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Protective effect of exercise and sildenafil on acute stress and cognitive function
Physiology & Behavior 151 (2015) 230–237
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Protective effect of exercise and sildenafil on acute stress and cognitive function Dilek Ozbeyli a, Ayse Gizem Gokalp b, Tolga Koral b, Onur Yuksel Ocal b, Berkay Dogan b, Dilek Akakin c, Meral Yuksel d, Ozgur Kasimay a,⁎ a
Department of Physiology, Marmara University School of Medicine, Istanbul, Turkey Undergraduate Medical Students, Marmara University School of Medicine, Istanbul, Turkey Department of Histology & Embryology, Marmara University School of Medicine, Istanbul, Turkey d Department of Medical Laboratory Techniques, Marmara University Vocational School of Health Services, Istanbul, Turkey b c
H I G H L I G H T S • • • • •
Effect of exercise and sildenafil on anxiety and cognition was examined. Protection from acute stress will improve cognitive functions. Chronic exercise and sildenafil improved stress status and cognitive functions. Sildenafil treatment protected from inflammatory changes induced by acute stress. Chronic exercise attenuated inflammatory status provoked by stress.
a r t i c l e
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Article history: Received 1 April 2015 Received in revised form 10 July 2015 Accepted 23 July 2015 Available online 28 July 2015 Keywords: Chronic exercise Sildenafil Acute stress
a b s t r a c t Introduction: There are contradictory results about the effects of exercise and sildenafil on cognitive functions. Aim: To investigate the effects of sildenafil pretreatment and chronic exercise on anxiety and cognitive functions. Method: Wistar rats (n = 42) were divided as sedentary and exercise groups. A moderate-intensity swimming exercise was performed for 6 weeks, 5 days/week, 1 h/day. Some of the rats were administered orogastrically with sildenafil (25 mg/kg/day) either acutely or chronically. Exposure to cat odor was used for induction of stress. The level of anxiety was evaluated by elevated plus maze test, while object recognition test was used to determine cognitive functions. Brain tissues were removed for the measurement of myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide levels, lucigenin-enhanced chemiluminescence, and for histological analysis. Results: Increased MPO and MDA levels in sedentary-stressed rats were decreased with sildenafil applications. Chronic exercise inhibited the increase in MPO levels. Increased nitric oxide and lucigenin chemiluminescence levels in sedentary-stressed rats, were diminished with chronic sildenafil pretreatment. The time spent in the open arms of the plus maze was declined in sedentary-stressed rats, while chronic sildenafil pretreatment increased the time back to that in non-stressed rats. Acute sildenafil application to exercised rats prolonged the time spent in open arms as compared to non-treated exercise group. The time spent with the novel object, which was decreased in sedentary-stressed rats, was increased with sildenafil pretreatment. Our results suggest that sildenafil pretreatment or exercise exerts a protective effect against acute stress and improves cognitive functions by decreasing oxidative damage. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
⁎ Corresponding author at: Department of Sports Physiology, Marmara University School of Medicine, Fevzi Çakmak Mah, Mimar Sinan Cad, No. 41, 34899 Ust Kaynarca, Pendik, Istanbul, Turkey. E-mail address: [email protected] (O. Kasimay).
http://dx.doi.org/10.1016/j.physbeh.2015.07.030 0031-9384/© 2015 Elsevier Inc. All rights reserved.
Sildenafil is a phosphodiesterase-5 (PDE-5) inhibitor that passes blood–brain barrier [1,2]. Sildenafil acts on intracellular cyclic guanosine monophosphate (cGMP) pathway, and in turn, leads to vascular smooth muscle relaxation, vasodilation, and overall an improvement in cerebral circulation [3]. Currently, sildenafil is used to treat erectile
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dysfunction and pulmonary hypertension by relaxing the vascular smooth muscle and allowing blood to flow easily [1,4]. Previous studies have shown that PDE5 inhibitors also have a role in nitric oxide (NO)/cGMP signaling pathway, which may affect the related parts of the brain that modulate the stress response and cognitive functions [5,6]. Although some studies have reported that acute administration of sildenafil might cause an anxiogenic effect, the effect of chronic sildenafil administration on anxiety is still unclear [7,8]. Solis et al. have indicated the anxiolytic effect of chronic sildenafil treatment based on the results of open-field test, which pointed out an increased number of center entries and a prolonged time spent in the center of the open field apparatus [9]. Additionally, in some animal studies, it was indicated that sildenafil consolidated the memory [10–14], and reported that injection of a cGMP analog into rat hippocampus improved the results of object recognition test, which is associated with memory consolidation [5]. Stress is an inevitable element in human life that can be defined as disruption of homeostasis. Following the perception of an acute stressful event, there is a cascade of changes in the nervous, cardiovascular, endocrine, and immune systems [15]. Stress augments oxidative damage and production of oxidants that may increase the risk of various diseases [16]. Furthermore, stress paradigms in animal models produce a variety of behavioral and physiological changes such as cognitive alterations, and numerous studies have reported the interactions of stress and cognitive changes at different stages of life in both animal models and in humans [17]. Moreover, acute psychological stress has different outcomes on variant cognitive functions such as improvement in declarative memory [18,19] along with a reduction in working memory [20]. The relationship between exercise and its possible changes in the body is partly understood. Although the effects of aerobic exercise on physiological functions were discussed extensively in previous studies, the effect of exercise on mental processes are still under investigation. Clinical trials have indicated that chronic physical exercise can have antidepressant and anxiolytic effects [21–23]. Furthermore, moderate intensity exercise also reduces oxidative stress [24,25], implying that exercise can be an effective and cost-efficient therapeutic alternative for a variety of anxiety and mood-related disorders. Although exercise has been shown as a therapeutic tool in most of the studies, nonvoluntary chronic exercise may be a stressor due to experimental design, housing or testing environment, rearing and handling situations in animal studies [26]. Up to now, researches on the relationship between exercise and cognitive function have yielded different results. In 1958, Clarke pointed out the physical benefits of exercise and its possible enhancing effects on cognitive functions [27]. Etnier et al. also emphasized the benefits of exercise on cognitive functions; however they concluded that the positive effects of exercise might be changed by duration and intensity of exercise, as well as the age/health/gender status of exerciser [28]. Moreover, the effect of chronic exercise on cognitive functions under acute stress conditions is not known yet. Eventually, there are contradictory results about the effects of sildenafil on anxiety. The effect of sildenafil pretreatment or chronic exercise on impaired cognitive functions due to acute stress is not clear and attracts attention. Thus, our aim was to investigate the effects of sildenafil pretreatment and chronic exercise on cognitive functions and anxiety-like behavior of rats exposed to acute psychological stress.
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pellets and tap water. All experimental protocols were approved by the Marmara University Animal Care and Use Committee. 2.2. Experimental design On the first day of the experiment, the rats were weighed, object recognition and plus maze tests were applied and blood samples were taken for the basal measurements (Fig. 1). The rats were divided into two main groups as sedentary and exercise groups. The exercise groups were exposed to a swimming protocol for 6 weeks. Rats in both groups were subjected to either acute or chronic sildenafil pretreatments, or received no treatments, and each of these subgroups consisted of 6 animals. In the last 2 weeks of the swimming protocol, the chronic sildenafil-pretreated group was administered sildenafil (25 mg/kg/day) by oro-gastric gavage at the end of the each exercise session. When the swimming protocol was completed, basal object recognition and plus maze tests were repeated. In the acute sildenafil-pretreated group, sildenafil was given at the same dosage and by the same route subsequent to object recognition and plus maze tests. Following a waiting period of 60 min, all groups except the control group were exposed to acute stress and all of the tests were repeated one last time. The rats were decapitated and trunk blood was taken for cortisol measurement. Additionally, brain tissues were removed and stored at −80 °C for the measurements of myeloperoxidase (MPO) activity, malondialdehyde (MDA), nitric oxide and lucigenin chemiluminescence levels. Additional samples were taken for histological analysis. 2.3. Swimming training protocol The rats were randomly assigned to sedentary and swimming groups and the swimming sessions were performed for 60 min/day, 5 days a week in warm water (30–32 °C) during the rats' dark cycle [29]. Sedentary rats were handled and dipped into water 5 times a week to mimic the same stress of touching the water that the exercised group had because of the training protocol. All rats were weighed weekly and followed closely. 2.4. Drug administration Sildenafil (Viagra; Pfizer, Istanbul) tablets (50 mg) was obtained from commercially available sources and dissolved in tap water. Sildenafil citrate (25 mg/kg/day) administration by oro-gastric gavage was performed only in one occasion in the acute sildenafil-pretreated group, while the dose was repeated daily for 2 weeks in the chronic sildenafil-pretreated group. After the administration of the drug, 1 h
2. Materials and methods 2.1. Animals Male Wistar rats (250–300 g, n = 42) supplied from the Marmara University Animal Center (DEHAMER; approval no: 14.2014.mar), were housed in a temperature-controlled (22 ± 2 °C) room and standardized light/dark (12/12 h) cycles. Rats were fed with standard rat
Fig. 1. Experimental protocol of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either nontreated or sildenafil (acute or chronic)-pretreated.
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was given for its absorption from the gastrointestinal tract before the rats were exposed to acute stress [30]. 2.5. Elevated plus maze test Elevated plus maze test is used to monitor the changes after anxiogenic/anxiolytic influences. There are 4 arms, two closed and two open, on the apparatus, which is 40–70 cm above the ground. Each rat was put in the apparatus facing the open arms and left there for 5 min, during which the process was recorded by a digital camera. The digital camera recordings were evaluated and the parameters for time spent in the open and closed arms were noted [31]. Percentage of the time spent in the open arms was calculated by the formula below. As the time spent in the open arms increases, the anxiety level decreases. Percentage of the time spent in the open arms (%): time spent in the open arms × 100 / (time spent in open + closed arms). 2.6. Acute stress model For acute stress exposure, the rats were put for 10 min into a cage containing 1 ml of fresh cat urine and 1 g of cat feces with no extra odor in it [32]. 2.7. Object recognition test The test was developed to evaluate cognitive functions. The test has familiarizing, sample object and novel object steps [33]. Dimensions of the boxes used for the test were 31 × 24 × 45.5 cm. The floor of the box was cleaned after each rat, and 3 objects (2 same and 1 different) were assigned to each box. The rats were familiarized with the researcher before the test. The subjects were kept in the test room for a while (10 min) in the 24-hour period before the test, so that the time that the rat would spend to explore the area was minimized to avoid the restriction of object recognition time. 2.7.1. Familiarizing Each rat was put into the boxes one by one for 10 min, during which the rat was familiarized with the environment. 2.7.2. Sample object (old object) Two identical objects were put and the rats, facing the opposite site, were released into the box. The test was recorded for 10 min by a video camera. It is important that no other stimulus was present in the environment that would distract the rat's attention. Before putting the next rat into the box, the apparatus and the objects were cleaned with 70% isopropyl alcohol and left to dry for 1 min. 2.7.3. Novel object (new object) The rats, which completed the familiarizing step, waited for about an hour and were taken to the next step. One sample (old) and one novel (new) object were put and the rats facing the opposite site were released into the box. The test was recorded for 3 min by a video camera. 2.7.4. Evaluation The results were determined by comparing the time spent with the sample and the novel objects. The nose of the subject touching the object was counted as ‘‘contact’’. Object recognition is reflected by spending more time in interacting with the novel than familiar object, and is given a positive difference score. The time difference spent with the novel object was calculated by the formula below. Difference score (centisecond): Time spent with the novel object − time spent with the sample object.
2.8. Measurement of tissue myeloperoxidase activity Myeloperoxidase is an enzyme located in azurophilic granules of neutrophils to kill the harmful microorganisms. Calculation of MPO activity is used to evaluate the polymorphonuclear leukocyte (PMNL) infiltration in tissue, because tissue MPO activity is proportional to histochemically calculated PMNL levels [34]. MPO activities were calculated in 0.2–0.5 g brain tissue samples. Thus, tissue-associated MPO activity was utilized as an indication of accumulation of neutrophils. All reagents for MPO assay were obtained from Sigma (St Louis, MO, USA). The tissue samples (0.2–0.3 g) were homogenized in 10 volumes of ice-cold potassium phosphate buffer (50 mM K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5% w/v). The homogenate was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then re-homogenized with an equivalent volume of 50 mM K2HPO4 containing 0.5% (w/v) HETAB and 10 mM EDTA (Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine·2 HCl. One unit (U) of enzyme activity was defined as the amount of the MPO present/g tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C. MPO activity is stated as unit/g. 1 unit MPO activity is defined as reduction of 1 mol H2O2 in 1 min at 25 °C [35]. 2.9. Malondialdehyde assay In the presence of oxygen, free radicals cause peroxidation of lipids in the membrane structure of the cell or its organelles. Lipid peroxides cause serious damage of the membrane, organelles and cell by starting an autocatalytic chain reaction. The most known end product of lipid peroxidation, which itself is also toxic for the cell, is malondialdehyde (MDA). In this experiment, lipid peroxidation is evaluated by measuring MDA levels in the brain tissues. Tissue samples (150–300 mg) were homogenized with 10% trichloroacetic acid (TCA) and these homogenates were centrifuged in 3000 rpm under 4 °C for 15 min. Supernatants were taken to a different tube and centrifuged again in 15,000 rpm at 4 °C for 8 min. Supernatant samples mixed with 0.67% thiobarbituric acid (TBA) were incubated in a 100 °C water bath. Their absorbance values were read using spectrophotometry in 535 nm wavelength against blank solution. Lipid peroxidation levels were stated as nmol MDA/g [36]. 2.10. Serum cortisol levels Blood cortisol levels were measured on the first day of the experiment and after acute stress application by an electrochemiluminescence immunoassay using cortisol-specific biotinylated antibody and streptavidin coated microparticles (Modular Analytics E170, Roche Diagnostics, Germany). Within-run and total precision values given by the manufacturer for 7.53–46 μg/dl concentration levels were between 1.1–1.3% and 1.6%, respectively. The measuring range was between 0.018 and 63.4 μg/dl. 2.11. Lucigenin chemiluminescence measurement The evaluation of the free radicals in the brain tissue samples was done by chemiluminescence method. For this purpose, the tubes containing tissue samples in phosphate buffered saline (PBS) plus HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic Acid) were monitored by lucigenin (Bis-N-methylacridinium nitrate, 0.2 mM) probe, which is sensitive to superoxide radical. Lucigenin-added tubes were counted for 5 min at 1 min intervals in a luminometer (Berthold EG & G Junior Minilumat LB 9509, Germany) and the results were stated as area under curve (AUC) relative light unit (rlu) [30]. 2.11.1. Nitric oxide levels Nitric oxide levels were evaluated in the hippocampal area of brain tissue and the results were expressed in relative light unit (rlu).
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2.11.2. Histological analysis Coronal sections from the brains of animals from each group were cut in order to perform histological analysis of the hippocampus. The coronal sections were then immersion fixed in 10% buffered formalin. After fixation, tissues were processed for routine paraffin embedding, sectioned (5 μm thick) by a rotary microtome and mounted on slides for light microscopic evaluation. The sections were stained with cresyl violet to visualize the general cytoarchitecture and the severity of neuronal damage in dentate gyrus (DG) and cornus ammonis region 3 of the hippocampus proper (CA3) was scored semi-quantitatively as follows: score 0 = no damage, score 1 = mild damage, score 2 = moderate damage, and score 3 = severe damage. Degeneration of neurons was assessed as pyknotic nuclei, deep basophilia in cytoplasm, increase in perineuronal space, and swelling or shrinkage of the cell. The average of the damage scores was calculated for each group and statistical analysis was performed [37]. 2.11.3. Statistical analysis The data was expressed as means ± SEM. Graph Pad Prism 5.0 statistics software was used. Groups of data were compared with Student ttest or analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of p b 0.05 were regarded as significant. 3. Results The cortisol values of the subjects are shown in Table 1. There was a significant increase in cortisol values in stress groups compared to the control group (p b 0.05–0.001). In exercised and acute sildenafilpretreated exercise groups, cortisol levels were increased compared to their corresponding groups (p b 0.05–0.01). Brain tissue MPO levels indicating neutrophil infiltration were increased in sedentary rats with acute stress (p b 0.05), and were decreased significantly with both acute and chronic sildenafil pretreatments (p b 0.05–0.001). Exercise prevented the elevation of MPO levels as compared to sedentary group (p b 0.01) (Fig. 2A). When compared to control group, the rise in MDA and lucigenin levels in the brain tissues of sedentary group with acute stress application (p b 0.05–0.001) were decreased significantly with acute (p b 0.001) and chronic (p b 0.05–0.001) sildenafil pretreatments, while the MDA and lucigenin levels were not significantly increased in the exercised group. Besides, the lucigenin levels were further declined with exercise as compared to sedentary group (p b 0.01). Furthermore, the existing MDA values in exercised group were decreased with both acute (p b 0.01) and chronic (p b 0.05) sildenafil pretreatments (Fig. 2B–C). Although there was an increase in brain tissue NO levels of sedentary and acute sildenafil-pretreated sedentary groups with acute stress exposure (p b 0.01–0.001), decrease in NO levels was seen in sedentary rats with chronic sildenafil pretreatment (p b 0.01). There was a further increase in NO values of exercised rats compared to sedentary groups, except acute sildenafil-pretreated group (p b 0.01–0.001) (Fig. 3). Compared to control group, a significant decrease was observed in the time spent at the open arms of plus maze task of sedentary rats exposed to acute stress (p b 0.05), while chronic sildenafil pretreatment prolonged this time significantly (p b 0.001). The time spent in the open arms was not decreased significantly in the exercised group as
Fig. 2. Myeloperoxidase (MPO) activity (A), malondialdehyde (MDA) level (B) and lucigenin chemiluminescence measurement (C) in brain tissue of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. *p b 0.05, **p b 0.01, ***p b 0.001, compared to control group; +p b 0.05, ++p b 0.01, +++p b 0.001, compared to corresponding group; ФФp b 0.01, compared to its own sedentary group. Each group consists of 6 animals.
compared to control group. When comparing acute sildenafil pretreated groups, exercised group spent more time in open arms than the sedentary ones (p b 0.001). In contrary, in the chronic sildenafil-pretreated groups, sedentary group spent more time in open arms than the exercised group (p b 0.001) (Fig. 4).
Table 1 Serum cortisol levels of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil- (acute or chronic) pretreated.
Cortisol (μg/dl)
Control
Sedentary
Sedentary + acute sildenafil
Sedentary + chronic sildenafil
Exercise
Exercise + acute sildenafil
Exercise + chronic sildenafil
3.88 ± 0.12
4.88 ± 0.12⁎⁎⁎
5.4 ± 0.61⁎
6.66 ± 0.37⁎⁎
7.39 ± 0.59⁎⁎⁎
7.65 ± 0.56⁎⁎⁎
7.38 ± 0.3⁎⁎⁎
⁎ p b 0.05, compared to control group. Each group consists of 6 animals. ⁎⁎ p b 0.01, compared to control group. Each group consists of 6 animals. ⁎⁎⁎ p b 0.001, compared to control group. Each group consists of 6 animals.
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Fig. 3. Nitric oxide (NO) levels in hippocampal area of brain tissue in non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. **p b 0.01, ***p b 0.001, compared to control group; ++p b 0.01, compared to corresponding group; ФФp b 0.01, ФФФp b 0.001, compared to its own sedentary group. Each group consists of 6 animals.
With acute stress application, difference score, the time difference spent with the novel and sample objects, was decreased significantly in sedentary rats (p b 0.01), showing impaired cognitive functions. The time spent with the novel object was prolonged by chronic sildenafil pretreatment in sedentary rats, and the difference score was increased as compared to sedentary group (p b 0.05). Exercise increased the time spent with the novel object both in exercise (p b 0.01) and in chronic sildenafil-pretreated exercise groups, when compared to their corresponding groups (p b 0.05). However, the time spent with the novel object was further decreased in acute sildenafil-pretreated rats that have exercised, as compared to acute sildenafil-pretreated sedentary group (p b 0.001) (Fig. 5). Histological analysis is shown in Fig. 6. Histologic damage score was exaggerated remarkably with stress application in each group, except the chronic sildenafil-pretreated exercise and sedentary groups (p b 0.05). Additionally, histologic damage score was alleviated in chronic sildenafil-pretreated exercise group, when compared to exercise group (p b 0.05) (Fig. 6A). In the present study, our morphological findings in light microscopic level revealed regular granular cells in the dentate gyrus and pyramidal cells of the hippocampal CA3 regions of the control rats. However, large numbers of pyknotic and shrunken granular cells in dentate gyrus and pyramidal cells in the CA3 regions were evident in sedentary group following stress exposure, probably due to a
Fig. 4. Analysis of elevated plus maze test of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. Percentage of the time spent in the open arms (%): time spent in the open arms × 100 / (time spent in open + closed arms). *p b 0.05, **p b 0.01, compared to control group; +++p b 0.001, compared to corresponding group; ФФФp b 0.001, compared to its own sedentary group. Each group consists of 6 animals.
Fig. 5. Object recognition test results of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. Difference score (centisecond): Time spent with the novel object − time spent with the sample object. Centisecond: cs. **p b 0.01, ***p b 0001 compared to control group; +p b 0.05, compared to corresponding group; Фp b 0.05, ФФp b 0.01, compared to its own sedentary group. Each group consists of 6 animals.
severe process of degeneration. The “dark” degenerated neurons were decreased in the hippocampi of chronic sildenafil-pretreated rats that were kept on exercise, when compared to sedentary stressed rats (Fig. 6B). 4. Discussion The present study was undertaken to evaluate the possible protective effects of sildenafil pretreatment and chronic exercise on cognitive functions and anxiety levels following acute psychological stress exposure. The results demonstrate that sildenafil pretreatment or exercise training improved cognitive function and anxiety-like behavior, protected against acute stress by decreasing oxidative damage parameters, such as lipid peroxidation, neutrophil infiltration and lucigenin activity, which indirectly represents the generation of superoxide radicals in the tissue. Present findings indicate that cortisol levels were increased by acute stress induction in all groups. As the acute stress model of this study is a potent psychological stress model, sildenafil pretreatment and exercise training were not able to inhibit the increase in cortisol levels. Similarly, in a previous study predator odor has induced an increase in corticosterone levels [38]. In another study, as the plasma cortisol levels were not changed following restraint stress application, they stated the alterations in adrenal sensitivity was responsible for unchanged cortisol levels [39]. According to previous data, stress model and adrenal sensitivity may affect the plasma cortisol levels. Increased MPO activity in sedentary rats by acute stress induction was decreased significantly with both acute and chronic sildenafil pretreatments. The attenuation of elevated MPO activity by sildenafil pretreatment implies that the drug might be effective in decreasing neutrophil infiltration. Although in a previous study of our group, the increased MPO levels of brain tissue were observed following electric foot shock stress induction [40]; our study is the first showing the effect of sildenafil pretreatment on elevated MPO levels in stressed conditions. However, several studies have reported the ameliorating effect of sildenafil on MPO levels of inflammation and ischemia–reperfusion (I/R) models [41–45]. Besides, in our study exercise training with or without sildenafil pretreatment prevented MPO levels from rising. Similar to our results, Cakir et al. demonstrated that chronic exercise training performed before induction of acute stress reduced MPO activities in brain tissue [40]. Our results display that acute and chronic sildenafil pretreatment declined acute stress-induced elevation of MDA and lucigenin levels in sedentary rats. Our findings support previously shown data that MDA
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Fig. 6. Histological analysis of the hippocampus in non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. A. Histologic damage score. *p b 0.05, compared to control group; +p b 0.05, compared to corresponding group. B. Cresyl Violet staining of the hippocampal dentate gyrus (DG) of all groups under light microscope. (A.), Control group. Regular granular cells in the DG of the hippocampus are observed. (B.), Rats under stress with no treatment or exercise. (C.), Nonpretreated exercise group. (D.), Acute sildenafil-pretreated sedentary group. (E.), Acute sildenafil pretreated exercise group. (F.), Chronic sildenafil-pretreated sedentary group. (G.), Chronic sildenafil pretreated exercise group. Degenerated “dark” neurons in the stratum granulosum of DG are shown with arrows in B–G. Note that only few degenerated neurons are seen in the hippocampus of chronic sildenafil-treated rats that have exercised. DG, stratum granulosum of the DG; H, hilus. Scale bar, 20 μm. Each group consists of 6 animals.
levels, the indicator of lipid peroxidation, increase by stress application [40]. However, there is no data about effect of sildenafil on MDA or lucigenin levels of stressed animals. Several studies have demonstrated the ameliorating effect of sildenafil on inflammatory processes and in I/ R models [30,42,45,46–52]. In recent studies, chronic swimming exercise has been shown to block the MDA increase in oxidative stress and electric foot shock stress models [40,53]. Our results regarding the effects of exercise from stress exposure are consistent with previous data. This is the first time that acute and chronic sildenafil
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pretreatments have been shown to augment the protective effect of exercise against lipid peroxidation. Lucigenin activity indirectly represents the superoxide radicals in the tissue, while superoxide dismutase is the enzyme that reduces these radicals [30]. When lucigenin activity increases in the tissue, level of superoxide dismutase enzyme declines [54]. Although there is no previous data regarding the effect of stress on lucigenin activity, decreased hippocampal superoxide dismutase levels in response to acute restraint stress support our results [55]. Moreover, according to our findings, increased lucigenin levels by acute stress were declined with acute and chronic sildenafil pretreatment in sedentary rats. Karakoyun et al. demonstrated that chronic sildenafil treatment decreased the elevated lucigenin levels in the colonic tissues of rats with colitis [30]. The attenuation of lucigenin levels points out that sildenafil has the potential to reduce the generation of oxidants, mostly superoxide anions, in tissues. In accordance with a previous study reporting that chronic exercise training suppresses the formation of superoxide anions, the findings of our study demonstrate a reduction in the lucigenin levels of the exercised group, [56]. Our results support the previous studies that the time spent by sedentary rats in the open arms is reduced following acute stress exposure, indicating an increase in anxiety-like behavior [57,58]. Additionally, in a previous study chronic sildenafil pretreatment in unstressed mice increased the time spent in open arms, suggesting decreased anxiety [59]. Similarly, in our study, time spent in open arms was increased in sedentary stressed rats, when chronic sildenafil pretreatment was given. Furthermore, in a previous study rats having genetically increased anxiety- and depression-like behavior were used and chronic sildenafil treatment has reversed the reduced social interactive behavior [60]. Likewise, our results suggest the anxiolytic effect of chronic sildenafil pretreatment upon acute stress exposure. However, there are contradictory findings that demonstrate anxiogenic or anxiolytic effects of acute sildenafil. Although an acute dose of intraperitoneally administered sildenafil, 1 mg/kg and higher, was shown to increase the anxietylike behavior in both unstressed and immobilization-stressed mice [8, 61], in recent studies 1 mg/kg dose of sildenafil had an anxiolytic effect in unstressed mice, and higher doses (5 to 20 mg/kg) have augmented the antidepressant activity in stressed rats [62,63]. Moreover, in our study the anxiety-like behavior was increased in stressed rats that have received acute sildenafil pretreatment. These variant results might be attributed to different times and routes of drug application in these studies. In previous studies, sildenafil was applied intraperitoneally at 15, 30 min and 6.5 h before anxiety levels were evaluated, but in our study sildenafil was given orally 60 min before elevated plus maze test. In our study the time spent in the open arms was not altered significantly in chronic exercised rats with acute stress exposure. Similarly, Sciolino et al. have indicated that chronic voluntary exercise did not change anxiety-like behavior in elevated plus maze task of pharmacologically stressed rats [64]. Our group previously has reported the anxiolytic effect of chronic swimming exercise in acute electric shock applied rats by analyzing anxiety-like behavior via hole-board test [40]. Although in several studies low nitric oxide levels were reported to increase anxiety-like behavior, in other studies it was reported that increasing the nitric oxide/cGMP cascade induces anxiety responses [65],.When nitric oxide levels are either above normal or below normal, they may induce anxiety responses. In our study, nitric oxide levels were elevated in exercised and chronic sildenafil-pretreated rats, which have expressed anxiety-like behavior in elevated plus maze task. Furthermore, it is known that chronic exercise training increases nitric oxide levels [66,67]. Consistent with previous data, in our study NO levels were increased with acute stress induction [8,68]. Moreover, NO levels in aged and young mice were decreased by chronic tadalafil treatment, another PDE-5 inhibitor, and by ameliorating lipid peroxidation levels a beneficial effect in neuroprotection was also reported. Similarly, we observed
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an increase in NO and MDA levels of stressed rats and a decline in NO levels and MDA levels of stressed sedentary rats with chronic sildenafil application [69]. The decreased time difference spent with the novel and sample objects, showing the declined cognitive functions in sedentary rats by acute stress exposure, was prolonged with chronic sildenafil pretreatment. There is a limited data about effects of PDE-5 inhibitors on cognitive functions of stressed rats. A previous study supported our findings that chronic tadalafil treatment ameliorated memory impairment induced by maternal-separation in rat pups [70]. Increase in the cGMP level through inhibition of PDE-5 improves object recognition memory in animals and auditory selective attention and verbal recognition memory in humans [13,71]. Enhanced cGMP levels may increase glucose metabolism and blood flow, thus may improve memory and cognition [72–74]. Although in our study acute sildenafil pretreatment was not able to improve the loss of cognitive function with stress, chronic sildenafil pretreatment improved cognitive functions, which may involve an increase in cGMP levels. In addition, exercise by itself and exercise training along with chronic sildenafil pretreatment provided an increase in the cognitive functions. In accordance with our results, previous data have indicated that running exercise improves memory functions and promotes neurogenesis and synaptogenesis in hippocampus [75]. On the other hand, in sildenafil-pretreated rats, exercise training augmented the changes that were observed in corresponding sedentary groups. Briefly, exercise may strengthen the improving effect of chronic sildenafil on cognitive functions. Acute stress increased the histologic damage score in each group, except the chronic sildenafil-pretreated groups. Among the exercised rats, the chronic sildenafil pretreated ones had further declined histologic damage score. In our study, we aimed to demonstrate the oxidative changes that have occurred in dentate gyrus and hippocampus following exposure to stress. Several studies have focused on cell proliferation and death following stress exposure, and have shown that tadalafil improved the depressive symptoms and ameliorated memory impairment in a psychological stress model by suppressing apoptosis and enhancing cell proliferation in the hippocampus [70,76]. Also in a recent study, running prevented stress-induced proliferation of hippocampal granule cells and pyramidal cells [77]. Ultimately, histological analysis in our study supported the aforementioned data indirectly. In conclusion, our results demonstrate that acute stress exposure results in oxidative damage and the underlying mechanisms of this damage appear to involve neutrophil migration, increased lipid peroxidation levels and increased lucigenin activity in brain tissue. Furthermore, our results demonstrate for the first time that sildenafil or chronic exercise training reduced stress-induced oxidative brain damage along with reductions in anxiety-like behavior and improvement in cognitive functions, supporting the potential protective role of sildenafil and exercise on anxiety and cognition. Acknowledgments The authors are grateful to Prof. Dr. Inci Alican for the donation of drug and to Prof. Dr. Goncagul Haklar for cortisol measurements.
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Protective effect of exercise and sildenafil on acute stress and cognitive function Dilek Ozbeyli a, Ayse Gizem Gokalp b, Tolga Koral b, Onur Yuksel Ocal b, Berkay Dogan b, Dilek Akakin c, Meral Yuksel d, Ozgur Kasimay a,⁎ a
Department of Physiology, Marmara University School of Medicine, Istanbul, Turkey Undergraduate Medical Students, Marmara University School of Medicine, Istanbul, Turkey Department of Histology & Embryology, Marmara University School of Medicine, Istanbul, Turkey d Department of Medical Laboratory Techniques, Marmara University Vocational School of Health Services, Istanbul, Turkey b c
H I G H L I G H T S • • • • •
Effect of exercise and sildenafil on anxiety and cognition was examined. Protection from acute stress will improve cognitive functions. Chronic exercise and sildenafil improved stress status and cognitive functions. Sildenafil treatment protected from inflammatory changes induced by acute stress. Chronic exercise attenuated inflammatory status provoked by stress.
a r t i c l e
i n f o
Article history: Received 1 April 2015 Received in revised form 10 July 2015 Accepted 23 July 2015 Available online 28 July 2015 Keywords: Chronic exercise Sildenafil Acute stress
a b s t r a c t Introduction: There are contradictory results about the effects of exercise and sildenafil on cognitive functions. Aim: To investigate the effects of sildenafil pretreatment and chronic exercise on anxiety and cognitive functions. Method: Wistar rats (n = 42) were divided as sedentary and exercise groups. A moderate-intensity swimming exercise was performed for 6 weeks, 5 days/week, 1 h/day. Some of the rats were administered orogastrically with sildenafil (25 mg/kg/day) either acutely or chronically. Exposure to cat odor was used for induction of stress. The level of anxiety was evaluated by elevated plus maze test, while object recognition test was used to determine cognitive functions. Brain tissues were removed for the measurement of myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide levels, lucigenin-enhanced chemiluminescence, and for histological analysis. Results: Increased MPO and MDA levels in sedentary-stressed rats were decreased with sildenafil applications. Chronic exercise inhibited the increase in MPO levels. Increased nitric oxide and lucigenin chemiluminescence levels in sedentary-stressed rats, were diminished with chronic sildenafil pretreatment. The time spent in the open arms of the plus maze was declined in sedentary-stressed rats, while chronic sildenafil pretreatment increased the time back to that in non-stressed rats. Acute sildenafil application to exercised rats prolonged the time spent in open arms as compared to non-treated exercise group. The time spent with the novel object, which was decreased in sedentary-stressed rats, was increased with sildenafil pretreatment. Our results suggest that sildenafil pretreatment or exercise exerts a protective effect against acute stress and improves cognitive functions by decreasing oxidative damage. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
⁎ Corresponding author at: Department of Sports Physiology, Marmara University School of Medicine, Fevzi Çakmak Mah, Mimar Sinan Cad, No. 41, 34899 Ust Kaynarca, Pendik, Istanbul, Turkey. E-mail address: [email protected] (O. Kasimay).
http://dx.doi.org/10.1016/j.physbeh.2015.07.030 0031-9384/© 2015 Elsevier Inc. All rights reserved.
Sildenafil is a phosphodiesterase-5 (PDE-5) inhibitor that passes blood–brain barrier [1,2]. Sildenafil acts on intracellular cyclic guanosine monophosphate (cGMP) pathway, and in turn, leads to vascular smooth muscle relaxation, vasodilation, and overall an improvement in cerebral circulation [3]. Currently, sildenafil is used to treat erectile
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dysfunction and pulmonary hypertension by relaxing the vascular smooth muscle and allowing blood to flow easily [1,4]. Previous studies have shown that PDE5 inhibitors also have a role in nitric oxide (NO)/cGMP signaling pathway, which may affect the related parts of the brain that modulate the stress response and cognitive functions [5,6]. Although some studies have reported that acute administration of sildenafil might cause an anxiogenic effect, the effect of chronic sildenafil administration on anxiety is still unclear [7,8]. Solis et al. have indicated the anxiolytic effect of chronic sildenafil treatment based on the results of open-field test, which pointed out an increased number of center entries and a prolonged time spent in the center of the open field apparatus [9]. Additionally, in some animal studies, it was indicated that sildenafil consolidated the memory [10–14], and reported that injection of a cGMP analog into rat hippocampus improved the results of object recognition test, which is associated with memory consolidation [5]. Stress is an inevitable element in human life that can be defined as disruption of homeostasis. Following the perception of an acute stressful event, there is a cascade of changes in the nervous, cardiovascular, endocrine, and immune systems [15]. Stress augments oxidative damage and production of oxidants that may increase the risk of various diseases [16]. Furthermore, stress paradigms in animal models produce a variety of behavioral and physiological changes such as cognitive alterations, and numerous studies have reported the interactions of stress and cognitive changes at different stages of life in both animal models and in humans [17]. Moreover, acute psychological stress has different outcomes on variant cognitive functions such as improvement in declarative memory [18,19] along with a reduction in working memory [20]. The relationship between exercise and its possible changes in the body is partly understood. Although the effects of aerobic exercise on physiological functions were discussed extensively in previous studies, the effect of exercise on mental processes are still under investigation. Clinical trials have indicated that chronic physical exercise can have antidepressant and anxiolytic effects [21–23]. Furthermore, moderate intensity exercise also reduces oxidative stress [24,25], implying that exercise can be an effective and cost-efficient therapeutic alternative for a variety of anxiety and mood-related disorders. Although exercise has been shown as a therapeutic tool in most of the studies, nonvoluntary chronic exercise may be a stressor due to experimental design, housing or testing environment, rearing and handling situations in animal studies [26]. Up to now, researches on the relationship between exercise and cognitive function have yielded different results. In 1958, Clarke pointed out the physical benefits of exercise and its possible enhancing effects on cognitive functions [27]. Etnier et al. also emphasized the benefits of exercise on cognitive functions; however they concluded that the positive effects of exercise might be changed by duration and intensity of exercise, as well as the age/health/gender status of exerciser [28]. Moreover, the effect of chronic exercise on cognitive functions under acute stress conditions is not known yet. Eventually, there are contradictory results about the effects of sildenafil on anxiety. The effect of sildenafil pretreatment or chronic exercise on impaired cognitive functions due to acute stress is not clear and attracts attention. Thus, our aim was to investigate the effects of sildenafil pretreatment and chronic exercise on cognitive functions and anxiety-like behavior of rats exposed to acute psychological stress.
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pellets and tap water. All experimental protocols were approved by the Marmara University Animal Care and Use Committee. 2.2. Experimental design On the first day of the experiment, the rats were weighed, object recognition and plus maze tests were applied and blood samples were taken for the basal measurements (Fig. 1). The rats were divided into two main groups as sedentary and exercise groups. The exercise groups were exposed to a swimming protocol for 6 weeks. Rats in both groups were subjected to either acute or chronic sildenafil pretreatments, or received no treatments, and each of these subgroups consisted of 6 animals. In the last 2 weeks of the swimming protocol, the chronic sildenafil-pretreated group was administered sildenafil (25 mg/kg/day) by oro-gastric gavage at the end of the each exercise session. When the swimming protocol was completed, basal object recognition and plus maze tests were repeated. In the acute sildenafil-pretreated group, sildenafil was given at the same dosage and by the same route subsequent to object recognition and plus maze tests. Following a waiting period of 60 min, all groups except the control group were exposed to acute stress and all of the tests were repeated one last time. The rats were decapitated and trunk blood was taken for cortisol measurement. Additionally, brain tissues were removed and stored at −80 °C for the measurements of myeloperoxidase (MPO) activity, malondialdehyde (MDA), nitric oxide and lucigenin chemiluminescence levels. Additional samples were taken for histological analysis. 2.3. Swimming training protocol The rats were randomly assigned to sedentary and swimming groups and the swimming sessions were performed for 60 min/day, 5 days a week in warm water (30–32 °C) during the rats' dark cycle [29]. Sedentary rats were handled and dipped into water 5 times a week to mimic the same stress of touching the water that the exercised group had because of the training protocol. All rats were weighed weekly and followed closely. 2.4. Drug administration Sildenafil (Viagra; Pfizer, Istanbul) tablets (50 mg) was obtained from commercially available sources and dissolved in tap water. Sildenafil citrate (25 mg/kg/day) administration by oro-gastric gavage was performed only in one occasion in the acute sildenafil-pretreated group, while the dose was repeated daily for 2 weeks in the chronic sildenafil-pretreated group. After the administration of the drug, 1 h
2. Materials and methods 2.1. Animals Male Wistar rats (250–300 g, n = 42) supplied from the Marmara University Animal Center (DEHAMER; approval no: 14.2014.mar), were housed in a temperature-controlled (22 ± 2 °C) room and standardized light/dark (12/12 h) cycles. Rats were fed with standard rat
Fig. 1. Experimental protocol of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either nontreated or sildenafil (acute or chronic)-pretreated.
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was given for its absorption from the gastrointestinal tract before the rats were exposed to acute stress [30]. 2.5. Elevated plus maze test Elevated plus maze test is used to monitor the changes after anxiogenic/anxiolytic influences. There are 4 arms, two closed and two open, on the apparatus, which is 40–70 cm above the ground. Each rat was put in the apparatus facing the open arms and left there for 5 min, during which the process was recorded by a digital camera. The digital camera recordings were evaluated and the parameters for time spent in the open and closed arms were noted [31]. Percentage of the time spent in the open arms was calculated by the formula below. As the time spent in the open arms increases, the anxiety level decreases. Percentage of the time spent in the open arms (%): time spent in the open arms × 100 / (time spent in open + closed arms). 2.6. Acute stress model For acute stress exposure, the rats were put for 10 min into a cage containing 1 ml of fresh cat urine and 1 g of cat feces with no extra odor in it [32]. 2.7. Object recognition test The test was developed to evaluate cognitive functions. The test has familiarizing, sample object and novel object steps [33]. Dimensions of the boxes used for the test were 31 × 24 × 45.5 cm. The floor of the box was cleaned after each rat, and 3 objects (2 same and 1 different) were assigned to each box. The rats were familiarized with the researcher before the test. The subjects were kept in the test room for a while (10 min) in the 24-hour period before the test, so that the time that the rat would spend to explore the area was minimized to avoid the restriction of object recognition time. 2.7.1. Familiarizing Each rat was put into the boxes one by one for 10 min, during which the rat was familiarized with the environment. 2.7.2. Sample object (old object) Two identical objects were put and the rats, facing the opposite site, were released into the box. The test was recorded for 10 min by a video camera. It is important that no other stimulus was present in the environment that would distract the rat's attention. Before putting the next rat into the box, the apparatus and the objects were cleaned with 70% isopropyl alcohol and left to dry for 1 min. 2.7.3. Novel object (new object) The rats, which completed the familiarizing step, waited for about an hour and were taken to the next step. One sample (old) and one novel (new) object were put and the rats facing the opposite site were released into the box. The test was recorded for 3 min by a video camera. 2.7.4. Evaluation The results were determined by comparing the time spent with the sample and the novel objects. The nose of the subject touching the object was counted as ‘‘contact’’. Object recognition is reflected by spending more time in interacting with the novel than familiar object, and is given a positive difference score. The time difference spent with the novel object was calculated by the formula below. Difference score (centisecond): Time spent with the novel object − time spent with the sample object.
2.8. Measurement of tissue myeloperoxidase activity Myeloperoxidase is an enzyme located in azurophilic granules of neutrophils to kill the harmful microorganisms. Calculation of MPO activity is used to evaluate the polymorphonuclear leukocyte (PMNL) infiltration in tissue, because tissue MPO activity is proportional to histochemically calculated PMNL levels [34]. MPO activities were calculated in 0.2–0.5 g brain tissue samples. Thus, tissue-associated MPO activity was utilized as an indication of accumulation of neutrophils. All reagents for MPO assay were obtained from Sigma (St Louis, MO, USA). The tissue samples (0.2–0.3 g) were homogenized in 10 volumes of ice-cold potassium phosphate buffer (50 mM K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5% w/v). The homogenate was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then re-homogenized with an equivalent volume of 50 mM K2HPO4 containing 0.5% (w/v) HETAB and 10 mM EDTA (Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine·2 HCl. One unit (U) of enzyme activity was defined as the amount of the MPO present/g tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C. MPO activity is stated as unit/g. 1 unit MPO activity is defined as reduction of 1 mol H2O2 in 1 min at 25 °C [35]. 2.9. Malondialdehyde assay In the presence of oxygen, free radicals cause peroxidation of lipids in the membrane structure of the cell or its organelles. Lipid peroxides cause serious damage of the membrane, organelles and cell by starting an autocatalytic chain reaction. The most known end product of lipid peroxidation, which itself is also toxic for the cell, is malondialdehyde (MDA). In this experiment, lipid peroxidation is evaluated by measuring MDA levels in the brain tissues. Tissue samples (150–300 mg) were homogenized with 10% trichloroacetic acid (TCA) and these homogenates were centrifuged in 3000 rpm under 4 °C for 15 min. Supernatants were taken to a different tube and centrifuged again in 15,000 rpm at 4 °C for 8 min. Supernatant samples mixed with 0.67% thiobarbituric acid (TBA) were incubated in a 100 °C water bath. Their absorbance values were read using spectrophotometry in 535 nm wavelength against blank solution. Lipid peroxidation levels were stated as nmol MDA/g [36]. 2.10. Serum cortisol levels Blood cortisol levels were measured on the first day of the experiment and after acute stress application by an electrochemiluminescence immunoassay using cortisol-specific biotinylated antibody and streptavidin coated microparticles (Modular Analytics E170, Roche Diagnostics, Germany). Within-run and total precision values given by the manufacturer for 7.53–46 μg/dl concentration levels were between 1.1–1.3% and 1.6%, respectively. The measuring range was between 0.018 and 63.4 μg/dl. 2.11. Lucigenin chemiluminescence measurement The evaluation of the free radicals in the brain tissue samples was done by chemiluminescence method. For this purpose, the tubes containing tissue samples in phosphate buffered saline (PBS) plus HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic Acid) were monitored by lucigenin (Bis-N-methylacridinium nitrate, 0.2 mM) probe, which is sensitive to superoxide radical. Lucigenin-added tubes were counted for 5 min at 1 min intervals in a luminometer (Berthold EG & G Junior Minilumat LB 9509, Germany) and the results were stated as area under curve (AUC) relative light unit (rlu) [30]. 2.11.1. Nitric oxide levels Nitric oxide levels were evaluated in the hippocampal area of brain tissue and the results were expressed in relative light unit (rlu).
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2.11.2. Histological analysis Coronal sections from the brains of animals from each group were cut in order to perform histological analysis of the hippocampus. The coronal sections were then immersion fixed in 10% buffered formalin. After fixation, tissues were processed for routine paraffin embedding, sectioned (5 μm thick) by a rotary microtome and mounted on slides for light microscopic evaluation. The sections were stained with cresyl violet to visualize the general cytoarchitecture and the severity of neuronal damage in dentate gyrus (DG) and cornus ammonis region 3 of the hippocampus proper (CA3) was scored semi-quantitatively as follows: score 0 = no damage, score 1 = mild damage, score 2 = moderate damage, and score 3 = severe damage. Degeneration of neurons was assessed as pyknotic nuclei, deep basophilia in cytoplasm, increase in perineuronal space, and swelling or shrinkage of the cell. The average of the damage scores was calculated for each group and statistical analysis was performed [37]. 2.11.3. Statistical analysis The data was expressed as means ± SEM. Graph Pad Prism 5.0 statistics software was used. Groups of data were compared with Student ttest or analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of p b 0.05 were regarded as significant. 3. Results The cortisol values of the subjects are shown in Table 1. There was a significant increase in cortisol values in stress groups compared to the control group (p b 0.05–0.001). In exercised and acute sildenafilpretreated exercise groups, cortisol levels were increased compared to their corresponding groups (p b 0.05–0.01). Brain tissue MPO levels indicating neutrophil infiltration were increased in sedentary rats with acute stress (p b 0.05), and were decreased significantly with both acute and chronic sildenafil pretreatments (p b 0.05–0.001). Exercise prevented the elevation of MPO levels as compared to sedentary group (p b 0.01) (Fig. 2A). When compared to control group, the rise in MDA and lucigenin levels in the brain tissues of sedentary group with acute stress application (p b 0.05–0.001) were decreased significantly with acute (p b 0.001) and chronic (p b 0.05–0.001) sildenafil pretreatments, while the MDA and lucigenin levels were not significantly increased in the exercised group. Besides, the lucigenin levels were further declined with exercise as compared to sedentary group (p b 0.01). Furthermore, the existing MDA values in exercised group were decreased with both acute (p b 0.01) and chronic (p b 0.05) sildenafil pretreatments (Fig. 2B–C). Although there was an increase in brain tissue NO levels of sedentary and acute sildenafil-pretreated sedentary groups with acute stress exposure (p b 0.01–0.001), decrease in NO levels was seen in sedentary rats with chronic sildenafil pretreatment (p b 0.01). There was a further increase in NO values of exercised rats compared to sedentary groups, except acute sildenafil-pretreated group (p b 0.01–0.001) (Fig. 3). Compared to control group, a significant decrease was observed in the time spent at the open arms of plus maze task of sedentary rats exposed to acute stress (p b 0.05), while chronic sildenafil pretreatment prolonged this time significantly (p b 0.001). The time spent in the open arms was not decreased significantly in the exercised group as
Fig. 2. Myeloperoxidase (MPO) activity (A), malondialdehyde (MDA) level (B) and lucigenin chemiluminescence measurement (C) in brain tissue of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. *p b 0.05, **p b 0.01, ***p b 0.001, compared to control group; +p b 0.05, ++p b 0.01, +++p b 0.001, compared to corresponding group; ФФp b 0.01, compared to its own sedentary group. Each group consists of 6 animals.
compared to control group. When comparing acute sildenafil pretreated groups, exercised group spent more time in open arms than the sedentary ones (p b 0.001). In contrary, in the chronic sildenafil-pretreated groups, sedentary group spent more time in open arms than the exercised group (p b 0.001) (Fig. 4).
Table 1 Serum cortisol levels of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil- (acute or chronic) pretreated.
Cortisol (μg/dl)
Control
Sedentary
Sedentary + acute sildenafil
Sedentary + chronic sildenafil
Exercise
Exercise + acute sildenafil
Exercise + chronic sildenafil
3.88 ± 0.12
4.88 ± 0.12⁎⁎⁎
5.4 ± 0.61⁎
6.66 ± 0.37⁎⁎
7.39 ± 0.59⁎⁎⁎
7.65 ± 0.56⁎⁎⁎
7.38 ± 0.3⁎⁎⁎
⁎ p b 0.05, compared to control group. Each group consists of 6 animals. ⁎⁎ p b 0.01, compared to control group. Each group consists of 6 animals. ⁎⁎⁎ p b 0.001, compared to control group. Each group consists of 6 animals.
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Fig. 3. Nitric oxide (NO) levels in hippocampal area of brain tissue in non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. **p b 0.01, ***p b 0.001, compared to control group; ++p b 0.01, compared to corresponding group; ФФp b 0.01, ФФФp b 0.001, compared to its own sedentary group. Each group consists of 6 animals.
With acute stress application, difference score, the time difference spent with the novel and sample objects, was decreased significantly in sedentary rats (p b 0.01), showing impaired cognitive functions. The time spent with the novel object was prolonged by chronic sildenafil pretreatment in sedentary rats, and the difference score was increased as compared to sedentary group (p b 0.05). Exercise increased the time spent with the novel object both in exercise (p b 0.01) and in chronic sildenafil-pretreated exercise groups, when compared to their corresponding groups (p b 0.05). However, the time spent with the novel object was further decreased in acute sildenafil-pretreated rats that have exercised, as compared to acute sildenafil-pretreated sedentary group (p b 0.001) (Fig. 5). Histological analysis is shown in Fig. 6. Histologic damage score was exaggerated remarkably with stress application in each group, except the chronic sildenafil-pretreated exercise and sedentary groups (p b 0.05). Additionally, histologic damage score was alleviated in chronic sildenafil-pretreated exercise group, when compared to exercise group (p b 0.05) (Fig. 6A). In the present study, our morphological findings in light microscopic level revealed regular granular cells in the dentate gyrus and pyramidal cells of the hippocampal CA3 regions of the control rats. However, large numbers of pyknotic and shrunken granular cells in dentate gyrus and pyramidal cells in the CA3 regions were evident in sedentary group following stress exposure, probably due to a
Fig. 4. Analysis of elevated plus maze test of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. Percentage of the time spent in the open arms (%): time spent in the open arms × 100 / (time spent in open + closed arms). *p b 0.05, **p b 0.01, compared to control group; +++p b 0.001, compared to corresponding group; ФФФp b 0.001, compared to its own sedentary group. Each group consists of 6 animals.
Fig. 5. Object recognition test results of non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. Difference score (centisecond): Time spent with the novel object − time spent with the sample object. Centisecond: cs. **p b 0.01, ***p b 0001 compared to control group; +p b 0.05, compared to corresponding group; Фp b 0.05, ФФp b 0.01, compared to its own sedentary group. Each group consists of 6 animals.
severe process of degeneration. The “dark” degenerated neurons were decreased in the hippocampi of chronic sildenafil-pretreated rats that were kept on exercise, when compared to sedentary stressed rats (Fig. 6B). 4. Discussion The present study was undertaken to evaluate the possible protective effects of sildenafil pretreatment and chronic exercise on cognitive functions and anxiety levels following acute psychological stress exposure. The results demonstrate that sildenafil pretreatment or exercise training improved cognitive function and anxiety-like behavior, protected against acute stress by decreasing oxidative damage parameters, such as lipid peroxidation, neutrophil infiltration and lucigenin activity, which indirectly represents the generation of superoxide radicals in the tissue. Present findings indicate that cortisol levels were increased by acute stress induction in all groups. As the acute stress model of this study is a potent psychological stress model, sildenafil pretreatment and exercise training were not able to inhibit the increase in cortisol levels. Similarly, in a previous study predator odor has induced an increase in corticosterone levels [38]. In another study, as the plasma cortisol levels were not changed following restraint stress application, they stated the alterations in adrenal sensitivity was responsible for unchanged cortisol levels [39]. According to previous data, stress model and adrenal sensitivity may affect the plasma cortisol levels. Increased MPO activity in sedentary rats by acute stress induction was decreased significantly with both acute and chronic sildenafil pretreatments. The attenuation of elevated MPO activity by sildenafil pretreatment implies that the drug might be effective in decreasing neutrophil infiltration. Although in a previous study of our group, the increased MPO levels of brain tissue were observed following electric foot shock stress induction [40]; our study is the first showing the effect of sildenafil pretreatment on elevated MPO levels in stressed conditions. However, several studies have reported the ameliorating effect of sildenafil on MPO levels of inflammation and ischemia–reperfusion (I/R) models [41–45]. Besides, in our study exercise training with or without sildenafil pretreatment prevented MPO levels from rising. Similar to our results, Cakir et al. demonstrated that chronic exercise training performed before induction of acute stress reduced MPO activities in brain tissue [40]. Our results display that acute and chronic sildenafil pretreatment declined acute stress-induced elevation of MDA and lucigenin levels in sedentary rats. Our findings support previously shown data that MDA
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Fig. 6. Histological analysis of the hippocampus in non-stressed or acute stress-induced rats, which were either sedentary or trained for 6 weeks before stress exposure and which were either non-treated or sildenafil (acute or chronic)-pretreated. A. Histologic damage score. *p b 0.05, compared to control group; +p b 0.05, compared to corresponding group. B. Cresyl Violet staining of the hippocampal dentate gyrus (DG) of all groups under light microscope. (A.), Control group. Regular granular cells in the DG of the hippocampus are observed. (B.), Rats under stress with no treatment or exercise. (C.), Nonpretreated exercise group. (D.), Acute sildenafil-pretreated sedentary group. (E.), Acute sildenafil pretreated exercise group. (F.), Chronic sildenafil-pretreated sedentary group. (G.), Chronic sildenafil pretreated exercise group. Degenerated “dark” neurons in the stratum granulosum of DG are shown with arrows in B–G. Note that only few degenerated neurons are seen in the hippocampus of chronic sildenafil-treated rats that have exercised. DG, stratum granulosum of the DG; H, hilus. Scale bar, 20 μm. Each group consists of 6 animals.
levels, the indicator of lipid peroxidation, increase by stress application [40]. However, there is no data about effect of sildenafil on MDA or lucigenin levels of stressed animals. Several studies have demonstrated the ameliorating effect of sildenafil on inflammatory processes and in I/ R models [30,42,45,46–52]. In recent studies, chronic swimming exercise has been shown to block the MDA increase in oxidative stress and electric foot shock stress models [40,53]. Our results regarding the effects of exercise from stress exposure are consistent with previous data. This is the first time that acute and chronic sildenafil
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pretreatments have been shown to augment the protective effect of exercise against lipid peroxidation. Lucigenin activity indirectly represents the superoxide radicals in the tissue, while superoxide dismutase is the enzyme that reduces these radicals [30]. When lucigenin activity increases in the tissue, level of superoxide dismutase enzyme declines [54]. Although there is no previous data regarding the effect of stress on lucigenin activity, decreased hippocampal superoxide dismutase levels in response to acute restraint stress support our results [55]. Moreover, according to our findings, increased lucigenin levels by acute stress were declined with acute and chronic sildenafil pretreatment in sedentary rats. Karakoyun et al. demonstrated that chronic sildenafil treatment decreased the elevated lucigenin levels in the colonic tissues of rats with colitis [30]. The attenuation of lucigenin levels points out that sildenafil has the potential to reduce the generation of oxidants, mostly superoxide anions, in tissues. In accordance with a previous study reporting that chronic exercise training suppresses the formation of superoxide anions, the findings of our study demonstrate a reduction in the lucigenin levels of the exercised group, [56]. Our results support the previous studies that the time spent by sedentary rats in the open arms is reduced following acute stress exposure, indicating an increase in anxiety-like behavior [57,58]. Additionally, in a previous study chronic sildenafil pretreatment in unstressed mice increased the time spent in open arms, suggesting decreased anxiety [59]. Similarly, in our study, time spent in open arms was increased in sedentary stressed rats, when chronic sildenafil pretreatment was given. Furthermore, in a previous study rats having genetically increased anxiety- and depression-like behavior were used and chronic sildenafil treatment has reversed the reduced social interactive behavior [60]. Likewise, our results suggest the anxiolytic effect of chronic sildenafil pretreatment upon acute stress exposure. However, there are contradictory findings that demonstrate anxiogenic or anxiolytic effects of acute sildenafil. Although an acute dose of intraperitoneally administered sildenafil, 1 mg/kg and higher, was shown to increase the anxietylike behavior in both unstressed and immobilization-stressed mice [8, 61], in recent studies 1 mg/kg dose of sildenafil had an anxiolytic effect in unstressed mice, and higher doses (5 to 20 mg/kg) have augmented the antidepressant activity in stressed rats [62,63]. Moreover, in our study the anxiety-like behavior was increased in stressed rats that have received acute sildenafil pretreatment. These variant results might be attributed to different times and routes of drug application in these studies. In previous studies, sildenafil was applied intraperitoneally at 15, 30 min and 6.5 h before anxiety levels were evaluated, but in our study sildenafil was given orally 60 min before elevated plus maze test. In our study the time spent in the open arms was not altered significantly in chronic exercised rats with acute stress exposure. Similarly, Sciolino et al. have indicated that chronic voluntary exercise did not change anxiety-like behavior in elevated plus maze task of pharmacologically stressed rats [64]. Our group previously has reported the anxiolytic effect of chronic swimming exercise in acute electric shock applied rats by analyzing anxiety-like behavior via hole-board test [40]. Although in several studies low nitric oxide levels were reported to increase anxiety-like behavior, in other studies it was reported that increasing the nitric oxide/cGMP cascade induces anxiety responses [65],.When nitric oxide levels are either above normal or below normal, they may induce anxiety responses. In our study, nitric oxide levels were elevated in exercised and chronic sildenafil-pretreated rats, which have expressed anxiety-like behavior in elevated plus maze task. Furthermore, it is known that chronic exercise training increases nitric oxide levels [66,67]. Consistent with previous data, in our study NO levels were increased with acute stress induction [8,68]. Moreover, NO levels in aged and young mice were decreased by chronic tadalafil treatment, another PDE-5 inhibitor, and by ameliorating lipid peroxidation levels a beneficial effect in neuroprotection was also reported. Similarly, we observed
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an increase in NO and MDA levels of stressed rats and a decline in NO levels and MDA levels of stressed sedentary rats with chronic sildenafil application [69]. The decreased time difference spent with the novel and sample objects, showing the declined cognitive functions in sedentary rats by acute stress exposure, was prolonged with chronic sildenafil pretreatment. There is a limited data about effects of PDE-5 inhibitors on cognitive functions of stressed rats. A previous study supported our findings that chronic tadalafil treatment ameliorated memory impairment induced by maternal-separation in rat pups [70]. Increase in the cGMP level through inhibition of PDE-5 improves object recognition memory in animals and auditory selective attention and verbal recognition memory in humans [13,71]. Enhanced cGMP levels may increase glucose metabolism and blood flow, thus may improve memory and cognition [72–74]. Although in our study acute sildenafil pretreatment was not able to improve the loss of cognitive function with stress, chronic sildenafil pretreatment improved cognitive functions, which may involve an increase in cGMP levels. In addition, exercise by itself and exercise training along with chronic sildenafil pretreatment provided an increase in the cognitive functions. In accordance with our results, previous data have indicated that running exercise improves memory functions and promotes neurogenesis and synaptogenesis in hippocampus [75]. On the other hand, in sildenafil-pretreated rats, exercise training augmented the changes that were observed in corresponding sedentary groups. Briefly, exercise may strengthen the improving effect of chronic sildenafil on cognitive functions. Acute stress increased the histologic damage score in each group, except the chronic sildenafil-pretreated groups. Among the exercised rats, the chronic sildenafil pretreated ones had further declined histologic damage score. In our study, we aimed to demonstrate the oxidative changes that have occurred in dentate gyrus and hippocampus following exposure to stress. Several studies have focused on cell proliferation and death following stress exposure, and have shown that tadalafil improved the depressive symptoms and ameliorated memory impairment in a psychological stress model by suppressing apoptosis and enhancing cell proliferation in the hippocampus [70,76]. Also in a recent study, running prevented stress-induced proliferation of hippocampal granule cells and pyramidal cells [77]. Ultimately, histological analysis in our study supported the aforementioned data indirectly. In conclusion, our results demonstrate that acute stress exposure results in oxidative damage and the underlying mechanisms of this damage appear to involve neutrophil migration, increased lipid peroxidation levels and increased lucigenin activity in brain tissue. Furthermore, our results demonstrate for the first time that sildenafil or chronic exercise training reduced stress-induced oxidative brain damage along with reductions in anxiety-like behavior and improvement in cognitive functions, supporting the potential protective role of sildenafil and exercise on anxiety and cognition. Acknowledgments The authors are grateful to Prof. Dr. Inci Alican for the donation of drug and to Prof. Dr. Goncagul Haklar for cortisol measurements.
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