by light
microscopy.
The measurements of total intima-media thickness
(IMT) were made using a photomicroscope (Carl
Q7
Zeiss, Oberkochen, West Germany) connected to a
millimeter eyepiece (Ernest leitz, Wetzlar, Germany,
12.5X), and using a millimeter ruler (Carl Zeiss
5þ100/100 mm) for calibration. To calculate the
IMT, 24 measurements were made in each section,
using a 25X objective lens and 1.6X optovar,
considering the IMT limits to be between the luminal
surface and external elastic lamina. The IMT results
are shown in micrometers (Neves et al. 2009).
Statistical analysis
Statistically significant differences were determined
by two-way analysis of variance (ANOVA) followed by
the Tukey test for multiple comparisons of means
(p,0.05). Stress and diet were considered the main
factors, and their interaction was also analyzed. Values
are presented as means ^SE of the means (SEM).
Results
Two-way ANOVA carried out on plasma corticoster-
one concentration evidenced a significant main effect
of stress [F(1,44) ¼60.14, p¼0.000, Table II],
without significant effect of diet ( p¼0.883) or
interaction ( p¼0.634). Stressed rats showed a higher
plasma corticosterone concentration in comparison
with non-stressed groups (Table II). There were no
significant differences in initial body weight
(p.0.05, Table II) among the four groups. However,
15 days after CMS, there was a significant main effect
of stress [F(1,44) ¼5.106, p¼0.029], and diet
[F(1,44) ¼30.198, p¼0.000] on final body weight,
without signifi cant interaction ( p¼0.306, Table II).
In the FP/BW
f
ratio, a significant stress/diet inter-
action [F(1,44) ¼18.174, p¼0.000, Table II] was
observed. The groups fed with a HD (HD and
HD þCMS) presented a higher FP/BW
f
value when
compared with the groups treated with the control diet
(p¼0.000, Tukey’s test, Table II). However, con-
sidering the effect of stress, stressed rats treated with a
HD (HD þCMS) showed a higher FP/BW
f
when
compared with non-stressed rats treated with the same
diet (HD) ( p¼0.000 with Tukey’s test, Table II]),
without difference between stressed (CMS) and non-
stressed (control) rats fed the control diet ( p¼0.392
with Tukey’s test, Table II).
Table II. Plasma concentrations of corticosterone and body weight of control and stressed rats, fed with control, or HD.
Variable Control CMS HD HD þCMS
Corticosterone, ng/ml 2.02 ^0.41 19.42 ^2.86*3.34 ^0.66 18.72 ^3.02
†
Initial body weight, g 319.1 ^2.1 324.3 ^3.0 319.9 ^3.6 324.4 ^2.5
Final body weight, g 437.5 ^2.4 426.0 ^1.6*453.1 ^4.5*448.8 ^4.5
‡
FP/BW
f
0.0303 ^0.0012 0.0269 ^0.0013 0.0372 ^0.0024*0.0472 ^0.0014
{
Control, non-stressed rats fed with control diet; CMS, chronic mild unpredictable stress; HD, hypercaloric diet; HD þCMS, hypercaloric
diet þchronic mild unpredictable stress. FP/BW
f
¼ratio of total (epididymal þretroperitoneal þperirenal) fat pad weight to final body
weight. N¼12 animals/group. *Significant difference vs. control ( p,0.05; two-way ANOVA þTukey).
†
Significant vs. HD ( p,0.05; two-
way ANOVA þTukey).
‡
Significant vs. CMS ( p,0.05; two-way ANOVA þTukey). Values are mean ^SEM.
GSTR 601369—13/7/2011—SHANMUGAM—395854
V.J. Neves et al.4
335
340
345
350
355
360
365
370
375
380
385
390
395
400
405
410
415
420
425
430
435
440

Two-way ANOVA carried out on TGL
values evidenced significant effects of stress
[F(1,44) ¼10.102, p¼0.003, Figure 1], and diet
[F(1,44) ¼7.251, p¼0.010], without significant
interaction ( p¼0.236). Significant main effects of
stress [F(1.44) ¼10.01, p¼0.003], and diet
[F(1.44) ¼9.86, p¼0.003, Figure 1] were also
observed for TC plasma concentration, without
significant effects for plasma concentration of HDL
(p.0.05). With regard to LDL, there was a
significant main effect of stress [F(1,44) ¼5.881,
p¼0.019, Figure 1], and diet [F(1,44) ¼8.777,
p¼0.005, Figure 1], but no interaction ( p¼0.222).
Similarly, for the AI, there was a significant effect of
stress [F(1,44) ¼10.782, p¼0.002, Figure 1], and
diet [F(1,44) ¼16.746, p¼0.000, Figure 1], without
interaction ( p¼0.526). Therefore, statistical analysis
showed that the groups fed with a HD (HD and
HD þCMS) presented higher plasma TGL concen-
trations, TC and LDL as well as higher AI values in
comparison with the groups treated with the control
diet (control and CMS). As regards the effects of
stress, CMS increased the plasma concentrations of
these lipids both in rats fed with the control and HD,
and this effect was more pronounced in the
HD þCMS group.
Figure 2 displays the mean fasting plasma glucose
(Panel A) and insulin (Panel B) concentrations, area
under the oral glucose tolerance test (OGTT) curve
(AUC) (Panel C), and HOMA index (Panel D) of
the four experimental groups. There was no signifi-
cant difference in fasting glucose among the
groups ( p.0.05), but two-way ANOVA revealed
a significant main effect of stress on fasting
insulin [F(1,36) ¼7.572, p¼0.009], AUC
[F(1,28) ¼38.065, p¼0.000], and on the HOMA
index [F(1,36) ¼9.169, p¼0.005], without signifi-
cant effect of diet or int eraction ( p.0.05). These
results showed that CMS increased the fasting insulin
concentration, AUC and HOMA indices, but not the
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Control CMS HD HD+CMS
*
†‡
*
TGL TC HDL LDL
**
**
†‡
†‡
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
**
†‡
AI
mmol/L
Atherogenic index
Figure 1. Effects of stress on plasma lipid concentrations of TGL,
TC, HDL, and LDL in control rats, stressed rats subjected to CMS,
non-stressed rats fed with HD, and rats fed with HD and subjected
to CMS (HD þCMS), 15 days after the end of stress protocol
(n¼12 rats per group). The AI for each of the four studied groups is
presented on the right axis of graph (n¼12 rats per group). Data are
mean ^SEM. *Significant difference vs. control ( p,0.05; two-
way ANOVA þTukey).
†
Significant vs. HD ( p,0.05; two-way
ANOVA þTukey).
‡
Significant vs. CMS ( p,0.05; two-way
ANOVA þTukey).
Control CMS HD HD+CMS
0
1
2
3
4
5
6
A
Fasting glucose (mmol/L)
Control CMS HD HD+CMS
0
5
10
15
20
*
†
B
Fasting insulin (pmol/L)
Control CMS HD HD+CMS
0
250
500
750
1000 *
†
C
AUC (mmol x min/L)
Control CMS HD HD+CMS
0
1
2
3
4
5
6
7†
*
D
HOMA index
Figure 2. Fasting plasma glucose (n¼10 rats/group), fasting plasma insulin (n¼10 rats/group) concentrations, area under the OGTT
curve (AUC) (7 –9 rats/group), and HOMA index (10 rats per group) carried out in control rats, stressed rats subjected to CMS, non-stressed
rats fed with HD, and rats fed with HD and subjected to CMS (HD þCMS), 10 days after the end of stress protocol. Data are presented as
mean ^SEM. *Significant difference vs. control ( p,0.05; two-way ANOVA þTukey).
†
Significant vs. HD ( p,0.05; two-way ANOVA þ
Tukey).
GSTR 601369—13/7/2011—SHANMUGAM—395854
Chronic stress impairs vascular function in rats 5
445
450
455
460
465
470
475
480
485
490
495
500
505
510
515
520
525
530
535
540
545
550

fasting glucose concentration in rats fed with the
control or HD, in comparison with the non-stressed
groups (control and HD) (Figure 2). The HD did not
change these parameters (Figure 2).
As the treatments did not modify the maximum
response to phenylephrine and dry weight of aortic
rings among the groups ( p.0.05; Table III),
the CEC were expressed as percentages of
maximum response (Figure 3). Considering pD
2
values of phenylephrine of endothelium-intact rings
(Table III), two-way ANOVA revealed a significant
main effect of stress [F(1,16) ¼37.923, p¼0.000],
without significant effect of diet or interaction
(p.0.05). Therefore, stress induced supersensitivity
to phenylephrine in the thoracic aorta with a 3.7-fold
shift to the left in endothelium-intact aortic rings of
Table III. Dr y weight of aorta rings and maximum contractile response (MR) to phenylephrine in endothelium-intact, endothelium-
denuded thoracic aortic rings and in the presence of L-NAME, isolated from control and stressed rats, fed with control or HD.
Groups Dry weight (mg) MR (mg/100 mg tissue) pD
2
Control Endothelium-intact 106.80 ^5.45 11.03 ^1.98 6.82 ^0.09
Endothelium-denuded 98.76 ^6.77 10.45 ^2.12 7.63 ^0.11
L-NAME 102.10 ^2.54 10.98 ^2.45 7.66 ^0.09
‡
CMS Endothelium-intact 98.05 ^3.43 12.09 ^1.66 7.45 ^0.26*
Endothelium-denuded 103.40 ^3.43 10.34 ^1.54 7.47 ^0.11
L-NAME 97.80 ^3.14 11.34 ^1.67 7.50 ^0.12
HD Endothelium-intact 101.02 ^4.45 9.89 ^0.87 6.76 ^0.11
Endothelium-denuded 98.40 ^5.13 10.34 ^1.78 7.23 ^0.10
L-NAME 102.00 ^8.44 10.65 ^1.54 7.34 ^0.11
‡
HD þCMS Endothelium-intact 99.45 ^2.42 11.34 ^1.34 7.32 ^0.06
†
Endothelium-denuded 97.56 ^5.61 12.34 ^1.67 7.36 ^0.11
L-NAME 101.40 ^3.66 11.35 ^0.98 7.40 ^0.09
Control, non-stressed rats fed with control diet; CMS, chronic mild unpredictable stress; HD, hypercaloric diet; HD þCMS, hypercaloric
diet þchronic mild unpredictable stress. N¼5 rats/group. pD
2
¼negative logarithm of the molar concentration of agonist producing 50%
of the maximum response. L-NAME ¼N
G
-nitro-L-arginine methyl ester. Data are presented as mean ^SEM. *Significant difference vs.
control ( p,0.05; two-way ANOVA þTukey).
†
Significant vs. HD ( p,0.05; two-way ANOVA þTukey).
‡
Significant vs. the CEC obtained
in the endothelium-intact aor tic ring in the same group ( p,0.05; two-way ANOVA þTukey).
–10 –9 –8 –7 –6 –5 –4
0
25
50
75
100
Control
CMS *
HD+CMS †
HD
A
Log [phenylephrine] M
% Maximum response
–10 –9 –8 –7 –6 –5 –4
0
25
50
75
100
HD
HD+CMS
Control
CMS
B
Log [phenylephrine] M
% Maximum response
–10 –9 –8 –7 –6 –5 –4
0
25
50
75
100
HD+CMS
HD
Control
CMS
C
Log [phenylephrine]M
% Maximum response
–10 –9 –8 –7 –6 –5 –4
0
25
50
75
100
Control
CMS *
HD
HD+CMS †
D
Log [acetylcholine] M
% Relaxation
Figure 3. CEC for phenylephrine in endothelium-intact (A), endothelium-denuded (B), and in the presence of L-NAME in the
endothelium-intact (C), and CEC to acetylcholine (D) obtained in the thoracic aorta isolated from control rats, stressed rats subjected to
CMS, non-stressed rats fed with HD, and rats fed with HD and subjected to CMS (HD þCMS), obtained 15 days after stress protocol
(N¼5–6 rats/group). Data are presented as mean ^SEM. *Significant difference vs. control ( p,0.05).
†
Significant vs. HD ( p,0.05).
GSTR 601369—13/7/2011—SHANMUGAM—395854
V.J. Neves et al.6
555
560
565
570
575
580
585
590
595
600
605
610
615
620
625
630
635
640
645
650
655
660

the CMS group when compared with the control
group (Figure 3A), and a 3.2-fold shift to the left of
the HD þCMS group when compared with HD
group (Figure 3A). Among the groups, there was no
difference in CEC obtained in aortic rings without
endothelium ( p.0.05; Figure 3B; Table III) or in
CEC obtained in the presence of L-NAME ( p.0.05;
Figure 3C; Table III). In the aorta isolated from the
control and HD groups, pD
2
values for phenylephrine
obtained in the presence of L-NAME were higher in
comparison with those pD
2
values obtained in the
endothelium-intact aor tic rings of the same
group. However, L-NAME did not change pD
2
for
phenylephrine in the aorta from the CMS and
HD þCMS groups when compared with the endo-
thelium-intact aorta (Table III). Considering the CEC
to acetylcholine, two-way ANOVA revealed a signifi-
cant main effect of stress [F(1,19) ¼8.522,
p¼0.009] in the maximal acetylcholine-induced
relaxation, with no effect of diet or interaction
(p.0.05). Aortic rings isolated from stressed
rats presented a markedly impaired maximal acetyl-
choline-induced relaxation, when compared with
non-stressed groups (Control ¼95.97 ^8.22; CMS
¼61.27 ^6.14; HD ¼94.21 ^6.14; HD þCMS
¼82.09 ^7.29%) ( p,0.05, Figure 3D).
When considering aortic morphology, there was a
significant main effect of stress on the aorta IMT
[F(1,16) ¼39.217, p¼0.000], without significant
effect of diet or interaction ( p.0.05). CMS
and HD þCMS groups presented greater aorta
IMT in comparison with the control and HD
groups, respectively (Control ¼91.3 ^0.7 mm;
CMS ¼96.9 ^1.1 mm; HD ¼90.6 ^1.2 mm;
HD þCMS ¼97.7 ^1.0 mm, p,0.05).
Discussion
This study showed that CMS induced insulin
resistance, high concentration of lipids in the
circulation, impaired the function and morphology
of the thoracic aorta from the stressed groups, fed with
control or high-fat but not high-sugar HD. The data
indicate that CMS seems to be the determinant factor
for these effects, since dyslipidemia induced by the
high-fat HD alone was not sufficient to induce
functional or morphological changes in the thoracic
aorta.
As reported in other studies (Willner 1997; Marin
et al. 2007), the high plasma corticosterone concen-
trations and lower final body weight observed after
the end of the stress protocol in stressed rats in
comparison with non-stressed rats, confirm the
efficacy of the CMS protocol and support the absence
of adaptation to stressors. The aim of this study was to
analyze long-term effects of CMS. According to
Moreau et al. (1994), CMS promotes a state of
anhedonia in rats, which can last for up to 20 days
after stress. Hence, the purpose of waiting 15 days
after the end of CMS was to analyze the level of
corticosterone within this 20-day period of anhedonia.
The sustained increase in the plasma corticosterone
concentration 15 days after the end of stress confirms
that CMS is an effective protocol to study the delayed
effects of chronic stress in rats.
The higher values observed for final body weight,
FP/BW
f
, and dyslipidemia with regard to the HD
versus control diet indicate the effectiveness of HD
treatment to disrupt the control of body weight. These
effects observed in the HD and HD þCMS when
compared with the control and CMS groups could be
related to the high-fat content in the HD. The oleic
acid present in soybean oil, and coconut fat in the
presence of sucrose (Bueno et al. 2008) can readily be
converted into acetate and then into cholesterol. The
Q4
SFA contained in coconut fat, mainly lauric, myristic,
and palmitic acids (Mendis et al. 2001), and in
Q3
soybean oil (palmitic) (Bueno et al. 2008), could
increase the production of TGL and cholesterol by
the liver, and could also decrease LDL catabolism
by repressing its receptors (Kamgang et al. 2005). In
addition, when associated with oleic acid, palmitic
acid (Black 2002) could increase the biosynthesis of
chylomicrons (rich in TGL) (Silva et al. 2006).
Unlike the effects of a HD on lipids, in the present
study, there were no differences in fasting plasma
glucose and insulin concentrations between non-
stressed rats fed with hypercaloric and control diets.
Moreover, the HD alone did not induce insulin
resistance. Although HD intake can be associated with
hyperglycemia and insulin resistance, different
authors have shown that these effects are often
controversial, and seem to be dependent on a high
supply of fat associated with high-sugar content.
While a sucrose-rich diet impaired glucose homeosta-
sis and induced insulin resistance before the increase
in adiposity was detected (Soria et al. 2001), a diet rich
Q3
in coconut oil and cholesterol without a significant
increase in sugar content induced an increase in blood
lipids accompanied by hypoglycemia and lower
concentrations of serum insulin in rats (Zulet et al.
1999). In healthy women, replacing 4% of the energy
in the form of palmitic or lauric acid by mono-
unsaturated fatty acids had only minor effects on
serum lipids and lipoproteins and caused no changes
in glucose metabolism (Schwab et al. 1995). It has
also been reported that the area under the curve
during the intravenous glucose tolerance test was
significantly lower, or did not change, after healthy
women were treated with a diet enriched with
monounsaturated fatty acid or SFA, respectively
(Uusitupa et al. 1994). Therefore, the fact that the
carbohydrate supply was not increased in the HD used
in the present study could explain the absence of
differences in glucose metabolism between the HD
and control groups.
GSTR 601369—13/7/2011—SHANMUGAM—395854
Chronic stress impairs vascular function in rats 7
665
670
675
680
685
690
695
700
705
710
715
720
725
730
735
740
745
750
755
760
765
770

The duration of treatment also influences the
metabolic effects of HDs. While the treatment of
rats with a high-fat diet rich in saturated and
unsaturated fatty acids for a period of 24 weeks
increased the blood concentration of insulin and
glucose, no differences were observed after 4 or 12
weeks of treatment in comparison with the
group treated with low-fat diet (Song et al. 2006).
Therefore, the results of the present study confirm
that the effect of a HD on glucose metabolism is
dependent on the association between high-fat and
high-sucrose content, since all the rats in the
present study were subjected to the same period
of treatment and the HD had a higher lipid content
but not a higher carbohydrate content in comparison
with the control diet.
When considering the metabolic effects of chronic
stress, in the present study, it was observed that CMS
increased the blood lipid concentration, and the
association between the HD and CMS potentiated
the dyslipidemia as well as the increase in FP/BW
f
observed in the HD þCMS group in comparison
with the control and HD groups. This effect could be
related to a possible stress-induced desensitization or
down-regulation of adrenoceptors in the fat cells
(Black 2003), leading to lower catecholamine-induced
lipolysis and increased adiposity in this group.
Another hypothesis is that the interaction between
HD and CMS could have an additive effect on CMS-
induced insulin resistance. In the present study, while
the HD alone did not change glucose metabolism,
CMS increased the fasting insulin concentrations,
HOMA index values, and OGTT responses in
stressed animals when compared with non-stressed
rats. Therefore, the high TGL and TC concentrations
in the circulation of the stressed groups may be a
consequence of the insulin resistance promoted by the
stress hormones. Under chronic stress conditions,
corticosteroids may cause insulin resistance (Black
2002; Kyrou and Tsigos 2009), which facilitates TGL
synthesis in the liver and may delay the clearance of
lipoproteins, also resulting in hypercholesterolemia
(Brindley et al. 1993). Moreover, the glucocorticoids
may decrease the binding and degradation of LDL by
liver cells (Brindley et al. 1993; Black 2002).
The metabolic effects of CMS observed in the
present study are in agreement with the evidence that
chronic stress interacts with dietary habits to
determine the occurrence of metabolic diseases. In
the chronic psychosocial stress model, it has been
observed that subordination is associated with
increased weight gain and dominance is associated
with lower weight gain or weight loss in mice fed on a
standard diet. However, opposite effects were
observed in mice fed on a high-fat diet (Bartolomucci
et al. 2009). It has been proposed that when chronic
stress to which animals and humans cannot easily
adapt is combined with high-fat high-sugar diets, it
stimulates the sympathetic nerves to upregulate the
expression of neuropeptide Y, which is an adrenergic
co-transmitter and stress mediator. Stress and a HD
also increase the glucocorticoid concentration in
visceral fat, which in turn upregulates the expression
of neuropeptide Y and its receptor Y2R, resulting in
fat growth, hyperinsulinemia and hyperlipidemia
(Kuo et al. 2008). In the present study, although
there was a cumulative effect of HD plus CMS on the
elevation of blood lipid concentration, it seems that
this effect did not influence the increase in fasting
insulin or lead to insulin resistance. This could be
explained by the duration of treatment because in
the present study the rats were treated for a period of
7 weeks.
Since dyslipidemia and insulin resistance are also
associated with vascular diseases and high risk of
atherogenesis, the function and morphology of the
thoracic aorta were also evaluated in the present study.
The use of adrenergic agonists, which are not
substrates of the catecholamine uptake systems, are a
tool for the study of the mechanisms involved in
alterations of vascular reactivity. In experiments
carried out in vitro, comparison among the responses
of endothelium-intact and endothelium-denuded
aortic rings, and CEC obtained in the presence and
in the absence of inhibitors of NO synthesis, are used
as pharmacological tools to investigate the role of the
endothelium and NO, respectively, in the modulation
of vascular activity. In addition, CEC to acetylcholine
may provide a direct evaluation of NO involvement in
vascular physiology. In the present study, CMS
induced supersensitivity to phenylephrine in aortic
rings with endothelium, and this effect was indepen-
dent of diet. The absence of difference in the CEC
obtained in the endothelium-denuded aortic rings
among the groups indicates that CMS-induced
supersensitivity to phenylephrine was endothelium
dependent. The absence of difference between
groups, in aortic rings incubated with L-NAME,
which is an NO synthesis inhibitor, indicates that
endothelial NO synthase (eNOS) seems to be
inhibited in vivo, by some other factor not related to
the addition of L-NAME in the incubation bath
(Neves et al. 2009). This effect seems to stem from the
exposure to CMS. The lower relaxation response to
acetylcholine observed in the thoracic aorta isolated
from stressed groups in comparison with control and
HD groups indicates that CMS decreased NO
bioavailability.
Considering that dyslipidemia induced by HD in
non-stressed rats did not change the sensitivity to
phenylephrine or acetylcholine in the thoracic aorta, it
seems that the increase in corticosterone levels and
insulin resistance induced by CMS could be the main
factors involved in the vascular changes observed in
the present study. Glucocorticoids reduce the
expression of guanosine triphosphate cyclohydrolase
GSTR 601369—13/7/2011—SHANMUGAM—395854
V.J. Neves et al.8
775
780
785
790
795
800
805
810
815
820
825
830
835
840
845
850
855
860
865
870
875
880

1 mRNA, which is necessary for the synthesis of the
cofactor tetrahydrobiopterin, essential for stabilizing
eNOS (Mitchell et al. 2004). Insulin stimulates
endothelium-derived NO production (Muniyappa
et al. 2008) and during insulin resistance, there is a
decrease in endothelial eNOS activation and an
increase in NO destruction by the reactive oxygen
species. These effects result in diminished vasodilata-
tion (Whaley-Connell and Sowers 2009) and
increased endothelin-mediated vasoconstriction
(Muniyappa and Quon 2007), and could also be
involved in the effects of CMS.
Other authors have shown that the vascular effects
of other stress protocols are dependent on the genetic
background of the animals. Bernatova and Csizma-
diova (2006) showed that chronic crowding stress
significantly reduced NO synthase activity in the
aorta, and acetylcholine-induced relaxation of the
femoral artery in borderline hypertensive adult rats
with normotensive mothers. Bernatova et al. (2007a)
did not observe change in the activity of NO synthase
in the aorta of Wistar normotensive rats subjected to
crowding stress. In addition, Bernatova et al. (2007b)
showed that crowding stress increased the acetyl-
choline-induced relaxation in the femoral and
mesenteric arteries from normotensive Wistar-Kyoto
rats. While borderline hyper tensive rats (BHR)
subjected to crowding stress showed a reduction in
NO synthase activity, suggesting a reduction in NO
production, when compared with the control BHR, no
changes were observed in Wistar normotensive rats
(Okruhlicova et al. 2008). Cordellini et al. (2006)
studied the effect of acute and chronic stress by
immobilization in Wistar and spontaneous hyperten-
sive rats (SHR). These authors showed that the
vascular adaptive response to stress involves hyper-
activity of the endothelial NO system in normotensive
rats. However, in the SHR strain, this adaptive
response is impaired, independently of the hyperten-
sive state.
In addition to the influence of genetic background,
the differences observed between crowding and the
vascular effects of CMS could also be related to stress
intensity. Since crowding is a mild stress protocol with
predictable stressors, and seems to be capable of
eliciting adaptive mechanisms to prevent cardiovas-
cular dysfunctions in normotensive rats, the repeated
and unpredictable characteristics of the stressors used
in the CMS protocol prevent the rodents from
adapting, and seem to impair endothelial function.
Q5 This hypothesis is supported by the high plasma
corticosterone concentrations observed in stressed
rats in the present study, and the absence of significant
difference in plasma corticosterone between rats
subjected to chronic crowding stress and control rats
(Bernatova et al. 2007a).
Diminished NO availability could also be related
to the increased aorta IMT in stressed groups
(Okruhlicova et al. 2008). Morphological evaluation
of IMT in the carotid artery is considered a marker of
target organ damage in human hypertension (Sierra
and de la Sierra 2008), and experimental studies
(Razuvaev et al. 2008). Considering that decreased
NO bioavailability may lead to the loss of mitogenic
quiescence of vascular smooth muscle cells and
consequent vessel wall hypertrophy (Costa and
Assreuy 2005), the results of this study suggest that
the aortic hypertrophy could be the result of the
possible lack of NO elicited by the higher plasma
corticosterone concentrations and insulin resistance
induced by CMS.
Therefore, the proatherogenic and prohypertensive
functional and morphological changes observed in the
thoracic aorta from stressed rats are in agreement with
the high AI values observed in both the CMS and
HD þCMS groups. However, in spite of the increase
in the AI value, non-stressed rats fed with the HD diet
did not develop any alteration in the thoracic aorta.
Another hypothesis to explain the absence of diet-
induced deleterious vascular effects is that dyslipide-
mia has to be associated with high concentration of
stress hormones or insulin resistance to be capable of
generating vascular damages in rats. Further studies
are needed to evaluate these hypotheses. These results
indicate that rats, although resistant to the athero-
sclerosis induced only by diet (Moghadasian 2002),
may present proatherogenic effects induced by CMS
via insulin resistance.
In conclusion, the present study showed that the
CMS protocol appears to be an appropriate model for
the investigation of the cardiovascular and metabolic
effects of stress. Investigation into the vascular damage
associated with dyslipidemia induced by stress but not
associated with dyslipidemia induced by the con-
sumption of a high-fat, but not high-sugar, HD could
help to clarify why some people present high
cholesterol concentrations in the circulation in the
absence of cardiovascular disease, while chronically
stressed individuals could suffer heart attacks, for
example. The association between glucocorticoids,
insulin resistance, and dyslipidemia in rats may be one
of the keys of association between chronic stress and
cardiovascular diseases, and may be investigated in the
CMS protocol providing insights for future clinical
studies of the relationship between chronic stress and
atherosclerosis.
Acknowledgements
The authors thank Margery Galbraith for English
editing.
Declaration of Interest: This work was supported
by FAPESP and CNPq, Brazil. The authors report no
conflicts of interest. The authors alone are responsible
Q6 for the content and writing of the paper.
GSTR 601369—13/7/2011—SHANMUGAM—395854
Chronic stress impairs vascular function in rats 9
885
890
895
900
905
910
915
920
925
930
935
940
945
950
955
960
965
970
975
980
985
990

References
Bartolomucci A, Cabassi A, Govoni P, Ceresini G, Cero C, Berra D,
Dadomo H, Franceschini P, Dell’Omo G, Parmigiani S,
Palanza P. 2009. Metabolic consequences and vulnerability to
diet-induced obesity in male mice under chronic social stress.
PLoS One 4:e4331.
Bernatova I, Csizmadiova Z. 2006. Effect of chronic social stress on
nitric oxide synthesis and vascular function in rats with family
history of hypertension. Life Sci 78:1726 – 1732.
Bernatova I, Csizmadiova Z, Kopincova J, Puzserova A. 2007a.
Vascular function and nitric oxide production in chronic social-
stress-exposed rats with various family history of hypertension.
J Physiol Pharmacol 58:487–501.
Bernatova I, Puzserova A, Navarova J, Csizmadiova Z, Zeman M.
2007b. Crowding-induced alterations in vascular system of
Wistar-Kyoto rats: Role of nitric oxide. Physiol Res 56:667– 669.
Black PH. 2002. Stress and the inflammatory response: A review of
neurogenic inflammation. Brain Behav Immun 16:622 – 653.
Black PH. 2003. The inflammatory response is an integral part of
the stress response: Implications for atherosclerosis, insulin
resistance, type II diabetes and metabolic syndrome X. Brain
Behav Immun 17:350–364.
Brindley DN, McCann BS, Niaura R, Stoney CM, Suarez E. 1993.
Stress and lipoprotein metabolism: Modulators and mechan-
isms. Metabolism 42:3–15.
Bueno AA, Oyama LM, de Oliveira C, Pisani LP, Ribeiro EB,
Silveira VL, Oller do Nascimento CM. 2008. Effects of different
fatty acids and dietary lipids on adiponectin gene expression in
3T3-L1 cells and C57BL/6J mice adipose tissue. Pflugers Arch
455:701– 709.
Cinti S. 2005. The adipose organ. Prostaglandins Leukot Essent
Fatty Acids 73:9– 15.
Cordellini S, Novo R, Lanza U, Jr. 2006. Exposure to stress:
Differential vascular adaptive response in spo ntaneously
hypertensive and Wistar rats: Role of nitric oxide, and
prohypertensive and hypertensive states. Life Sci 79:646– 653.
Costa RS, Assreuy J. 2005. Multiple potassium channels mediate
nitric oxide-induced inhibition of rat vascular smooth muscle cell
proliferation. Nitric Oxide 13:145–151.
Cunha TS, Moura MJCS, Bernardes CF, Tanno AP, Marcondes
FK. 2005. Vascular sensitivity to phenylephrine in rats submitted
to anaerobic training and nandrolone treatment. Hypertension
46:1010– 1015.
Friedewald WT, Levy RI, Fredrickson DS. 1972. Estimation of the
concentration of low-density lipoprotein cholesterol in plasma
without use of the preparative ultracentrifuge. Clin Chem 18:
499– 502.
Gu H, Tang C, Peng K, Sun H, Yang Y. 2009. Effects of chronic
mild stress on the development of atherosclerosis and expression
of tol l-like receptor 4 s ignaling pathway in adolescent
apolipoprotein E knockout mice. J Biomed Biotechnol 613879:
10–13.
Kamgang R, Mboumi RY, N’dille
´GP, Yonkeu JN. 2005. Cameroon
local diet-induced glucose intolerance and dyslipidemia in adult
Wistar rat. Diabetes Res Clin Pract 69:224 – 230.
Kaplan JR, Manuck SB, Clarkson TF, Lusso FM, Taub DM, Miller
EW. 1983. Social stress and atherosclerosis in normocholester-
olemic monkeys. Science 220:733– 735.
Karnovsky MJ. 1965. A formaldehyde– glutaraldehyde fixative of
high osmolarity for use in electron microscopy. J Cell Biol 27:
137A– 138A.
Kuo LE, Czarnecka M, Kitlinska JB, Tilan JU, Kvetnansky
´R,
Zukowska Z. 2008. Chronic stress, combined with a high-fat/
high-sugar diet, shifts sympathetic signaling toward neuropep-
tide Yand leads to obesity and the metabolic syndrome. Ann NY
Acad Sci 1148:232– 237.
Kyrou I, Tsigos C. 2007. Stress mechanisms and metabolic
complications. Horm Metab Res 39:430–438.
Kyrou I, Tsigos C. 2009. Stress hormones: Physiological stress and
regulation of metabolism. Curr Opin Pharmacol 9:787 – 793.
Lauterio TJ, Bond JP, Ulman EA. 1994. Development and
characterization of a purified diet to identify obesity-susceptible
and resistant rat populations. J Nutr 124:2172–2178.
Levin BE, Richard D, Michel C, Servatius R. 2000. Differential
stress responsivity in diet-induced obese and resistant rats.
Am J Physiol Regul Integr Comp Physiol 279:R1357– R1364.
Ludwigh DS. 2003. Novel treatments for obesity. Asia Pac J Clin
Nutr 12:S8.
Luft JH. 1961. Improvements in epoxy resin embedding methods.
J Biophys Biochem Cytol 9:409– 414.
Marin MT, Cruz FC, Planeta CS. 2007. Chronic restraint or
variable stresses differently affect the behavior, corticosterone
secretion and body weight in rats. Physiol Behav 90:29– 35.
Mendis S, Samarajeewa U, Thattil RO. 2001. Coconut fat and
serum lipoproteins: Ef fects of pa rtial replacement with
unsaturated fats. Br J Nutr 85:583 – 589.
Mitchel l BM, Dorrance AM, Mack EA, Webb RC. 2004.
Glucocorticoids decrease GTP cyclohydrolase and tetrahydro-
biopterin-dependent vasorelaxation through glucocorticoid
receptors. J Cardiovasc Pharmacol 43:8– 13.
Moghadasian MH. 2002. Experimental atherosclerosis: A historical
overview. Life Sci 70:855–865.
Moreau JL. 1997. Validation of an animal model of anhedonia, a
major symptom of depression. Encephale 23:280–289.
Moreau JL, Bourson A, Jenck F, Martin JR, Mortas P. 1994.
Curative effects of the atypical antidepressant mianserin in
the chronic mild stress-induced anhedonia model of depression.
J Psychiatry Neurosci 19:51 – 56.
Moura MJCS, Marcondes FK. 2001. Influence of estradiol and
proges terone on the sensitivity of rat thoracic aor ta to
noradrenaline. Life Sci 68:881– 888.
Muniyappa R, Quon MJ. 2007. Insulin action and insulin resistance
in vascular endothelium. Curr Opin Clin Nutr Metab Care 10:
523–530.
Muniyappa R, Iantorno M, Quon MJ. 2008. An integrated view of
insulin resistance and endothelial dysfunction. Endocrinol
Metab Clin North Am 37:685 – 711.
Neves VJ, Moura MJCS, Tamascia ML, Ferreira R, Silva NS, Costa
R, Montemor PL, Narvaes EAO, Bernardes CF, Novaes PD,
Marcondes FK. 2009. Proatherosclerotic effects of chronic stress
in male rats: Altered phenylephrine sensitivity and nitric oxide
synthase activity of aorta and circulating lipids. Stress 12:
320–327.
Okruhlicova L, Dlugosova K, Mitasikova M, Bernatova I. 2008.
Ultrastructural characteristics of aortic endothelial cells in
borderline hypertensive rats exposed to chronic social stress.
Physiol Res 57:S31– S37.
Razuvaev A, Lund K, Roy J, Hedin U, Caidahl K. 2008.
Noninvasive real-time imaging of intima thickness after rat
carotid artery balloon injury using ultrasound biomicroscopy.
Atherosclerosis 199:310– 316.
Reeves PG, Nielsen FH, Fahey GC, Jr. 1993. AIN-93 purified diets
for laboratory rodents: Final report of the American Institute of
Nutrition ad hoc writing committee on the reformulation of the
AIN-76A rodent diet. J Nutr 123:1939–1951.
Schwab US, Niskanen LK, Maliranta HM, Savolainen MJ,
Kesa¨niemi YA, Uusitupa MI. 1995. Lauric and palmitic acid-
enriched diets have minimal impact on serum lipid and
lipoprotein concentrations and glucose metabolism in healthy
young women. J Nutr 125:466– 473.
Sheril A, Jeyakumar SM, Jayashree T, Giridharan NV, Vajreswari A.
2009. Impact of feeding polyunsaturated fatty acids on
cholesterol metabolism of dyslipidemic obese rats of WNIN/
GR-Ob strain. Atherosclerosis 204:136– 140.
Shively CA, Register TC, Clarkson TB. 2009. Social stress, visceral
obesity, and coronary artery atherosclerosis: Product of a
primate adaptation. Am J Primatol 71:742–751.
GSTR 601369—13/7/2011—SHANMUGAM—395854
V.J. Neves et al.10
995
1000
1005
1010
1015
1020
1025
1030
1035
1040
1045
1050
1055
1060
1065
1070
1075
1080
1085
1090
1095
1100

Sierra C, de la Sierra S. 2008. Early detection and management of
the high-risk patient with elevated blood pressure. Vasc Health
Risk Manag 4:289– 296.
Silva AP, Guimara
˜es DE, Mizurini DM, Maia IC, Ortiz-Costa S,
Sardinha FL, do Carmo MG. 2006. Dietary fatty acids early in
life affect lipid metabolism and adiposity in young rats. Lipids
41:535– 541.
Søndergaard LG, Stoltenberg M, Doering P, Flyvbjerg A, Rungby J.
2006. Zinc ions in the endocrine and exocrine pancreas of zinc
deficient rats. Histol Histopathol 21:619–625.
Song GY, Gao Y, Di YW, Pan LL, Zhou Y, Ye JM. 2006. High-fat
feeding reduces endothelium-dependent vasodilation in rats:
Differential mechanisms for saturated and unsaturated fatty
acids? Clin Exp Pharmacol Physiol 33:708–713.
Soria A, D’Alessandro ME, LombardoYB. 2001. Duration of feeding
on a sucrose-rich diet determines metabolic and morphological
changes in rat adipocytes. J Appl Physiol 91:2109–2116.
Tran TT, Kahn CR. 2010. Transplantation of adipose tissue and
stem cells: Role in metabolism and disease. Nat Rev Endocrinol
6:195–213.
Uusitupa M, Schwab U, Sari Makimattila S, Karhapaa P, Sarkkinen
E, Maliranta H, Agren J, Penttila I. 1994. Effects of two high-fat
diets with different fatty acid compositions on glucose and lipid
metabolism in healthy young women. Am J Clin Nutr 59:
1310– 1316.
Vahl TP, Ulrich-Lai YM, Ostrander MM, Dolgas CM, Elfers EE,
Seeley RJ, D’Alessio DA, Herman JP. 2005. Comparative
analysis of ACTH and corticosterone sampling methods in rats.
Am J Physiol Endocrinol Metab 289:E823–E828.
Whaley-Connell A, Sowers JR. 2009. Hypertension and insulin
resistance. Hypertension 54:462 – 464.
Willner P. 1997. Validity, reliability and utility of the chronic mild
stress model of depression: A 10-year review and evaluation.
Psychopharmacology (Berl) 134:319–329.
Zulet MA, Barber A, Garcin H, Higueret P, Martı
´nez JA. 1999.
Alterations in carbohydrate and lipid metabolism induced by a
diet rich in coconut oil and cholesterol in a rat model. J Am Coll
Nutr 18:36–42.
GSTR 601369—13/7/2011—SHANMUGAM—395854
Chronic stress impairs vascular function in rats 11
1105
1110
1115
1120
1125
1130
1135
1140
1145
1150
1155
1160
1165
1170
1175
1180
1185
1190
1195
1200
1205
1210