526269

research-article2014
JRA0010.1177/1470320314526269Journal of the Renin-Angiotensin-Aldosterone SystemWincewicz and Braszko

Original Article

Journal of the Renin-Angiotensin-

Angiotensin II AT1 receptor blockade Aldosterone System

2015, Vol. 16(3) 495­–505

by telmisartan reduces impairment of

© The Author(s) 2014
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spatial maze performance induced by DOI: 10.1177/1470320314526269
jra.sagepub.com

both acute and chronic stress

Dominik Wincewicz and Jan J Braszko

Abstract
Introduction: Despite recognition of stress as a causation of severe neuropsychological dysfunctions, no casual and
clinically effective anti-stress therapeutic strategy has yet been found. We have previously shown that blockade of initial
stress response by angiotensin receptor blockers alleviates the negative effect of prolonged stress on cognitive non-spatial
functions of rats. Here we aimed to find whether telmisartan reduces stress-related memory decline in spatial hippocampal-
dependent learning tasks conditioned upon differences in level of stress induced by aversive nature of memory tests.
Methods: Male Wistar rats were exposed to chronic restraint stress for three weeks and daily treated with either
vehicle or telmisartan (1 mg/kg). Afterwards rats were tested in three spatial learning and memory paradigms: Morris
water maze (MWM), radial arm maze (RAM), and Barnes maze (BM).
Results: Stressed animals demonstrated significantly impaired performance in all the tests, which was normalized in the
animals stressed and treated with telmisartan. Interestingly, despite the fact that MWM and RAM are more stressful,
which affects animal behavior, therefore considered less sensitive than BM, more significant effect of telmisartan was
found in MWM and RAM than BM.
Conclusions: AT1 angiotensin receptor blockade attenuates negative effect of both acute and chronic stress on spatial
memory.

Keywords
Angiotensin II, Barnes maze, Morris water maze, radial arm maze, stress, telmisartan

Date received: 5 September 2013; accepted: 26 January 2014

Abbreviations However, this is an example of adaptive behavior in

ACE-I Angiotensin-converting enzyme inhibitor
Ang II Angiotensin II
ANOVA I One-way analysis of variance
ARB Angiotensin II type 1 receptor blocker
AT1 Angiotensin II type 1 receptor
BM Barnes maze
CNS Central nervous system
HPA Hypothalamic-pituitary-adrenal axis
LTP Long-term potentiation
MWM Morris water maze
PPARγ Peroxisome proliferator-activated receptor γ
RAM Radial arm maze
SEM Standard error of mean
Introduction
healthy individuals and deregulation of the underlying
mechanism might result in cognitive decline.3 Chronic and
acute stress modulates learning and memory processes,
acquisition, consolidation, retention, and retrieval.
Prolonged experience of continuous physiological arousal
negatively affects cognitive performance.4 Acute stimulus
might enhance memory performance,5 but acute intense
event in previously stressed subjects results in amplifica-
tion of hypothalamic-pituitary-adrenal (HPA) axis deregu-
lation, which might trigger memory loss.6
Stress exceeding adaptation mechanisms is potentially
harmful due to over activity of three major neuroendocrine
Department of Clinical Pharmacology, Medical University of Bialystok,
Poland

Corresponding author:
Stressful experiences are generally well remembered,1 Dominik Wincewicz, Department of Clinical Pharmacology, Medical
especially if the context of perceived stress matches the University of Bialystok, Waszyngtona 15a, Bialystok, 15-274, Poland.
context of the information being encoded.2 Email: d.wincewicz@gmail.com
496 Journal of the Renin-Angiotensin-Aldosterone System 16(3)
systems – the sympatho-adrenergic system (SAS), the
HPA axis, and the renin–angiotensin system (RAS).7
However, this effect might be reduced by suppression of
the brain RAS system, as we have previously shown using
highly selective angiotensin type one (AT1) receptor block-
ade, which effectively prevented deleterious effects of
chronic stress on retrieval of memory in rats.8,9
In this study, we attempted to evaluate the effect of AT1
receptor blockade using telmisartan in hippocampal-
dependent memory tasks. Spatial and non-spatial cogni-
tion are two important aspects of cognitive function.
Various types of behavioral tasks have been designed to
evaluate specific type of memory in rodents.10–13 Most of
the spatial memory tasks use positive or negative rein-
forces, such as food for radial arm maze (RAM) or water
immersion for a Morris water maze (MWM) test, however
those effects are minimized in Barnes maze (BM).
Therefore, we decided to use all three paradigms to evalu-
ate potential protective effect of telmisartan on memory
loss due to chronic stress and learning abilities under
stressful conditions in a variety of intensification.
Moreover, using mazes with diverse reinforces seems use-
ful to incorporate results of animal studies with the human
population.
Materials and methods
Animals
The experiments were conducted on male Wistar rats,
weighing approximately 150 g at the beginning. Animals
were housed five to a cage in a temperature (22°C) and
humidity (50–60%) controlled room, on a 12 h: 12 h light/
dark cycle with light on from 6:00 a.m. Free access to
standard laboratory food and tap water was provided. All
animals were handled daily for 2 min each until the day of
experiment. All the procedures were conducted between
1:00 p.m. and 6:00 p.m. A 30 min adaptation period in the
experimental room preceded all the tests. To minimize the
influence of hemodynamic changes, we did not study
hypertensive animals. All procedures involving animals
were approved by the local Ethics Commission for Animal
Experimentation.
Drugs
The specific AT1 receptor antagonist – telmisartan – was
suspended in 0.5% methylcellulose (vehicle) at concentra-
tion of 1 mg/ml. Telmisartan or its vehicle was dosed by oral
gavage at 1 mg/kg body weight, considered to be a non-
hypotensive dose in rats.14,15 One hundred and twenty male
Wistar rats were randomly assigned to four groups: (1) 30
control rats – receiving 0.5% methylcellulose as a vehicle;
(2) 30 rats receiving telmisartan suspended in 0.5% methyl-
cellulose; (3) 30 rats receiving 0.5% methylcellulose
subjected to repeated restraint stress procedure; (4) 30 rats
receiving telmisartan suspended in 0.5% methylcellulose
subjected to repeated restraint stress procedure. The subjects
received either telmisartan or vehicle each day approxi-
mately 30 min before the beginning of stressing procedure.
Animal weight was measured each day prior to drug
administration.
Stress procedure
Two groups of animals (30 rats each) were subjected to
chronic restraint stress, 2 h daily for 21 days.16,17 The
restraint was imposed during the light phase from 1:00
p.m. to 3:00 p.m. The restrainer was made of transparent
perforated plastic tube, 20 cm long, and 7 cm in diameter.
A rat was eased into the restrainer, head first, and once in
the tube it was closed with a Plexiglass lid. The animals fit
tightly into the restrainers and it was not possible for them
to move or turn around. At the same time not stressed con-
trol rats were briefly handled and returned to their home
cages. Randomly chosen animals subjected to stress were
checked for gastric ulceration on the next day after ending
the behavioral tests. Rats were anaesthetized with the mix-
ture of ketamine (50 mg/kg) and xylazine (7.5 mg/kg)
injected intraperitoneally and sacrificed. Exposed gastric
mucosa was visually examined under the 5× magnification
lens for gastric ulceration. No visible signs of injury were
found, that could potentially interfere with the results of
behavioral tests.
Behavioral tests
Hippocampal-dependent cognitive performance of rats
was estimated the next day after ending stress and drug
administration procedures. Three different paradigms with
three different levels of stress induced by the procedure
were used to assess its influence on working memory.
Moreover this work has benefited from evaluating spatial
perception, spatial cognition, and spatially directed action
in both allocentric (MWM, BM) and egocentric (RAM)
representations. Each animal would participate in one
behavioral paradigm only (40 animals assigned to MWM,
40 participating in BM and 40 tested in RAM, from total
number of 120 rats).
Morris water maze.  Forty rats (different from those used
for RAM and BM) randomly assigned to four groups as
described above were used. This task was adapted from
the paradigm originally described by Morris.13 The water
maze was a black circular pool (180 cm in diameter, 60
cm high), filled with water (26±1°C) made opaque with
a grain instant coffee, to the depth of 30 cm, placed in a
room rich in consistently located spatial cues (door,
shelves, illumination lights, posters). The pool was
divided into four quadrants designated Northeast (NE),
Wincewicz and Braszko 497
Northwest (NW), Southeast (SE), and Southwest (SW).
An escape platform (9 cm in diameter) was placed in the
middle of one quadrant (NW), 1.5 cm below the water
surface, equidistant from the sidewall and middle of the
pool. The platform provided the only escape from the
water and was located in the same quadrant conducted
through trials each day, but its location was changed
daily. Four different starting points for rats were equally
spaced around the perimeter of the pool (N, E, S, and
W). On each of the three training days, three start points
were used once each in a pseudo-random sequence. A
trial began by placing the animal in the water facing the
wall of the pool at one of the starting points. If the ani-
mal failed to escape on the platform within 60 s it was
gently placed there by the experimenter and allowed to
stay for 15 s.
The interval was 5 min. After each trial, the rats were
towel and fan dried and returned to their home cages. All
animals were trained one session of three trials daily for
three consecutive days. The latency to reach the original
position of the platform, the number of crossings over its
previous location during 1 min, and time spent by rat in the
target quadrant and in the opposite quadrant and the border
zone were measured manually.
Eight-arm radial maze.  Forty rats (different from those used
for MWM and BM) randomly assigned to four groups as
described above were used. The apparatus was an elevated
eight arm radial-maze which was made of 0.5 cm thick
gray wooden planks. Each arm (68 × 10 cm2) extended
from an octagonal-shaped center hub (30 cm in diameter).
A small cup (4.5 cm in diameter) was placed at the end of
each arm. The maze was elevated 80 cm above the floor
and placed in a sound attenuated room with a masking
noise of 70 dB above the human thresholds.18 The rats
were submitted to three habituation sessions (one per day).
They were put on the central platform and allowed to
explore the maze entries for 10min.At the end of habitua-
tion stage, they were deprived of food for 24 h before the
beginning of the training phase. Rats were given 12 ses-
sions (one per day). The training session lasted until the rat
made eight choices or 15 min elapsed. The cups at the end
of each arm contained arachnid peanuts. The experimenter
recorded order of the eight choices made by each rat. One
hour after the completion of the session, rats were given
access to food for 10 min. The following data was consid-
ered: mean number of errors – an error was an entrance in
a previously visited arm; mean number of correct entries
– correct choices made in sequence before the first error in
each session; mean time spend by rats to visit eight maze
arms within each session.
Barnes maze. Forty rats (different from those used for
MWM and RAM) randomly assigned to four groups as
described above were used. On the first day of testing, the
animals (were brought in the testing suite) and allowed to
enter the goal box through one of the holes in the circular
surface (120 cm diameter) of the maze. Once the animal
entered the hole, a black Plexiglass cover was positioned
over the hole to prevent escape. A 4-min habituation in the
goal box was given prior to the first training trial. Next,
the animal was placed for 30 s in a 20 cm diameter by 30
cm high round nontransparent holding box that was posi-
tioned in the center of the maze. The holding box was then
removed, a timer begun, and the experimenter moved
behind the curtain. An escape was counted when all four
paws of the animal were in the goal box. Following suc-
cessful location of the goal box, the animals were allowed
to stay there for 60 s. A maximum of 4 min was allowed
for each trial and if an animal did not locate in the goal
box during this 4 min period it was removed from the
maze and placed in the goal box for 60 s. During the 60 s
period in the goal box, the maze was wiped with a paper
towel to remove any feces or urine prior to the beginning
of the next trial. The test paradigm consisted of two trials
per day for 3 days. Before the start of the second trial, the
animal was returned to its home cage for 1 min and then
placed again in the holding box in the center of the maze
to start the second trial as described above. The maze was
then rotated a random number of holes (between 1 and 8
holes using a random numbers table) to prevent use of
odor trails in solving the task. The hole under which was
the escape tunnel was changed to assure its constant posi-
tion relative to extra-maze cues (shelves, posters, and
laboratory bench) for 3 days. Following the rotation of
the table, the procedure was repeated with the next ani-
mal. Two measures were recorded on each trial. The first
was the latency to find the goal box. The second was the
number of errors committed by each animal. An error
was defined as a head poke or exploration of any hole
other than the hole above the goal box and including pre-
servative investigations of the same hole. At the end of
each trial the maze was cleaned using a 70% ethanol
solution.11
Statistical analysis
Data were presented as means of ± standard error of mean
(SEM). A two-way analysis of variance (ANOVA II)
(treatment × days) with repeated measures followed by the
post hoc Bonferroni test for multiple comparisons was
used for latencies to reach platform in the MWM and mean
number of errors, latencies, and number of correct entries
in RAM. One-way analysis of variance (ANOVA I), fol-
lowed by post hoc Bonferroni test for chosen group com-
parisons, was applied for calculating performance of the
rats in MWM (savings ratios, border zone time, and swim-
ming speed), Barnes maze (escape latencies and number of
errors), and body weight gain. Levels were deemed signifi-
cant at p < 0.05.
498 Journal of the Renin-Angiotensin-Aldosterone System 16(3)
Figure 1.  Effects of chronic stress (daily restraint for 2 h, 21
days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or both
in combination on mean ± SEM escape latency in the Morris
water maze over three days (three trials per day), n = 10. *p
< 0.05 stress + telmisartan vs. stress, **p < 0.01 control vs.
stress, ***p < 0.001 telmisartan vs. stress.
Results
Effects of chronic stress, chronic telmisartan
treatment, or both in combination on rats’
performance in the Morris water maze
Figure 1 presents mean ± SEM escape latency in the
MWM. The stressed subjects reached the platform signifi-
cantly later than the control (p < 0.01), telmisartan (p <
0.001), and stress + telmisartan (p < 0.05) group of rats.
Telmisartan administration not only abolished adverse
effects of stress on spatial working memory, but also
improved performance of not stressed subjects. ANOVA II
of the latencies to reach platform in the water maze
revealed significant treatment effect (F3,36 = 7.226; p <
0.001), but no significant treatments × days interaction
(F6,72 = 1.367; p > 0.05). Nevertheless, the fact that most of
the rats effectively learned the task meant the day effect
was not significant (F2,6 = 1.818; p > 0.05).
Figure 2 presents mean ± SEM of the time spent in bor-
der zone in the MWM. ANOVA I of the time spent in the
border zone from nine trials yielded F3,36 = 4.663; p < 0.05,
indicating that stressed subjects spent significantly more
time in the border zone than non-stressed rats. This effect
was abolished by telmisartan treatment of stressed rats.
Figure 3 presents performance of rats in MWM
expressed as mean ± SEM savings ratio, a proportion of
second trial latency to total trial time for the session.
Figure 2.  Effects of chronic stress (daily restraint for 2 h,
21 days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or
both in combination on the task-solving strategy in the Morris
water maze. Bars represent means + SEM of the time spend in
the border zone from nine trials (three trials per day for three
days), n = 10. *p < 0.05 vs. control and telmisartan.
According to the interpretation suggested by Glenn and
Mumby,19 the smaller savings ratios the better retention of
the platform position. ANOVA I of the calculated savings
ratios from MWM test yielded F3,36 = 4.595; p < 0.05,
showing statistically significant differences between
stressed rats in comparison to control and telmisartan
groups.
Figure 4 presents mean ± SEM swimming speed, calcu-
lated as number of line crossings divided by escape latency.
The results suggest that all rats were performing the task
with equal swimming speed and this parameter did not
influence the outcome of their performance. ANOVA I of
the results obtained in MWM test revealed no significant
(F3,36 = 3.265; p > 0.05) differences between the groups.
Effects of chronic stress, chronic telmisartan
treatment, or both in combination on rats’
performance in the radial arm maze
Figure 5(a) presents the mean ± SEM numbers of errors,
cumulated in three four-day blocks. ANOVA II of these
results yielded F2,6 = 20.523; p < 0.001, showing significant
Wincewicz and Braszko 499
Figure 3.  Effects of chronic stress (daily restraint for 2 h, 21
days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or both
in combination on working memory in the Morris water maze
expressed in mean savings ratios. Bars represent means + SEM,
n = 10. *p < 0.05 vs. control and telmisartan.
decrease of the errors made by all rats across 12 days. No
significant treatments × days interaction was found (F6,72 =
0.576; p > 0.05). ANOVA I of the total number of errors
made by the rats over 12 days yielded F2,6 = 7.496, p < 0.01
showing significant treatment effect. These results demon-
strate that impairment of working memory displayed by
chronically stressed rats that is partially diminished by
telmisartan.
Figure 5(b) presents mean number ± SEM of correct
and sequential entries to arms of the radial maze, prior to
first error. ANOVA II of the daily numbers of the correct
entries made by each rat over 12 days yielded F2,6 =
18.791; p < 0.001, showing significant time effect, i.e.
increase of the correct entries made by all rats across 12
days. No significant treatments × days interaction was
found (F6,72 = 0.664; p > 0.05). ANOVA I of the total num-
ber of the correct entries made by each rat over 12 days
yielded F3,36 = 5.592; p < 0.01 control vs stress; p < 0.05
telmisartan vs stress.
Figure 5(c) presents the mean ± SEM of the time spent
by rats visiting eight arms. ANOVA II of the times used by
all rats yielded F6,72 = 4,38; p < 0.001, pointing to the sig-
nificant increase of the rats performance in time of visiting
Figure 4.  Effects of chronic stress (daily restraint for 2 h,
21 days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or
both in combination on swimming speed (line crossed/s) in the
Morris water maze. Bars represent means + SEM, n = 10. No
statistically significant differences were found.
maze arms. Significant treatments × days interaction was
found (F6,72 = 4.380; p < 0.001). ANOVA I of the times
spent by rats visiting all arms over 12 days yielded F3,36 =
3.726; p < 0.05, showing significant differences among
stressed and not stressed subjects.
Effects of chronic stress, chronic telmisartan
treatment, or both in combination on rats’
performance in the Barnes maze
Figure 6 presents mean ± SEM number of errors made by
rats in the Barnes maze. ANOVA I of the results reviled
significant difference between stressed and control sub-
jects (F3,36 = 2.867; p < 0.05) in selected pair comparison.
Telmisartan treatment of stressed rats reduced this effect
but further post hoc comparisons made with Bonferroni
revealed that the difference is not statically significant.
Figure 7 presents mean ± SEM of escape latency in the
Barnes maze. Exploration time was extended in the
stressed subjects and this effect was lowered in telmisartan
treated group, but ANOVA I of the results from all the tri-
als and post hoc comparisons made with Bonferroni
revealed that the differences are not statically significant.
500 Journal of the Renin-Angiotensin-Aldosterone System 16(3)

Figure 5.  Points represent means ± SEM from 4 days each of the values obtained from 10 animals. (a) Mean number of errors,
**p < 0.01 control and telmisartan vs. stress; (b) mean number of correct sequential entries before the first error, **p < 0.01 control
vs. stress, *p < 0.05 telmisartan vs. stress; (c) mean time of eight arms exploration, *p < 0.05 control and telmisartan vs. stress.

Effects of stress and telmisartan on body

weight gain
As shown in Table 1, all of the rats gained weight during
the course of the study, but growth rate differed among the
groups. Restraint induced a significant weight loss (p <
0.001) that was even greater in the rats receiving telmisar-
tan. The effect became apparent during the first week of
restraint. Moreover, animals that were not subjected to
stress procedure and received telmisartan also gained sig-
nificantly (p < 0.005) less weight than control group; how-
ever, this effect appeared after two weeks of the study.

Figure 6.  Effects of chronic stress (daily restraint for 2 h,
21 days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or
both in combination on working memory in the Barnes maze.
Bars represent mean + SEM number of errors from six trials
(two trials per day for three days), n = 10. *p < 0.05 stress vs.
control.
Discussion
Despite rapid development of technology and biochemical
methods, behavioral tests are still recognized as very sen-
sitive tools in testing animal cognition, which might reveal
minor hippocampal-induced impairment before, or even in
the absence of, neuron death.20 Although the Morris water
maze, the radial arm maze and the Barnes maze are well
established means of testing spatial memory,11,13,21,22 nei-
ther might be considered optimal for studying the behavio-
ral effects of stress. The MWM is itself highly stressful to
the animals.23 The RAM relies on a food reward to moti-
vate the animals, and glucocorticoids have profound
effects on hunger and satiety.24 While strong aversive stim-
uli (i.e. water or food deprivation) are likely to produce
Wincewicz and Braszko 501
stress in the animal that influences the performance in the
task, weak aversive stimulation might result in lack of
motivation to escape from the circular platform in the
Barnes maze.25 Performance of rodents with hippocampal
damage in all the mazes mentioned above is impaired,
which strongly supports hypothesis of the spatial nature of
all the tasks.26,27 All the paradigms are challenging tasks
for rodents and employ a variety of sophisticated mne-
monic processes.28 These processes comprise the acquisi-
tion and spatial localization of relevant visual cues that are
subsequently processed, consolidated, retained, and then
retrieved in order to successfully navigate using allocen-
tric (MWM, BM) or egocentric (RAM) representations.
Figure 7.  Effects of chronic stress (daily restraint for 2 h,
21 days), chronic telmisartan (1 mg/kg p.o., daily, 21 days), or
both in combination on working memory in the Barnes maze.
Bars represent mean + SEM time of escape latency from six
trials (two trials per day for three days), n = 10. No statistically
significant differences were found.
Table 1.  Effects of stress and telmisartan (1 mg/kg) on body weight gain. Values are means ± SEM of body weight (g). *p < 0.05,
**p < 0.005, ***p < 0.001 in comparison to control group, n = 10 rats in each group.
Groups Day 1
Control 150.4 ± 1.41
Telmisartan 151 ± 2.28
Stress 152.7 ± 2.37
Stress + telmisartan 155 ± 1.27
The general processes used for visuospatial navigation in
rats also contribute considerably to human day-to-day cog-
nitive processes.29 Therefore, to broaden utility of this
study all three paradigms were used (MWM, RAM, and
BM) as it might become relevant to neurodegenerative and
neuropsychiatric illnesses where cognition is impaired.30–32
As a continuation of our previous studies,9 and screen-
ing for compounds with potential cognitive enhancing
effects, as well as delineating deleterious effects of stress
on cognition, we proceeded with development studies of
telmisartan. The results of the present study clearly dem-
onstrate that pre-treatment with telmisartan significantly
reduces stress-related cognitive impairment in rats.
Parameters of cognitive performance (latencies, errors and
correct choices, savings ratios) in MWM, RAM, and BM
were significantly deteriorated in chronically stressed sub-
jects. This effect was reduced in stressed subjects that
received telmisartan. Interestingly, the most profound
effect was reviled in the MWM, which is the most stressful
paradigm therefore, capitalizes upon aversive motivation.
Moreover, the MWM allows for the assessment of spatial
memory without potential experimental manipulation
incorporating food reward. Rats exhibit a robust increase
in corticosterone on the first day of water maze testing that
is stable and only slightly diminished throughout days of
testing.33 No such data are available for the RAM and BM.
According to inverted-U-shaped fashion, suggesting that
an optimal level of corticosterone will enhance memory
and higher levels may impair it,26 telmisartan treatment
presumably diminish excessive release of corticosterone in
stressed subjects, suppressing over-activation of the HPA
axis and enhanced corticotrophin-releasing hormone, as
has been recently described by Braszko et al.8 Noteworthy,
time latencies in the MWM seems to efface among the
stressed and non-stressed subjects on the second and the
third day of testing. It is possible that control rats sooner
than stressed rats switch from spatial to non-spatial strate-
gies with overtraining.4 The least profound effect of pro-
tective outcome of telmisartan treatment in stress induced
cognitive decline was reviled in the Barnes maze, the least
stressful of all three mazes. Number of errors made by
stressed subjects in the BM was significantly higher and
telmisartan treatment reduced this effect, but no statisti-
cally significant differences in the escape latency were
found. However, unitary measures such as latency and
Day 7 Day 14 Day 21
181.6 ± 3.21 225.5 ± 4.20 271.5 ± 5.25
179.4 ± 2.60 211.5 ± 4.03* 252.6 ± 3.48**
169.2 ± 3.08* 188.8 ± 2.91*** 222 ± 3.61***
167.4 ± 2.81** 184.4 ± 3.54*** 213 ± 3.81***
502 Journal of the Renin-Angiotensin-Aldosterone System 16(3)
path length are more susceptible to non-mnemonic influ-
ences and are not the best indicators of cognitive perfor-
mance in learning tasks.34 Moreover, the BM induces a
modest increase in corticosterone,35 which might be insuf-
ficient for motivation of animals to perform in the task.36
In our study, adaptation procedure was not effective
enough, to exert animals for more readily exploration of
the holes on the surface of circular platform of the BM.
Rats often hesitated to enter the escape hole and started to
inspect other holes, what clearly had a direct effect on
latency and distance measures. Control rats needed more
than 60 s to find the escape hole in the BM, which is rela-
tively long when compared to the MWM performance
(<45 s) in which the diameter of the apparatus is much
longer. Execution of the task in the RAM was impaired in
the stressed subjects and expressed by mean number of
errors. Interestingly mean number of correct entries was
slightly lower in rats receiving telmisartan, as those rats
chose more often not to enter arms sequentially, but with-
out committing more errors than controls. Despite the fact
that RAM is generally used to measure spatial memory,
using this paradigm has notable limits. For example, even
blind rats can learn to enter arms in one direction (i.e. con-
stantly turn left), which might be a failure in long-term
testing procedure, when hippocampal mediated processes
become independent from the hippocampus over weeks.37
Another limitation for using RAM in stressed subjects, is
the fact that chronic stress may influence motivation with
clearly interferes with the results of the test. The effects of
stress and telmisartan treatment tested in the RAM were
slightly less profound than in MWM, but more expressed
in comparison to the BM. The data presented here, indi-
cating the differences among three paradigms, are
assumed to be caused by the negative correlation between
learning and level of corticosterone release. However, cer-
tain causative direction cannot be drawn and needs further
investigation.
Repeated restraint is used in this study as a model of
moderate psychological stress, which is a complex
response that also affects metabolism, therefore commonly
considered as an animal model of anorexia nervosa.38
Repeated stressors in rats generally seem to reduce food
intake and body weight.39 Stress-induced effects on metab-
olism and energy balance have been demonstrated as a
result of repeated daily restraint and as an acute response
in the hours immediately after a single restraint.40 In our
study weight loss induced by repeated restraint appeared
within the first week of stress sessions and became more
pronounced after each exposure to stress, which indirectly
indicates the effectiveness of the restraint procedure.
Significant attenuation of weight gain was also a result of
telmisartan administration, which is not very surprising
since reduced adipogenesis and weight gain have already
been reported as the effects of this ARB in rodents.41 In
addition to being a blocker of AT1 receptor, telmisartan is
also a partial agonist of the peroxisome proliferator–
activated receptor-gamma (PPAR-γ), a well-known target
of insulin sensitizing drugs used to treat type 2 diabetes.
Only telmisartan, and to a lesser extent candesartan, are
the only commercially available ARBs producing PPAR-γ
activation, when tested at concentrations typically achieved
in plasma with conventional oral dosing.42,43 Telmisartan
influences the expression of PPAR target genes involved in
carbohydrate and lipid metabolism and reduction of glu-
cose, insulin, and triglyceride levels,44 but the significance
of PPAR-γ activation in this study goes beyond its meta-
bolic effects. These nuclear receptors are considered to be
attractive targets for the treatment of cognitive impairment
due to their ability to suppress microglial-mediated inflam-
matory responses,45 promote tissue repair,46 and induce the
degradation of the Aβ peptides in the brain by activating
genes responsible for reverse cholesterol transport.47
Nevertheless, PPAR-γ are expressed in the fetal brain as a
factor involved in cellular development, its expression is
rather limited in the healthy mature brain.48 Moreover, the
use of the PPAR-γ activators (i.e. rosiglitazone, pioglita-
zone) for CNS-targeted disease treatments is compromised
due to their poor blood brain barrier (BBB) penetrance.49
Telmisartan, however, effectively penetrates BBB in a
dose- and time-dependent manner, to inhibit centrally
mediated effects of Ang II for over 4 hours following
peripheral administration of a dose as low as 1 mg/kg.50
However, it is essential that a non-hypotensive dose of 1
mg/kg does not result in a significant reduction in blood
pressure and does not affect cerebral blood flow.51
Although blood pressure (BP), even in normotensive rats,
as a continuous, not a static, variable might be altered due
to restraint, which clearly is a factor for development and
progression of cognitive decline, the negative effects of
stress are simultaneously mediated through angiotensin II
and glucocorticoids, affecting both circadian BP rhythm
and cognitive performance. Therefore, stress induced
alterations of BP cannot be considered as an independent
trigger of memory loss. Stress induces an increase in BBB
permeability,52 and disruptions of BBB are casually asso-
ciated with cognitive decline.53 Interestingly, treatment
with an ARB ameliorates cognitive impairment in type-2
diabetic subjects,54 which is caused most likely due to
decrease in BBB permeability.55 Moreover, recent studies
suggest that salt-dependent hypertension and activation of
the brain RAS contributes to BBB malfunction and cogni-
tive decline, which is reversed with an ARB treatment
independent of blood pressure changes, by preventing
BBB permeability.56 These data indicates importance of
the brain RAS in the pathogenesis of impaired cognitive
performance. The role of the Ang II is well established in a
complexity of sympathoadrenal response.57 In rats, high
density of AT1 receptors is present in brain regions that are
involved in the regulation of sympathetic tone, such as the
endothelial cells and neurons of the circumventricular
Wincewicz and Braszko 503
organs outside the BBB (e.g. SFO, OVLT), cerebrovascu-
lar endothelial cells of the BBB, and neuronal AT1 recep-
tors selectively localized in many brain areas located
inside the BBB (e.g., PVN, SCh, and MnPO).58 Significant
numbers of AT1 receptors are located in higher stress-
regulatory structures: hippocampus, septum and amyg-
dala. AT1 receptors are also expressed in parvocellular
corticotrophin-releasing factor (CRF) forming neurons
controlling CRF release during stress.59 These brain struc-
tures are important therapeutic targets for ARBs, espe-
cially those lipophilic like telmisartan that readily crosses
BBB to inhibit the brain RAS,50 as a treatment of stress
related CNS disorders. It has been reported that sartans
normalize cerebrovascular remodeling and autoregulation
in hypertension, prevents and reverses cerebrovascular
inflammation, reduces vulnerability to brain ischemia, and
protects from stroke,60 but explanation of many positive
effects of ARBs would go beyond AT1 receptor blockade.
Besides central functions of Ang II, other naturally occur-
ring neuroactive peptides of the RAS are also involved in
memory processing.61 The effects of angiotensin IV (Ang
IV) and des-Phe(6)Ang IV include improvement of several
memory aspects (i.e. recall of appetitively and aversively
motivated behaviors and learning of spatial tasks).
Moreover, angiotensin metabolites modulate central func-
tions by increasing levels of some of the other brain pep-
tides, including oxytocin, which is associated with
anxiolytic effects.62
As a consequence of AT1 blockade, ARBs increase not
only Ang II levels, but also levels of Ang metabolites.
Interestingly, some data suggest that angiotensin neuro-
peptide release, occurring in mild stressed subjects,
results in memory enhancement that could be attenuated
by both ACE-inhibitors and ARBs.63 ACE-inhibitors
decreases the formation of Ang II, Ang III, and Ang IV
from Ang I, while ARBs selectively block AT1 receptor,
thus diminishing Ang II-mediated biological actions
without affecting the actions of other angiotensin neuro-
peptides, suggesting the key role of Ang II not only in
stress-induced memory impairment but also enhance-
ment. This indicates flexibility and complexity of brain
RAS system demanding further evaluation conditioned
upon different levels of stress.
Nevertheless, accumulating preclinical studies and
clinical reports call attention on the therapeutic and neuro-
protective effects of ARBs,64,65 including stress attenuating
properties of telmisartan,14,66–68 although at this point nar-
rowing the group of ARBs, excluding those that are hydro-
philic (i.e. losartan, eprosartan),69,70 to indicate the most
potent of withdrawing negative effects of stress, seems
unreasonable.
In conclusion, data presented in this study indicate that
AT1 angiotensin receptor blockade with telmisartan attenu-
ates negative effect of both, acute and chronic stress on
memory, which is a novelty in the pursuit of effective ther-
apeutic approach to stress induced cognitive decline.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This work was supported by the Medical University of Bialystok.
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