Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223

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Autonomic Neuroscience: Basic and Clinical

journal homepage: www.elsevier.com/locate/autneu

Does exposure to chronic stress influence blood pressure in rats?

Larisa Bobrovskaya ⁎, 1, Daniel Beard 1, Evgeny Bondarenko, Mirza I. Beig, Phillip Jobling, Frederick R. Walker,
Trevor A. Day 2, Eugene Nalivaiko
School of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW, Australia

a r t i c l e i n f o a b s t r a c t

Article history: The principal aim of this study was to determine whether prolonged chronic footshock stress can evoke
Received 30 November 2012 sustained changes in blood pressure in rats and to elucidate possible underlying neurochemical mechanisms
Received in revised form 2 May 2013 as mediated by the sympathoadrenal system. Adult male Wistar rats instrumented for telemetric recording of
Accepted 4 May 2013
arterial pressure, heart rate and locomotor activity were subjected to six weeks of inescapable unpredictable
electrical footshocks (FS+) or were exposed to shock chambers but were not shocked (FS−). Compared to
Keywords:
Footshock stress
FS− animals, FS+ animals had significantly reduced body weight gain (by 30%), locomotor activity (by 25%)
Blood pressure and social interaction time (by 30%) — symptoms commonly induced by chronic stress and depression in
Sympathetic ganglia humans. These changes were associated with small, but significant increases in systolic blood pressure (by 7%)
Adrenal gland and pulse pressure (by 11%) in FS+ rats relative to FS− rats. We have also found neurochemical alterations in
Tyrosine hydroxylase sympathoadrenal pathways (that lasted for at least one week post-stress) including about 2–3 fold increases
Angiotensin receptor in the levels of tyrosine hydroxylase phosphorylation in the sympathetic ganglia and adrenal gland and a
1.8-fold increase in the expression of the Angiotensin II receptor type 1 protein in the adrenal gland of FS+
rats relative to FS− rats. We conclude that uncontrollable and unpredictable footshock stress can lead to eleva-
tion in systolic blood pressure when applied for an extended period of time (six weeks) in Wistar rats, and that
these changes could be mediated by stress-induced modifications in sympathoadrenal pathways.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction how acute challenges provoke rapid physiological alterations, the

Epidemiological studies have demonstrated that individuals exposed
to chronically stressful life events frequently exhibit persistent hyperten-
sion (Henry and Cassel, 1969; Pickering, 2004; Steenland et al., 2000;
Timio et al., 1988). Importantly, studies investigating stress-linked hy-
pertension have observed that elevations in arterial pressure (AP) can
persist long after the termination of stress events, and in many instances
continue during sleep (Schnall et al., 1992). Together these findings sug-
gest that psychological stressors alter the regulation of AP during stress
exposure and that these alterations persist well beyond the duration of
stressor.
Thus far the large majority of research that examined the relation-
ship between stress and AP has focused on acute stress. While these
studies have been informative from the perspective of understanding
Abbreviations: FS, footshock; AP, arterial pressure; TH, tyrosine hydroxylase; Ang II,
Angiotensin II; AT1R, Angiotensin II receptor type 1; Ser, serine residue.
⁎ Corresponding author at: School of Pharmacy and Medical Sciences, University of
South Australia, Adelaide, SA 5000, Australia. Tel.: +61 8 830 21218; fax: +61 8 830
22389.
E-mail address: Larisa.Bobrovskaya@unisa.edu.au (L. Bobrovskaya).
1
Both authors contributed equally to this work.
2
Current address: Faculty of Science, Engineering and Built Environment, Deakin
University, Victoria, Australia.
models used do not replicate the conditions under which stress leads
to sustained hypertension in humans. Certainly, there have been several
studies that have examined the impact of chronic stress on AP but the
results have been difficult to interpret (see Nalivaiko (2011) for recent
review). For instance, several studies investigating effects of chronic
stress on AP have used the tail-cuff procedure to measure blood pressure
in experimental animals. It is now well recognised that this procedure
causes substantial pressor and tachycardic responses (Van Acker et al.,
2001), as well as elevation in plasma noradrenaline and Angiotensin II
(Ang II) concentrations, even following recommended habituation
(Grundt et al., 2009). It is quite possible therefore that the changes in
AP following tail cuff recordings are attributable to the approach taken
to acquire the measurements.
Several studies have, nevertheless, employed the more reliable bio-
telemetry method or indwelling catheters to measure blood pressure.
Reports using this more refined approach have consistently shown
that chronic stress does not influence AP (Gelsema et al., 1994; Grippo
et al., 2003; Lemaire and Mormède, 1995). Critically, however, these re-
ports have used relatively mild forms of stress. Moreover, the amount of
stress experienced by the animals subjected to interventions such as so-
cial defeat, social isolation or chronic mild stress is difficult to quantify.
As such, it is not entirely clear whether the reported negative results
should be interpreted as indicating that chronic stress cannot alter AP
in rodents or that the intensity of the stress was simply insufficient to

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218 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223
cause a measurable change. This latter possibility seems considerably
more likely as it is widely recognised that principle determinant of a
stressful experience is the degree to which it is unpredictable and uncon-
trollable (Maier, 1984). Having actual or perceived control over a stress-
ful situation powerfully reduces its negative physiological consequences.
It is possible therefore that in previous studies in which animals were
exposed to relatively mild stressors and also allowed the animal a signif-
icant degree of latitude with respect to behavioural modification of the
experience may have mitigated the overall impact of the exposure
regimen.
Consequently, the primary aim of the current study was to determine
whether uncontrollable and unpredictable footshock, an extremely well
characterised and easily quantifiable stressor, could evoke a sustained
elevation in AP if applied for an extended period of time (six weeks). Im-
portantly, we used constant telemetric monitoring of arterial pressure
and heart rate. Additionally, some established stress-linked behavioural
changes were monitored using the social interaction and locomotor
activity tests. These tests were chosen for confirming that our stress pro-
tocol was potent enough to elicit symptoms typically present in chroni-
cally stressed or depressed patients — reduced motivation and reduced
motor drive (Grippo and Johnson, 2009; Hammen, 2005). We also
intended to investigate the impact of chronic footshock stress on activa-
tion of the sympathoadrenal system, as chronic activation of this system
can lead to increased catecholamine release from the sympathetic nerve
terminals and adrenal gland and therefore may contribute to a number
of cardiovascular disorders in humans and experimental animals includ-
ing hypertension (Grassi et al., 2008; Grassi et al., 2010; Lee et al., 1991;
Parati and Esler, 2012). Specifically, we assessed the expression and
phosphorylation of tyrosine hydroxylase (TH) and the levels of Ang II re-
ceptor type 1 (AT1R) in the adrenal gland and in the stellate ganglia —
the indices previously associated with chronic stress and/or sympathetic
activation (Baruchin et al., 1990; Cierco and Israel, 1994; Dendorfer et al.,
2002; Fluharty et al., 1983; Kvetnansky et al., 2004; Ma et al., 2001;
Nankova et al., 1996; Powis and Obrien, 1991; Stromberg et al., 1991).
2. Materials and methods
2.1. Ethical approval
All animal procedures were approved by the Ethics Committee of
the University of Newcastle (Australia) and animals were cared for
according to the principles of the Australian code of practice for the
care and use of animals for scientific purposes (7th edition 2004).
2.2. Experimental animals
Experiments were conducted on adult male Wistar rats (between 7
and 10 weeks of age). All animals were housed individually in an animal
holding facility, with rat chow and water available ad libitum. The room
temperature was maintained at 21 °C ± 1 °C and animals were kept on
a reversed 12 h:12 h light/dark cycle (lights on at 19.00 h).
2.3. Telemetry blood pressure recording
Telemetry probes (TA11PA- C40, Data Sciences International USA)
were implanted one week prior to the commencement of the experimen-
tal protocol. Surgery was conducted under 2% isoflurane in 100% oxygen
anaesthesia as described previously (Beig et al., 2011). The transmitter's
catheter was inserted into the left femoral artery via a small incision;
with the catheter tip advanced into the abdominal aorta. Animals recov-
ered from anaesthesia were returned to their home cage where they
were left for one week. Telemetric transmitters were configured and
calibrated both before implantation and after explantation. When we
compared calibrations made before implantation and after explantation,
we did not find any detectable changes in gain. We assessed not only
base level, but also sensitivity (by placing probes in a 50 ml syringe and
increasing pressure in the syringe, with the syringe output connected to
a manometer). Furthermore, the DSI system incorporated the Ambient
Pressure Monitor that made automatic correction for changes in the baro-
metric ambient pressure.
The radiotelemetry system employed in this study allowed recording
of AP, heart rate and locomotor activity in freely-moving animals. These
parameters were continuously acquired 24 h a day 7 days a week
during 7 weeks of the experimental protocol (except 40 min footshock
sessions). Cardiovascular parameters (heart rate and mean AP) were
acquired using the Dataquest ART software (Data Sciences International,
USA). The Data Sciences program compressed the data into 10 second
averages for systolic blood pressure, diastolic blood pressure and subse-
quently calculated pulse pressure and mean arterial pressure from these
values. Due to the sheer size of the data we then further compressed the
raw data into daily averages and weekly averages.
2.4. Chronic footshock stress protocol
Footshock procedure was performed by placing animals into a
perspex footshock cylinder (22 cm long × 9 cm diameter) located in-
side an enclosed box. At the commencement of the footshock session
the programmable animal shocker (San Diego Instruments, USA) de-
livered an electrical current (1.5 mA) through the grids to the feet of
the animal for 1 s. Each footshock stress session lasted 40 min during
which time the animal received six shocks, with a randomly allocated
interval between each shock to avoid predictability. Following each
session the animals were returned to their home cage. The condition
of the animal feet was monitored regularly to ensure that there were
no adverse effects of the footshock procedure.
Animals were randomly assigned to two experimental groups:
footshock-sham (FS − group; n = 6) and footshock-stress (FS +
group; n = 6). FS + animals were placed in the footshock apparatus
and received footshocks as described above. FS − animals were
placed in the footshock apparatus but did not receive footshocks.
The experiment lasted for seven weeks in total for each group. During
week 0 the animals were left undisturbed in order to obtain a mea-
sure of resting baseline parameters. During weeks 1–6, FS + animals
were exposed to the footshocks three times a week (weeks 1–4) or
seven times a week (weeks 5–6). During the post-stress week 7,
animals were re-exposed to shock chambers (without shocks) for
5 min, with concurrent recording of ultrasound vocalisation. Animals
were weighed once a week. Animals were euthanized at the end of
week 7 by an anaesthetic overdose, sodium pentobarbital (Lethabarb,
80 mg/kg i.p.) as reported in our previous study (Damanhuri et al.,
2012). Stellate ganglia and adrenal glands were collected for bio-
chemical assessment of TH and AT1R expression.
2.5. Assessment of animal behaviour
2.5.1. Ultrasonic vocalisation
During week 0 (pre-stress) experimental animals were placed in the
footshock apparatus and remained within these boxes for 1 min with-
out being shocked. During this time 22-kHz ultrasonic vocalisations
were recorded using a MiniBat ultrasound microphone (Ultra Sound
Advice, UK) and acquired using lab chart software (ADI instruments,
Australia). During week 7 (post-stress) all experimental animals were
once again placed into the same footshock apparatus that they were ex-
posed to throughout the experimental protocol. Animals remained
within these boxes for 1 min without being shocked. During this time
22-kHz ultrasonic vocalisations were recorded in order to detect alter-
ations in animal vocalisation following chronic stress.
2.5.2. Social interaction test
The test was performed during week 7. Social interaction was tested
in a square arena 50 × 50 × 40 cm all painted in black under low light
(Overstreet and Griebel, 2004). Rats were paired up with a naive, age-
 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223
and size-matched target rat (this naive rat never came into contact with
the experimental animals and was not exposed to the footshock proto-
col or sham procedure). Both animals were placed simultaneously into
the arena and were allowed to interact for a period of 10 min. Social in-
teraction was videotaped using an infrared camera mounted above the
arena. Social behaviour was manually scored offline by an observer
blind to treatment. The duration of social interaction for each animal
was defined as time during which the test rat was sniffing, grooming
or following the target rat (Cecchi et al., 2002; File and Seth, 2003;
Starr et al., 2007).
2.6. Measurement of TH phosphorylation, TH protein and AT1R protein in
the stellate ganglia and the adrenal glands
Tissues were homogenised and the samples were run on 10% SDS-
PAGE and then transferred to nitrocellulose as described previously
(Bobrovskaya et al., 2010). Membranes were immunoblotted with
total TH, phospho-Ser40-specific TH or AT1R antibodies overnight at
4 °C. AT1R specific antibody (N-10; sc-1173) was from Santa Cruz Bio-
technology (USA). Total TH and phospho-Ser40-specific TH antibodies
were generated and tested for specificity as previously described
(Gordon et al., 2009). Secondary antibodies (goat anti-rabbit or rabbit
anti-sheep immunoglobulin, Peirce (USA)) were applied to the mem-
branes for 1 h at room temperature. The immunoblots were visualised
and quantified on the Fuji LAS 4000 imaging system (GE Health Care,
Little Chalfont, UK) using ECL-advanced detection reagents. The density
of the bands was measured using Fujifilm Multigauge v3.0 software
(Tokyo, Japan) and expressed as a fold increase relative to FS− animals.
The loading controls were performed by analysis of the total TH protein
and β-actin protein. TH phosphorylation at Ser40 (as an index of TH
activity (Dunkley et al., 2004)) was expressed as the ratio of TH phos-
phorylation at Ser40 (pSer40TH) to total TH protein, to account for
variability in total TH protein between samples. Total TH protein and
AT1R protein levels were expressed as the ratio of TH or AT1R protein
to β-actin.
2.7. Statistical analysis
Student's unpaired t-tests were used for comparison of heart rate,
systolic blood pressure, diastolic blood pressure, pulse blood pressure
and locomotor activity between groups during both the stress and
stress-free periods. Student's unpaired t-tests were also used for
comparison of AT1R expression, pSer40TH, TH protein expression
and social interaction between groups. P values b 0.05 were deemed
significant.
3. Results
3.1. Effect of chronic footshock stress on body weight, locomotor activity
and behaviour
The mean weekly values for body weight and locomotor activity for
baseline (“B”), stress period (“On”) and post-stress period (“Off”) are
presented in Fig. 1A & C, respectively. The average delta values for the
whole stress period (“On”) versus post-stress period (“Off”) are pres-
ented in Fig. 1B & D, respectively. Chronic stress evoked substantial re-
duction in the body weight gain in the FS+ group relative to the FS−
group during both stress and post-stress periods (p b 0.01; Fig. 1B).
Likewise, locomotor activity of the FS+ group was significantly reduced
compared to the FS− group during both stress (“On”) and post-stress
(“Off”) periods (Fig. 1D).
The time of social interaction was substantially reduced in FS +
rats compared to FS − rats when assessed in week 7 (post-stress) of
the experimental protocol (p b 0.05; Fig. 1E). Emission of 22-kHz
ultrasonic vocalisations was measured to assess the degree of distress
during re-exposure to context. Pre-stress, none of the FS + or FS −
219
rats emitted ultrasonic vocalisations when exposed to the footshock
apparatus in the absence of stress. Post-stress, none of FS− rats emitted
22-kHz ultrasonic vocalisations when exposed to the footshock appara-
tus in the absence of stress. In contrast, all rats in the FS+ group emitted
ultrasonic vocalisations when exposed to the footshock apparatus in the
absence of stress.
3.2. Effect of chronic footshock stress on cardiovascular parameters
The mean weekly values for systolic, diastolic and mean arterial
pressure, pulse blood pressure and heart rate for baseline (“B”), stress
period (“On”) and post-stress period (“Off”) are presented in Fig. 2A,
C, E, G & I, respectively. The average delta values for the same param-
eters for the whole stress period (“On”) versus post-stress period
(“Off”) are presented in Fig. 2B, D, F, H & J respectively. Our results
demonstrate that the average change in systolic blood pressure was
significantly higher in FS + rats in comparison to FS − rats during
stress period (p b 0.05) and this increase seemed to sustain during
post-stress week 7 but did not reach statistical significance (p = 0.053)
(Fig. 2B). There were no significant differences in the average changes
in diastolic blood pressure (Fig. 2D) or mean arterial pressure (Fig. 2F) be-
tween groups during stress period (“On”) or post-stress period (“Off”).
The average change in pulse blood pressure was significantly higher
(p b 0.01) in FS+ rats in comparison to FS− rats during stress period
(“On”) and this change was sustained (p b 0.01) during post-stress
week (“Off”) (Fig. 2H). We were unable to perform telemetric recordings
during footshock sessions, and for this reason we compared cardiovascu-
lar parameters immediately after the footshock sessions. We found that
differences in AP and heart rate between FS+ and FS− animals were
present only during the first 2–3 min after the footshock sessions.
Although subsequent return to the baseline took more than 1 h, the
time course of this return was similar in both groups; therefore all data
throughout the experimental protocol were included in the analysis.
There was a decrease in average heart rate over time for both experi-
mental groups (Fig. 2I & J) but there was no significant difference in
the heart rate between the groups. There was no habituation to stress
procedure in the FS+ group throughout the experimental protocol:
maximal values of tachycardic and pressor changes and their time
course to the baseline in week 1 were not significantly different from
that in week 6 (not shown).
3.3. Effect of chronic footshock stress on the AT1R protein, TH protein and
pSer40TH in the stellate ganglia and adrenal gland
Representative immunoblots for the levels of TH protein, pSer40TH
and AT1R protein in stellate ganglia and adrenal gland are presented in
Fig. 3A & C. In the stellate ganglia there were no significant differences
in the expression of the TH protein or AT1R protein between the experi-
mental groups (Fig. 3B). However, there was a 2.3-fold increase
(p b 0.05) in pSer40TH in the FS+ group relative to FS− group
(Fig. 3B). In the adrenal gland there was a significant increase in
pSer40TH (3.3-fold, p b 0.05) and AT1R protein (1.8-fold, p b 0.05) in
the FS+ group relative to FS− group (Fig. 3D).
4. Discussion
Our major finding is that chronic stress caused pronounced behav-
ioural and neurochemical changes, which were associated with some in-
creases in systolic and pulse blood pressure without effects on mean
blood pressure. Indeed, reduced locomotor activity and reduced social
activity in our stressed rats closely resembled symptoms induced by
chronic stress and depression in humans. These changes were associated
with neurochemical alterations in the sympathoadrenal system, namely
increases in AT1R expression and pSer40TH in the adrenal gland and
pSer40TH in sympathetic ganglia. These data provide some evidence
220 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223

A B On Off B
500
25 FS- **
475 FS+

Body Weight (g)

20

Δ Body Weight
(% Baseline)
450 15 **
425 10

400 5

375 0
0 1 2 3 4 5 6 7 On Off

C 3.5
D 30
*

Δ Locomotor Activity
Locomotor Activity

3.0 20
(Arbitray Units)

(% baseline)
10 *
2.5
0
2.0
-10
1.5
-20

1.0 -30
0 1 2 3 4 5 6 7 On Off
Time (Weeks) Stress

E *
200
Social Interaction (s)

150

100

50

0
FS- FS+
Post-stress week

Fig. 1. Effect of chronic footshock stress on body weight (A, B), locomotor activity (C, D) and social interaction time (E) in chronically stressed (n = 6) and control (n = 6) animals.
(A, C) illustrate weekly averages for stressed (black circles) and non-stressed (white circles) rats during the baseline week (“B”), the stress period (“On”) and the final stress-free
week (“Off”). (B, D) illustrate the average changes for stressed (black columns) and control (white columns) rats during the entire 6 week stress period (“On”) and 1 week
post-stress period immediately following termination of the stress protocol (“Off”). (E) illustrates social interaction time in chronically stressed and control animals measured dur-
ing post-stress period (week 7). Data are presented as mean ± S.E.M. *p b 0.05; **p b 0.01 compared to control during the same period.

that exposure to chronic unpredictable and uncontrollable stress in the 4.2. Chronic stress did not cause alterations in mean arterial pressure,
rat can have some impact on arterial blood pressure. but significantly increased systolic and pulse blood pressure

The inability of pre-clinical studies to consistently recapitulate epide-

4.1. Chronic footshock stress leads to reduction in body weight gain and
stress-related behaviours in FS + animals
Chronic stress significantly reduced body weight gain, locomotor
activity and social interaction of the FS + rats relative to FS − rats.
These observed symptoms are commonly found in depressed and
chronically stressed humans, and we consider such behavioural vali-
dation of our model as one of the strength of our experimental design
(Grippo and Johnson, 2009; Hammen, 2005). In addition, we have
found that FS + rats (but not FS − rats) emitted 22-kHz vocalisations
when re-exposed to the footshock apparatus at the end of experimen-
tal protocol during week 7 (stress-free week). Emission of 22-kHz
ultrasonic vocalisations is strongly regarded as a correlate for stress
and anxiety (Cuomo et al., 1992). Our findings therefore confirm that
our stress protocol was indeed stressful to rats and was able to induce
anxiety/depression-like behaviours (Cullen and Rowan, 1994).
miological findings demonstrating that hypertension arises as a result of
prolonged exposure to psychological stress has been an enduring puzzle
within the field of cardiovascular research. Recently, the many possible
contributing factors to the situation have been reviewed (Nalivaiko,
2011). Briefly a few plausible explanations were proposed: 1) using the
tail-cuff method for assessing AP; 2) using stressors that were relatively
mild in nature and were difficult to accurately quantify; and 3) the ability
of animals to develop coping strategies to avoid stressful events during
chronic stress protocols. In the present study we tried to address these
limitations and have demonstrated for the first time that severe, long-
lasting and unpredictably applied chronic stress which altered animal
behaviour and some markers of sympathoadrenal activity caused signif-
icant increases in systolic and pulse blood pressure without affecting
mean arterial and diastolic pressure. The principal determinant of the
diastolic pressure is vascular tone, while the systolic and the pulse pres-
sure depend on both vascular tone and stroke volume. Thus, one possible
 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223 221

A 150
B On Off B 4 FS-
*
FS+
145

Δ Systolic BP
(% baseline)
2

Systolic BP
(mmHg)
140
0

135
-2
130
0 1 2 3 4 5 6 7 On Off

C 110 D 8

Δ Diastolic BP
105
Diastolic BP

(% baseline)
4
(mmHg)

100
2

95 0

-2
90
0 1 2 3 4 5 6 7 On Off

E 120
F 2

1
(% baseline)
115
(mmHg)

Δ MAP
MAP

110
-1

105 -2
0 1 2 3 4 5 6 7 On Off

G 45 H 20
**
**
10
(% baseline)
Δ Pulse BP

40
Pulse BP
(mmHg)

-10
35
-20

30 -30
0 1 2 3 4 5 6 7 On Off

I 425
J -2

400 -4
Δ Heart Rate
(% baseline)
Heart Rate

375
(BPM)

-6

350
-8
325
-10
300
0 1 2 3 4 5 6 7 On Off
Time (Weeks) Stress

Fig. 2. Effect of chronic footshock stress on systolic blood pressure (A, B), diastolic blood pressure (C, D), mean arterial blood pressure (E, F), pulse blood pressure (G, H) and heart
rate (I, J) in chronically stressed (n = 6) and control (n = 6) animals. (A, C, E, G, I) illustrate weekly averages for stressed (black circles) and non-stressed (white circles) rats dur-
ing the baseline week (“B”), the stress period (“On”) and the final stress free week (“Off”). (B, D, F, H, J) illustrate the average changes for stressed (black columns) and control
(white columns) rats during the entire 6 week stress period (“On”) and 1 week post-stress period immediately following termination of the stress protocol (“Off”). Data are
presented as mean ± S.E.M. *p b 0.05; **p b 0.01 compared to control during the same period.
222 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223

A Stellate ganglia C Adrenal gland

TH TH
pSer40 pSer40

AT1R AT1R

β-actin β-actin
FS- FS+
FS- FS+

B D
5 5 * FS-
FS-
4 FS+ 4 FS+

Fold increase
Fold increase

3
* 3
*
2 2

1 1

0 0
TH pSer40 AT1R TH pSer40 AT1R

Fig. 3. Effect of chronic footshock stress on TH protein expression, TH phosphorylation at Ser40 (pSer40) and AT1R expression in stellate ganglia (A, B) and adrenal gland (C, D) in
chronically stressed (n = 6) and control (n = 6) animals. (A, C) Representative immunoblots showing the levels of expression of TH, pSer40TH and AT1R in stellate ganglia (A) and
adrenal gland (B) measured at the end of the experimental protocol (week 7). (B, D) Histograms showing quantitated data for total TH/β-actin, pSer40/TH and AT1R/β-actin levels
in stellate ganglia (B) and adrenal gland (D) of chronically stressed (black columns) and control (white columns) rats. Data are expressed as the mean of the fold increase ± S.E.M.
*p b 0.05.

explanation of hypertensive changes observed in our study could be
sympathetically-induced increase in myocardial contractility, without
effects on the peripheral vascular resistance. This is however unlikely,
as heart rate remained unaffected by chronic stress. Another possibility
is that sympathetic vascular outflow did increase, but only to the minor
extent, so that blood vessels became less compliant without reduction
of their diameter (Alicandri et al., 1980). As a result, the mild/minor in-
crease in the vascular sympathetic activity might make the arterial wall
more rigid, without reducing vessel diameter. In this situation, the same
stroke volume would cause larger pulse pressure and systolic pressure
changes but diastolic pressure would not be affected.
There was a reduction (although not significant) in systolic pressure
and pulse pressure between week 0 (baseline) and week 1 for the con-
trol group (Fig. 2A & G), which can be explained by a change in the level
of novelty associated with the testing environment. A similar reduction,
although of less magnitude, can be seen with the locomotor activity
data (Fig. 1C).
Average heart rate decreased over time for both experimental
groups which is an age related alteration, as has been shown previously
(Goldberg et al., 1988; Minami et al., 1989).
4.3. Chronic stress leads to activation of the sympathoadrenal system
In this study we observed a clear activation of the sympathoadrenal
system as evidenced by increases in phosphorylation of TH and the
expression of AT1R in sympathetic ganglia and adrenal gland. The in-
volvement of AT1R in pressor and tachycardic responses to acute
stressors is well documented, and comprises both central and peripheral
mechanisms (Castren and Saavedra, 1989; Head and Mayorov, 2001;
Stromberg et al., 1991). It is well established that Ang II can enhance
sympathetic nervous system function by activating prejunctional AT1R
located on sympathetic nerve terminals. In particular, activation of
AT1R facilitates the release of catecholamines from the adrenal medulla
(Bunn and Marley, 1989; Powis and Obrien, 1991) and from sympathet-
ic nerve terminals in the myocardium (Dendorfer et al., 2002; Ma et al.,
2001). Therefore, Ang II can modulate cardiovascular function during
acute stress by directly increasing the release of catecholamines from
sympathetic nerves and adrenal gland. Less is known about the involve-
ment of Ang II signalling in chronic stress. Several rodent studies
reported stress-induced increases in renin and/or Ang II in the brain,
plasma and various peripheral tissues (Aguilera et al., 1995; Yang et
al., 1993). Chronic stress also leads to the increase of Ang II receptors
in the brain (Dumont et al., 1999). Substantial increase in AT1R expres-
sion in the adrenals and a trend to increase in sympathetic ganglia found
in these studies suggest increased sympathetic nerve activity and adre-
nal activity in response to chronic footshock stress. This conclusion is
further supported by our findings of increased TH phosphorylation at
Ser40 in both stellate ganglia and adrenal gland. With regards to TH,
chronic stress has consistently been shown to increase TH protein and/
or the amount of mRNA encoding the enzyme in the adrenal medulla
and sympathetic ganglia (Baruchin et al., 1990; Kvetnansky et al.,
2004; Nankova et al., 1996). However, increases in TH mRNA and/or
TH protein do not always lead to TH activation. Phosphorylation of TH
at Ser40 represents a more direct measure of TH activity both in vitro
and in vivo (Bobrovskaya et al., 2007; Bobrovskaya et al., 2010;
Dunkley et al., 2004). The effects of psychological stress on this regulato-
ry mechanism have not been studied before. We observed that chronic
footshock stress produced 2–3 fold increases in pSer40TH in the adrenal
gland and stellate ganglia, which persisted for at least one week
post-stress, suggesting increased TH activity and as a result, increased
capacity to synthesise catecholamines.
5. Conclusion
We provide evidence that severe, long-lasting and unpredictably ap-
plied chronic stress in rats alters animal behaviour, some markers of
sympathoadrenal activity and has some impact on arterial blood pressure.
We would like to acknowledge that while stress-induced behavioural
(reduced locomotor activity and social interaction) and biochemical
(pSer40TH, AT1R) effects were quite robust, pressor changes were rather
small (although still statistically significant). This difference suggests that
biochemical markers of sympathoadrenal activation are possibly more
sensitive indices of pro-hypertensive effects of chronic stress.
 L. Bobrovskaya et al. / Autonomic Neuroscience: Basic and Clinical 177 (2013) 217–223 223

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