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Modulatory effects of caffeine on oxidative stress and
anxiety-like behavior in ovariectomized rats
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2015-0502.R2
Manuscript Type: Article
Date Submitted by the Author: 24-Mar-2016
Complete List of Authors: Caravan, Ionut; University of Medicine and Pharmacy, Physiology Sevastre Berghian, Alexandra; Univeristy of Medicine and Pharmacy, Physiology Moldovan, Remus; University of Medicine and Pharmacy , Physiology Decea, Nicoleta; University of Medicine and Pharmacy, Physiology Orasan, Remus; University of Medicine and Pharmacy , Physiology
Filip, Gabriela; University of Medicine and Pharmacy, Physiology
Keyword: caffeine, ovariectomy, brain, blood, oxidative stress, behavior, neuroprotection, rats.
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Modulatory effects of caffeine on oxidative stress and anxiety-like behavior in
ovariectomized rats
Ionut Caravan, Alexandra Sevastre Berghian*, Remus Moldovan, Nicoleta Decea,
Remus Orasan, Gabriela Adriana Filip
1 Department of Physiology, ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy, 1 Clinicilor Street,
400006 Cluj-Napoca, Romania
* Corresponding author
Alexandra Sevastre Berghian
Address: Clinicilor Street, no 1, Cluj-Napoca, Romania
Phone: 0040-744692075 Fax: 0040-264-598575
e-mail: [email protected];
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Abstract
Menopause is accompanied by enhanced oxidative stress and behavioral changes, effects
attenuated by antioxidants. The aim of this study was to evaluate the effects of caffeine on behavior and
oxidative stress in an experimental model of menopause. Female rats were divided in: sham-operated
(CON), sham-operated and caffeine-treated (CAF), ovariectomized (OVX), ovariectomized and caffeine-
treated (OVX+CAF). Caffeine (6 mg/kg) and vehicle were administered for 21 days (subchronic) and 42
days (chronic), using 2 experimental subsets. Behavioral tests and oxidative stress parameters in the
blood, whole brain and hippocampus were assessed. The subchronic administration of caffeine
decreased the lipid peroxidation and improved the antioxidant defense in the blood and brain. The
GSH/GGSG ratio in the brain was improved by chronic administration, with reduced activities of
antioxidant enzymes and enhanced nitric oxide and malondialdehyde levels. In particular, the lipid
peroxidation in the hippocampus decreased in both experiments. The rats became hyperactive after 21
days of treatment, but no effect was observed after chronic administration. In both experimental
subsets, caffeine had anxiolytic effects as tested in EPM. The administration of low doses of caffeine, for
a short period of time, may be a new therapeutic approach to modulating the oxidative stress and
anxiety in menopause.
Keywords: caffeine, ovariectomy, brain, blood, oxidative stress, behavior, neuroprotection, rats.
1. Introduction
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Menopause, defined as the permanent cessation of menstruation, is a physiological process that
occurs in women around the age of 45-55 years, being linked to the loss of ovarian activity. The
estrogens have regulatory roles in many organs, therefore their decreased secretion during menopause
triggers many pathological reactions: several changes in the plasmatic lipid fractions (Maynar et al.
2001) and in the circulating catecholamine levels (Knonenberg et al. 1984), the activation of the renin-
angiotensin system (Yung et al. 2011), the decreased endothelial nitric oxide (NO) synthesis (Gavin et al.,
2009) and the enhanced oxidative activity (Sanchez-Rodriguez et al. 2012). The estrogen depletion at
menopause was also reported to be associated with cognitive decline, depression and anxiety (Walf and
Frye 2006).
Generally, the reactive oxygen species (ROS) are produced during normal metabolism and have
useful functions. The involvement of OS in several menopause-related conditions has gained more and
more interest and the elevated level of ROS was reported to be responsible, or at least to contribute to
the pathogenesis, of weight gain (Mittal and Kant 2009), osteoporosis (Rao et al. 2009), hypertension
(Yanes and Reckelhoff 2011), insulin resistance (Bloch-Damti and Bashan 2005), skin aging (Bottai et al.
2013), depression (Michel et al. 2012) and cognitive decline (Droge and Schipper 2007). Moreover, the
impaired oxidant/antioxidant profile and oxidative stress-related anxiety in menopause can be reversed
by dietary polyphenols, suggesting the beneficial effects of natural antioxidants in anxiety (Halliwell
2006; Salim 2011).
The hormone replacement therapy was widely used to prevent or to treat the symptoms and
the conditions associated with menopause. Unfortunately, the hormonal replacement therapy has
important adverse effects. The increased risk of cancer, especially breast cancer, after long-term
estrogens exposure, is limiting the clinical use (Stahlberg et al. 2004). In the past 10 years, the scientists
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have searched for alternative therapies to reduce the OS level, to improve the quality of life in
postmenopausal women.
Caffeine (1, 3, 7-trimethylxanthine), a key component of many beverages and food (coffee, tea,
cocoa) is widely consumed for its stimulating effect on the central nervous system. Due to the ability to
block the adenosine receptors (ARs), especially the A1 and A2A subtypes, it has many pharmacological
actions (Fredholm et al. 1999). Caffeine and its metabolites are used in clinical medicine as
myorelaxants, analgesics, diuretics and bronchodilators (Kaiser and Quinn 1999). Moreover, caffeine
proved to be effective as an adjuvant in the treatment of obesity (Molnar et al. 2000), Parkinson’s
disease (Barbier 2001) and had preventive effects on memory impairment (Leite et al. 2011). It may also
regulate the NO production in the brain by modulating the ARs and the arginase activity (Nikolic et al.
2003).
Despite all the aforementioned beneficial effects, the exact influence of caffeine on the
oxidant/antioxidant status in postmenopausal conditions was never studied before. The present study
was designed to investigate the effects of subchronic and chronic caffeine administration on the
oxidative stress in the blood and in the brain of ovariectomized rats. In addition, the locomotion and the
anxiety-like behavior of animals were evaluated.
2. Methods
2.1 Reagents
Trichloroacetic acid, o-phthalaldehyde, t-butyl hydroperoxide, glutathione reductase, reduced
glutathione, Bradford reagent, cytochrome c, xanthine, xanthineoxidase, β–nicotinamide adenine
dinucleotide phosphate reduced tetrasodium salt (NADPH) and caffeine were purchased from Sigma-
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Aldrich Chemicals GmbH (Munich, Germany). Guanidine hydrochloride, 2-thiobarbituric acid, and EDTA-
Na2 were obtained from Merck KGaA (Darmstadt, Germany), while absolute ethanol, hydrogen peroxide
and n-butanol were purchased from Chimopar (Bucharest, Romania).
2.2 Animals and experimental design
The study was performed on 104 female Wistar rats (3-month-old, weighing 180 ± 15 g). The
animals were supplied by the Animal Department of “Iuliu Hatieganu” University of Medicine and
Pharmacy, Cluj-Napoca. They were housed in a 12 h light/dark cycle at room temperature (24 ± 2 °C). All
animals were fed a standard normocaloric pellet diet and received water ad libitum. All experiments
were approved by the Ethics Committee on Animal Welfare of the "Iuliu Hațieganu" University and were
performed in accordance with the European Convention for the Protection of Vertebrate Animals used
for Experimental and other Scientific Purposes, Council of Europe no. 123, Strasbourg 1985.
In order to evaluate the effects of subchronic and chronic caffeine administration, the
experiment was divided into two subsets (Figure 1). Each of the two experimental subsets included 4
groups of treated animals, 13 rats each. The caffeine and the drinking water were administered orally. In
subset 1, two groups underwent sham surgery (opening of the abdominal cavity, exposure of the
ovaries, and sewing it back), and then were given 0.2 ml of drinking water (CON) or caffeine solution
(CAF). The amount of caffeine administrated daily in rats was 6 mg/kg b.w. The animals in the others
groups underwent bilateral ovariectomy (surgical removal of the ovaries) and were given drinking water
(OVX) or a caffeine solution, in a dose of 6 mg/kg b.w./day (OVX+CAF). After 21 days, the behavioral
tests were conducted. Twenty-four hours after the last behavioral test, under anesthesia with an
intraperitoneal injection of ketamine/xylazine cocktail (90 mg/kg b.w. ketamine and 10 mg/kg b.w.
xylazine), all animals were euthanized. The blood and the whole brain tissues from 8 rats in each group
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were harvested and frozen. From the other 5 rats in the group, only the hippocampus was harvested for
the oxidative stress assays.
In subset 2, the same groups were used: two sham groups, one received drinking water (CON)
and the other one was treated with caffeine in a dose of 6 mg/kg b.w./day (CAF); two groups underwent
bilateral ovariectomy, one which received drinking water (OVX) and the other one was treated with the
same caffeine once daily (OVX+CAF). After 42 days, the behavioral tests were conducted. Twenty-four
hours after the last behavioral test, anesthesia with an intraperitoneal injection of ketamine/xylazine
cocktail was induced. Then, all animals were euthanized and the same samples as in subset 1 were
harvested and frozen.
2.3 Behavioral tests
The locomotor activity and the anxiety-like behavior were measured in Open Field Test (OFT)
and Elevated Plus Maze (EPM). The behavior sessions, recorded by a visual tracking system (Smart Basic
Software v3.0 Panlab Harvard Apparatus; Ugo Basil Animal Mazes for Video-Tracking), were carried out
between 09:00 and 14:00.
The next day, after the last dose of caffeine, animals were placed in the center of the OFT
apparatus (100 x 100 x 40 cm) for rats, with gray non-reflecting base and walls. The floor of the open
field arena was electronically divided into 9 equal squares. Normally, rats tend to walk close to the walls,
a behavior called thigmotaxis. The index of activity (total travelled distance, travelled distance in
periphery, total number of entries, number of entries in periphery) and the index of emotionality
(travelled distance in center, number of entries in center, time spent in center divided by the total time),
during each 5-min testing session, were automatically recorded. At the end of each session, the open
field arena was cleaned with 70 % ethanol and animals returned to the home cage (Carrey et al. 2000).
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One day later, the rats were placed in the center of the EPM, which consists of a plus-shaped
maze, 60 cm elevated from the floor with two open and two closed arms (length 50 cm, width 10 cm,
closed wall height 40 cm) opposite to each other and inter-connected by a central platform. Time spent
in the unprotected open arms divided by the total time spent in all arms of the maze (time ratio) was
taken as an experimental measure of anxiety. Therefore, the smaller these ratios, the more "anxious"
the rats (Walf and Frye 2007).
2.4 Oxidative and nitrosative stress parameters
For preparing the cytosolic fractions, the brain tissue and the hippocampus were homogenized
as previously described by Filip et al (2011) and the protein content in homogenates was measured with
Bradford method (Noble and Bailey 2009). In order to evaluate the oxidative/anti-oxidative status, the
glutathione reduced/glutathione oxidized (GSH/GSSG) ratios were assessed in the serum and brain
tissue homogenate, while the malondialdehyde (MDA) levels were assessed in the serum, hippocampus
and whole brain tissue homogenate. The MDA levels were considered as markers of oxidative attack on
lipids and the glutathione reduced/glutathione oxidized (GSH/GSSG) ratio was interpreted as an
antioxidant biomarker. In the whole brain homogenate, we assessed the activity of antioxidant enzymes
such as catalase (CAT) and glutathione peroxidase (GPx) and the NO level. The antioxidant enzymes
were also assessed in the hippocampus as markers of the antioxidant defense.
The MDA levels in the blood, whole brain homogenates and hippocampus were determined by
spectrofluorimetry, using the method with 2-thiobarbituric acid. The values were expressed as
nanomoles/mg of protein (Conti et al. 1991). The blood and brain tissue homogenate levels of GSH and
GSSG levels were fluorimetrically measured using o-phtalaldehyde. Their concentrations were
determined using standard curves and the results were expressed as GSH/GSSG ratios (Hu 1994).
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The CAT activity was determined in the whole brain homogenates and hippocampus by using
the method described by Pippenger et al. (1998) and the values were expressed as units/mg of protein.
The GPx activity was determined with a slightly modified Flohe and Gunzler method (1984) and the
values were expressed as micromoles of NADP produced/min/mg of protein. The NO level in the whole
brain homogenates was determined by Griess reaction for nitrite plus nitrate in a two-step procedure.
The concentration of nitrite was calculated in comparison with a nitrite-standard curve and the results
were expressed as nanomoles/mg of protein (Titheradge 1998).
2.5 Statistical analysis
Statistical analysis of results was conducted by using ANOVA GraphPad Prism software, version
6.0 (GraphPad, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM).
The comparisons between the groups were made by unpaired t-test and one-way ANOVA analysis of
variance, using a Turkey’s Multiple Comparison Test. A p value lower that 0.05 was considered to be
statistically significant.
3. Results
3.1 Oxidative stress assessment in blood
The influence of caffeine administration for 21 days and 42 days respectively on MDA levels and
GSH/GSSG ratios in blood is illustrated in Figure 2. In subset 1, MDA level in blood was significantly
higher in the OVX group (2.65 ± 0.18 nmoles/mg protein) as compared to the CON group (1.74 ± 0.18
nmoles/mg protein; p = 0.01) and decreased significantly after caffeine administration (1.84 ± 0.15
nmoles/mg protein in OVX+CAF group vs. 2.65 ± 0.18 nmoles/mg protein in OVX group; p=0.01). In
subset 2, the serum MDA evolution had the same pattern. Thus, the values were significantly increased
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in OVX group (4.03 ± 0.32 nmoles/mg protein) as compared to CON group (3.08 ± 0.22 nmoles/mg
protein; p=0.03) and were reduced significantly after caffeine exposure (2.77 ± 0.17 nmoles/mg protein
in OVV+CAF group; p=0.004).
The GSH/GSSG ratio in blood diminished significantly in the OVX group (2.11 ± 0.16) as
compared to the CON group (3.18 ± 0.22; p=0.003) and was increased in the OVX+CAF group (3.29 ±
0.41) as compared to the OVX group (p=0.01) in subset 1 of experiments. Also, in subset 2, the
GSH/GSSG ratio in blood increased significantly in OVX+CAF group (6.93 ± 0.65) as compared to OVX
group (2.09 ± 0.28; p
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0.42 nmoles/mg protein) as compared to the OVX group (5.37 ± 0.33 nmoles/mg protein; p=0.02) in
chronic administration of caffeine.
The effect of caffeine administration for 21 days and 42 days respectively on the MDA levels,
GPx and CAT activities in the brain is illustrated in Figure 4.
In the subset 1, the MDA level assessed in the whole brain homogenate, was significantly lower
in OVX+CAF group (0.11 ± 0.01 nmoles/mg protein) as compared to OVX group (0.19 ± 0.02 nmoles/mg
protein; p=0.009). Interestingly, after 42 days of caffeine administration in ovariectomized rats, the MDA
increased significantly as compared to OVX group treated with vehicle (0.16 ± 0.02 nmoles/mg protein
vs. 0.09 ± 0.008 nmoles/mg protein in OVX group; p=0.01).
After 21 days of caffeine administration, the MDA level in the hippocampus was also
significantly lower in the OVX+CAF group (0.22 ± 0.02 nmoles/mg protein) as compared to the OVX
group (0.45 ± 0.05 nmoles/mg protein; p=0.02). The same tendency was observed after 42 days of
caffeine treatment: OVX+CAF group (0.19 ± 0.02 nmoles/mg protein) and OVX group (0.38 ± 0.04
nmoles/mg protein; p=0.003) (Figure 4A, 4B).
Regarding the GPx activity in the whole brain homogenates, in the subset 1 we did not observe
significant differences between the OVX + CAF group (20.98 ± 1.22 µmoles NADP/min/mg protein) and
the OVX group (22.00 ± 1.28 µmoles NADP/min/mg protein; p>0.05). A significantly reduced activity in
OVX + CAF group (19.61 ± 0.41 µmoles NADP/min/mg protein) as compared to OVX group (24.51 ± 0.74
µmoles NADP/min/mg protein; p=0.001) was observed in the subset 2. In the both experimental
subsets, there were no significant differences between the groups regarding the GPx activities in
hippocampus (Figure 4C, 4D).
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The CAT activity in the tissue homogenates increased significantly in the group which underwent
bilateral ovariectomy and received caffeine 21 days (0.27 ± 0.03 U/mg protein) as compared to the OVX
group (0.17 ± 0.01 U/mg protein; p=0.009) and decreased significantly after 42 days of caffeine
administration (0.26 ± 0.06 U/mg protein in OVX + CAF group vs.0.50 ± 0.02 U/mg protein in OVX group;
p=0.01). Regarding the enzyme activity in hippocampus, in the subset 1, we observed a lower activity in
the OVX+CAF group (0.20 ± 0.001 U/mg protein) as compared to the OVX group (0.42 ± 0.002 U/mg
protein; p=0.001). In the subset 2, there were not significant differences between the OVX and OVX+CAF
groups (Figure 4E, 4F).
3.3 Anxiety-like behavior tests
The influence of subchronic and chronic caffeine administration on general locomotor activity,
tested in OFT, is illustrated in Figure 5 and Figure 6.
The OFT is the most commonly used to examine locomotor activity in rodents (Carrey et al.
2000). It seems that OF is a good model to assess the effects of classical benzodiazepines and 5-HT1A
receptor agonists, but not other compounds displaying anxiolytic-like effects (Prut and Belzung 2003). In
the OFT, there was no significant effect of ovariectomy as compared to the control group on general
locomotion (total travelled distance, total number of entries, both travelled distance and number of
entries in periphery) in the 21-days experiment. Conversely, the rats became hyperactive with the
subchronic caffeine administration both as compared to control (CAF vs CON; p0.05).
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The influence of 21 and 42 days of caffeine administration on emotionality, tested in OFT, is
illustrated in Figure 7 and Figure 8A, 8B.
Regarding the emotionality (travelled distance in center, number of entries in center, time spent
in center/total time), we observed no significant differences between the OVX and CON groups, both for
subchronic and chronic caffeine administration (p>0.05). The ovariectomized rats that were
subchronically treated with caffeine travelled a greater distance, made more entries and spent
significantly more time in the center of the open field arena as compared to ovariectomized group
(OVX+CAF vs OVX; p0.05). Both
subchronic and chronic administration of caffeine in OVX groups increased significantly the time spent in
the open arms (OVX+CAF vs OVX; p
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4. Discussion
The results of the current work showed that both subchronic (21 days) and chronic (42 days)
administration of small doses of caffeine (6 mg/kg b.w.) significantly reduced the oxidative stress in the
blood of ovariectomized rats. In the whole brain homogenate, the subchronic administration decreased
the oxidative stress and the NO level, while the chronic treatment diminished the activity of the
antioxidant enzymes and increased the MDA and NO levels in parallel with increased GSH/GSSG ratio. In
the hippocampus of ovariectomized rats, both subchronic and chronic administration of caffeine
reduced the peroxidation of lipids (lower MDA levels). Regarding the behavioral effects of caffeine, the
locomotor activity was increased in subchronic administration, but no change was observed with the
chronic treatment. Moreover, based on the EPM test results, caffeine administration had anxiolytic
effects in ovariectomized rats, in both experimental subsets.
We have chosen the experimental ovariectomy in rats to simulate the menopause in women.
The menopausal age strongly correlates with important pathological changes in women’s body leading
to high morbidity (Gavin et al. 2009; Kronenberg et al. 1984; Maynar et al. 2001; Sanchez-Rodriguez et
al. 2012; Walf and Frye 2006; Yung et al. 2011). Thus, the postmenopausal women are at a high risk of
obesity, osteoporosis, hypertension, insulin resistance, cognitive decline, depression, anxiety and other
diseases, mainly due to the enhanced OS levels (Bloch-Damti and Bashan 2005; Bottai et al. 2013; Droge
and Schipper 2007; Michel et al. 2012; Mittal et al. 2009; Rao et al. 2007; Salim 2011; Sanchez-Rodriguez
et al. 2012; Yanes and Reckelhoff 2011).
In the present study, we provide evidence that ovariectomy in rats led to early (after 21 days)
increase of the OS parameters. Therefore, we observed enhanced serum MDA levels, reduced
GSH/GSSG ratios in blood and brain, reduced activities or no changes in antioxidant enzymes and
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enhanced NO levels in the brain. Moreover, in ovariectomized rats (OVX group), we observed decreased
locomotor activity in OFT (the most used test to examine locomotor activity in rodents) and increased
anxiety-like behavior in EPM test (the gold standard test to examine the anxiolytic effect in rodents)
after 42 days of vehicle administration. These observations are consistent with the literature. Several
studies found a correlation between the estrogen depletion and the increased OS level in women
(Bloch-Damti and Bashan 2005; Sanchez-Rodriguez et al. 2012) and also in rats (Dilek et al. 2010; Patki et
al. 2013). They reported an increased level of MDA and a reduced GSH/GSSG ratio in the blood of
ovariectomized rats (Dilek et al. 2010) and in postmenopausal women (Bloch-Damti and Bashan 2005).
Several changes in the brain antioxidant profile were also identified in menopause: reduced GSH/GSSG
ratio (Abbas and Elsamanoudy 2011; Dilek et al. 2010), increased MDA levels (Abbas and Elsamanoudy
2011) and high levels of nitrated proteins (Patki et al. 2013). It seems that hippocampus is the most
susceptible region to ovariectomy-induced estrogen depletion, in terms of generation of oxidative stress
(Patki et al. 2013). We have also observed enhanced MDA levels in the hippocampus of the
ovariectomized rats, especially 21 days after the ovariectomy. Regarding the antioxidant enzymes, some
studies showed decreased activities (Abbas et al. 2011) while others reported no changes in their levels
(Dilek et al. 2010). The behavioral changes observed were also comparable with previous studies which
reported anxiety-like behavior and inhibitory effect on locomotion after ovariectomy in rats (Patki et al.
2013).
Although many therapies have been tried to counteract these changes, none of them had fully
proved efficiency. Caffeine was chosen to be studied in this experimental model due to the ability to
block the A1 and A2A ARs (Fredholm et al. 1999). It has been demonstrated that adenosine receptors are
involved in the regulation of ROS production, thus having protective effects in neurons and glial cells
(Schubert et al. 1997). We have chosen a low dose of caffeine (6 mg/kg b.w.) taking into consideration
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several adverse effects of large doses administration: insomnia, tremor, anxiety, cardiac arrhythmias,
hypertension and gastrointestinal disturbances (Bhattacharya et al. 1997; Chou and Benowitz 1994).
Furthermore, the dose used is equivalent to approximately 100 mg of caffeine in humans, which is the
amount contained in a cup of coffee (Allometric Scaling Calculator; Fredholm et al. 1999).
We have evaluated the effect of caffeine on blood oxidative stress markers as the general
indicator of oxidant/antioxidant status. The brain is highly biochemically sensitive to oxidative damage
(Halliwell 2006). It is rich in polyunsaturated fatty acids which are substrates for oxidation and due to
the considerable oxygen consumption; the production of ROS in brain mitochondria is higher. In
addition, the brain has modest antioxidant mechanisms. As previously discussed, the hippocampus
seems to be the most susceptible area to the ovariectomy-induced estrogen depletion. For these
reasons we have evaluated the effect of caffeine administration on ovariectomy-induced OS in rats, by
assessing the NO levels and the OS markers in the whole brain homogenate, along with the MDA levels
and antioxidant enzymes activities in the hippocampus.
In this study, we noticed that caffeine improved the GSH/GSSG ratio in blood and brain, in both
experimental subsets. Given that cysteine is the limiting amino acid for the glutathione synthesis (Chung
et al. 1990) and caffeine may stimulate the cysteine uptake by cells (Aoyama et al. 2011), probably the
GSH/GSSG ratio was improved, at least partially, by this mechanism. After subchronic caffeine
administration in ovariectomized rats, the GSH/GSSG ratios in blood and brain increased, close to
control levels. Interestingly, the chronic treatment led to higher ratios as compared to the control
groups. These results suggest there is excessive free radicals generation early after ovariectomy or other
mechanisms involved in the GSH synthesis are activated only after a long period of caffeine
administration.
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GPx and CAT activities had a different pattern in the brain and hippocampus, in the two
experimental subsets. The subchronic treatment increased the CAT activity while the GPx activity was
not changed. It is known that the stimulation of A3 receptors by adenosine is cytoprotective and
enhances the activity of antioxidant enzymes (Maggirwar et al. 1994). The high free-adenosine levels
due to A1 and A2A ARs blockade by caffeine (Conlay et al. 1997) and the low affinity of caffeine for the A3
receptors (Fredholm et al. 1999) are two conditions that may allow endogenous adenosine to stimulate
the A3 ARs. Conversely, the chronic treatment decreased both enzyme activities and at the same time
the brain MDA levels were enhanced, probably due to reduced ability of GPx to remove the lipid
peroxides.
Another explanation is related to the increased production of NO and protein nitration reactions
(Radi 2013). In normal concentration, NO has beneficial effects in the brain, but pathological levels
inhibit nearly all enzyme-catalyzed reactions through tyrosine residues’ oxidation, resulting in nitrated
proteins. In our study, the chronic administration of caffeine in ovariectomized rats enhanced the NO
level in brain and at the same time the antioxidant enzymes had decreased activities. Previous studies
showed caffeine had an inhibitory effect on the activity of brain arginase (Nikolic et al. 2003). Thus, it
may contribute to the NO synthesis by increasing the available arginine pool. Although after chronic
treatment the NO level in the OVX group was similar to control, at 21 days after ovariectomy we
observed a 2-fold higher brain NO level in the OVX group as compared to control. This is consistent with
the literature data which reported increased levels of nitrated proteins after the same period of time
(Patki et al. 2013). Through bilateral ovariectomy, the rat’s body needs to manage not only the abrupt
estrogens depletion, but also the high level of pro-inflammatory cytokines. The activation of inducible
nitric oxide synthase (iNOS) in response to these cytokines could be another mechanism behind the
enhanced NO secretion following ovariectomy (Garry et al. 2015).
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Interestingly, the subchronic caffeine administration in ovariectomized rats decreased the NO to
the control levels. The ability of caffeine to increase the GSH synthesis was previously discussed and it is
also known that the pathological NO level can be scavenged by the GSH, resulting in inactive molecules
called nitrosothiols (Fang et al. 2002). Furthermore, it was reported that caffeine, via adenosine A1
receptors blockade, increased the cAMP production and inhibited pre-transcriptional TNF-α production
in cord blood monocytes (Chavez-Valdez et al. 2009). Thus, the decreased NO level close to control, in
subchronic caffeine treatment, could be also due to the inhibition of iNOS by the reduced cytokines
level. In addition, Han YJ et al (2001) reported that antioxidant enzymes, especially CAT, can suppress
the NO production in macrophages through the inhibition of NF-kB activation. Moreover, the fact that
after 42 days of caffeine administration the NO level was enhanced and at the same time the CAT
activity was decreased supports this theory. However, further studies are necessarily to clarify the
mechanisms behind these changes.
The acute and chronic administration of ARs modulators has opposite effects, a phenomenon
called “the effect inversion”. It was firstly described on behavior but the mechanisms involved are not
yet fully understood. Some authors suggested that it may be dependent upon the duration of exposure
and the selectivity of ARs ligands (Jacobson et al. 1996). In our study, the subchronic caffeine
administration had antioxidant effects, while the chronic exposure exacerbated the oxidative stress in
the brain of ovariectomized rats. The stimulation of the A2A receptors by adenosine leads to injurious
effects, mainly due to the glutamate release, while the activation of the A1 receptors is neuroprotective
(Cunha 2005). Several A2A receptors antagonists (including caffeine) reduced the injury in animal models
of ischemic stroke (Phillis 1995), Parkinson’s disease (Chen et al. 2001) and Huntington’s disease (Fink et
al. 2004). Furthermore, the stimulation of adenosine A2A receptors potentiated the nitric oxide release
and glutamate efflux from the glial cells (Saura et al. 2005), which in turn may stimulate the activity of
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iNOS and amplify the NO production. The effect can be inhibited by A2A ARs antagonists (Garry et al.
2015; Li et al. 2001). In addition, as a response to the chronic exposure to caffeine the increased
expression of A1 ARs may lead to a shift in the A1/A2A ARs balance (Ferre 2008; Shi et al. 1993).
Moreover, the A2A receptors have a higher affinity for caffeine compared with A1 receptors (Fredholm et
al. 1999) and this could explain the neuroprotective effects of caffeine after subchronic treatment. In
the chronic administration of caffeine, the ARs tended to heteromerize and thereby decreased the
affinity of caffeine for A2A receptors (Ciruela et al. 2006) and enhanced the oxidative damage in brain.
The increased locomotor activity in rats as a response to small or moderate doses of caffeine
was previously reported (Fredholm et al. 1999), but the behavioral effects of caffeine in ovariectomized
rats were never studied. Garrett et al (1994) demonstrated that caffeine inhibited the negative effects of
adenosine from dopamine receptors and produced locomotor stimulant effects. Both A1 and A2A
receptors are involved in the motor-activating effects of caffeine (Karcz-Kubicha et al. 2003). Moreover,
the caffeine had the ability to induce glutamate-dependent and glutamate-independent release of
dopamine (Ferre 2008). In our study, after 21 days of caffeine administration in ovariectomized rats,
they became hyperactive as seen in OFT. This effect was not observed after chronic caffeine treatment
and it may be due to the tolerance of the ARs in chronic exposure.
The mechanisms involved in ARs tolerance are still under debate. Some changes in the A1-A2A
heteromers (Ciruela et al. 2006), the tolerance to the effects of A1 receptor blockade (Karcz-Kubicha et
al. 2003), specific gene expression alterations in the striatum (Svenningsson et al. 1999), the pushed-up
GABAergic activity in the brain (Mukhopadhyay and Poddar 1998) and changes in dopamine receptors
(Powell et al. 2001) were proposed as underlying mechanisms for this process.
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In order to assess some changes in anxiety and emotionality, we chose two behavioral tests: the
OFT and the EPM. In OFT, an increase in the central locomotion (travelled distance in center, number of
entries in center) or in the time spent in the central part of the device (time spent in the center/total
time), can be considered as reduced emotionality behavior (Ramos 2008). Our results showed that
subchronic administration of caffeine in ovariectomized rats increased significantly the center time ratio
and the central locomotion (distance and entries in center). Conversely, the chronic administration of
caffeine showed a significant decrease in central locomotion as compared to OVX group. Thus, the
subchronic, but not the chronic, administration of caffeine decreased the emotionality index of the
ovariectomized rats.
In addition, the EPM test was used in our experiments. It has been validated to assess the
anxiety-like behavior in rats, based on the natural aversion of rodents for open spaces (Walf and Frye
2007) and it is recognized as the gold standard for anxiety-like behavior evaluation in rodents. The time
spent in the unprotected open arms calculated as a ratio of the total time is considered a relevant
parameter of anxiety (Walf and Frye 2007). Both the subchronic and chronic administration of caffeine
in ovariectomized rats had anxiolytic effects in EPM, sustained by increased time spent in the open
arms. As long as the hippocampus was reported to be involved in anxiety-like behavior (Shin 2006), the
reduced MDA levels in the hippocampus after caffeine administration could explain the anxiolytic effect
of caffeine. The decline in catalase activity in subchronic administration was probably due to the direct
destruction by free radicals (Hellemans et al. 2003).
The effects of caffeine administration on anxiety are still debated in literature. Some authors
have claimed caffeine induces anxiety (Bhattacharya et al. 1997) while others reported an anxiolytic
effect (Tang et al. 1989), but all these studies were carried out on healthy subjects. The A2A ARs,
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responsible for the anxiety-like effect, may become tolerant to the long-term caffeine exposure. Thus,
the anxiolytic effect is becoming predominant with chronic administration. The involvements of the A2A
receptors in the genesis of anxiety effect, the A2A antagonists capacity to ameliorate anxiety-like
behaviors in rodents and the increased densities of A1, but not A2A ARs after chronic caffeine treatment,
support this idea (Shi et al. 1993; Yamada et al. 2014).
Even though, the OFT is considered the most popular test used in behavioral research, its
meaning in the field of emotionality/anxiety still remains unclear. OFT has been compared with EPM and
it seems they measure different aspects of emotionality in rodents, a fact sustained by Ramos (Ramos
2008). It has been mentioned that environments, such as open spaces, brightly illuminated or elevated
areas, may influence the animals behavioral responses (Anchan et al. 2014; Ramos 2008). Thus, the
difference, seen in regard to these tests, underscores the importance of using more than one test in the
assessment of anxiety-like behavior in rodents.
In conclusion, our study demonstrates that the subchronic administration of caffeine in low dose
(6 mg/kg b.w.) decreased the oxidative stress and had anxiolytic effects in ovariectomized rats. The
improved GSH/GGSG ratio and increased the activity of antioxidant enzymes in parallel with decreasing
the nitric oxide levels, could be the mechanisms behind the neuroprotective effects of caffeine.
However, probably due to the specific changes in the adenosine receptors, the chronic caffeine
administration exacerbated the oxidative stress in the whole brain homogenate, but not in the
hippocampus, demonstrating also anxiolytic effects. The oxidative stress parameters in the brain of
ovariectomized rats were almost the same as in the control group after 42 days of experiment and at
the same time, the chronic treatment enhanced the overall oxidative stress. These observations, led us
to the idea that there should be no reason for long-term caffeine administration. Taken together, these
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experimental findings suggest that the administration of low doses of caffeine for a short period of time
may be a new therapeutic approach to decrease the enhanced oxidative stress and the heightened
anxiety in menopause. Moreover, this study suggests the necessity for additional studies to investigate
the effects of caffeine and specific adenosine receptors antagonists on the brain oxidant/antioxidant
status in relation to the behavioral changes in menopause.
Authors’ contributions
IC designed the experiments and wrote the paper. AB conducted the behavioral tests, performed the
statistical analysis for behavioral parameters and drafted the manuscript. RM designed and
implemented the research study. ND carried out and analyzed the biochemical parameters. RO
contributed to the implementation of the study and critically revised the manuscript for important
intellectual content. AF did the statistical analysis for oxidative stress parameters, wrote the paper and
gave the final approval. All authors read and approved the manuscript.
Acknowledgements
We wish to thank the members of the Oxidative Stress Laboratory and Physiology Department of ‘‘Iuliu
Hatieganu’’ University of Medicine and Pharmacy, Cluj-Napoca, for providing the materials and
supporting this work.
Competing interests
The authors declare that they have no conflict of interest.
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List of abbreviations used:
ARs – adenosine receptors
CAT – catalase
EPM – elevated plus maze
GPx – glutathione peroxidase
GSH – reduced glutathione
GSSG – oxidized glutathione
iNOS – inducible nitric oxide synthase
MDA – malondialdehyde
NO – nitric oxide
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OFT – open field test
OS – oxidative stress
ROS – reactive oxygen species
SEM – standard error of mean
Figures legends
Figure 1. Experimental design. The two subsets of experiments (subchronic-21 days; chronic-42 days)
are illustrated as a diagram. OVX-ovariectomized; OFT-Open Field Test; EPM-Elevated Plus Maze.
Figure 2. Oxidative stress parameters assessment in blood. A) In subset 1 of experiments, MDA level
increased in OVX group as compared to CON (p=0.01) and decreased after CAF administration (p=0.01).
B) In subset 2, lipid peroxidation enhanced in OVX group as compared to CON (p=0.03) and decreased
after caffeine administration (p=0.004). C) GSH/GSSG ratio diminished in OVX group as compared to
CON (p=0.003) and increased after 21 days of caffeine administration (OVX+CAF vs. OVX group p=0.01)
D) while in subset 2 of experiments, GSH/GSSG ratio increased in OVX+CAF (p
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compared to OVX group (p=0.03) in subset 1 while in subset 2 of experiments D) NO enhanced after
caffeine administration (OVX+CAF vs. OVX group, p=0.02). Each group consisted of 8 rats. Results are
expressed as mean ± SEM; *p
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entries increased in OVX+CAF group as compared to OVX group (p0.05). Each group consisted of 13 rats. Results are expressed
as mean ± SEM; *p
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Figure 8. The effects of caffeine on emotionality in OFT and anxiety in EPM. A) The center time/total
time ratio increased in OVX+CAF group as compared to OVX group (p=0.004) in subset 1 of experiments
while in subset 2 B) the same parameter was not significantly different. C) The open arms time/total
time ratio increased in OVX+CAF group as compared to OVX group (p=0.006) in subset 1 of experiments
while in subset 2 D) the same parameter increased (p
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Figure 1. Experimental design. The two subsets of experiments (subchronic-21 days; chronic-42 days) are illustrated as a diagram. OVX-ovariectomized; OFT-Open Field Test; EPM-Elevated Plus Maze.
338x190mm (300 x 300 DPI)
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Canadian Journal of Physiology and Pharmacology
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Figure 2. Oxidative stress parameters assessment in blood. A) In subset 1 of experiments, MDA level increased in OVX group as compared to CON (p=0.01) and decreased after CAF administration (p=0.01). B) In subset 2, lipid peroxidation enhanced in OVX group as compared to CON (p=0.03) and decreased after
caffeine administration (p=0.004). C) GSH/GSSG ratio diminished in OVX group as compared to CON (p=0.003) and increased after 21 days of caffeine administration (OVX+CAF vs. OVX group p=0.01) D) while in subset 2 of experiments, GSH/GSSG ratio increased in OVX+CAF (p
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Figure 3. The effects of caffeine administration on the GSH/GSSG ratios and NO levels in the brain A) GSH/GSSG ratio was reduced in the OVX group as compared to CON (p=0.008) and increased in OVX+CAF group as compared to OVX group (p=0.01) in subset 1 of experiments. B) GSH/GSSG ratio increased in
OVX+CAF group as compared to OVX group (p=0.008) in subset 2 of experiments. C) NO level increased in OVX group as compared to CON group (p
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Figure 4. The effects of caffeine administration on the MDA levels and the antioxidant enzymes activity in brain. A) The level of MDA decreased after 21 days of caffeine administration in OVX+CAF group as
compared to OVX group in the whole brain homogenate (p=0.009) and also in the hippocampus (p=0.02). B) After 42 days of caffeine administration, the MDA level in the whole brain homogenate increased in the OVX+CAF group as compared to OVX (p=0.01), while in the hippocampus it was decreased (p=0.003). C) GPx activity was not significantly different between the OVX+CAF and OVX groups (p>0.05) in subset 1 of experiments while in subset 2 D) GPx activity decreased in the whole brain homogenate, but not in the hippocampus, after caffeine administration (OVX+CAF vs. OVX group, p=0.001). E) In the whole brain
homogenate, the CAT activity increased in OVX+CAF group as compared to OVX group (p=0.009), while in the hippocampus it was decreased. F) The CAT activity in the brain homogenate diminished in OVX+CAF
group as compared to OVX group (p=0.01), but these differences were not observed in the hippocampus. In each groups, 8 rats were used for the whole brain homogenate analysis and 5 rats for the hippocampus
assessments. Results are expressed as mean ± SEM; *p
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Figure 5. The effects of caffeine on the total travelled distance and the number of entries in OFT. A) The total travelled distance was increased in CAF group as compared to CON group (p
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Figure 6. The effects of caffeine on the travelled distance and the number of entries in periphery in OFT. A) In subset 1 of experiments the travelled distance in periphery increased in CAF group as compared to CON
group (p
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Figure 7. The effects of caffeine on emotionality in OFT. A) The travelled distance in center increased in OVX+CAF group as compared to OVX group (p=0.03) in subset 1 of experiments while in subset 2 B) the
same parameter decreased after caffeine administration (p=0.01 vs. OVX group). C) The number of entries in center enhanced in OVX+CAF group as compared to OVX group (p=0.02) after 21 days of caffeine
administration while D) after 42 days decreased (p=0.01 vs. OVX group). Each group consisted of 13 rats. Results are expressed as mean ± SEM; *p
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Figure 8. The effects of caffeine on emotionality in OFT and anxiety in EPM. A) The center time/total time ratio increased in OVX+CAF group as compared to OVX group (p=0.004) in subset 1 of experiments while in
subset 2 B) the same parameter was not significantly different. C) The open arms time/total time ratio
increased in OVX+CAF group as compared to OVX group (p=0.006) in subset 1 of experiments while in subset 2 D) the same parameter increased (p