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Protective effect of atorvastatin on d -galactose-induced aging model in mice
Accepted Manuscript Title: Protective Effect of Atorvastatin on D-galactose-Induced Aging Model in Mice Authors: Elham Kaviani, Mohammadreza Rahmani, Ayat Kaeidi, Ali Shamsizadeh, Mohamad Allahtavakoli, Nazanin Mozafari, Iman Fatemi PII: DOI: Reference:
S0166-4328(17)30662-9 http://dx.doi.org/doi:10.1016/j.bbr.2017.07.029 BBR 11001
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
22-4-2017 17-7-2017 22-7-2017
Please cite this article as: Kaviani Elham, Rahmani Mohammadreza, Kaeidi Ayat, Shamsizadeh Ali, Allahtavakoli Mohamad, Mozafari Nazanin, Fatemi Iman.Protective Effect of Atorvastatin on D-galactose-Induced Aging Model in Mice.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective Effect of Atorvastatin on D-galactose-Induced Aging Model in Mice Elham Kaviani1, Mohammadreza Rahmani1,2, Ayat Kaeidi1, 2, Ali Shamsizadeh1, 2, Mohamad Allahtavakoli1, 2, Nazanin Mozafari1, Iman Fatemi1, 2* 1
Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences,
Rafsanjan, Iran 2
Department of Physiology and Pharmacology, School of Medicine, Rafsanjan University of
Medical Sciences, Rafsanjan, Iran Running title: Atorvastatin and Aging * Corresponding author: Iman Fatemi, Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Postal code: 7717684884 Tel: +9834-31315083 Mobile: +989133431737 Fax: +983431315003 Email: [email protected], Alternate Email: [email protected] Highlights
Chronic exposure to d-galactose induces oxidative damage and cognitive impairment. Atorvastatin exerted substantial benefit in preventing neurodegeneration. Atorvastatin improves aging-related disorders such as cognitive impairments, anxiety and sarcopenia.
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Abstract Atorvastatin (Ator), competitive inhibitors of 3-hydroxymethyl-3-glutaryl-coenzyme-A reductase, is a cholesterol lowering drug. Ator has been shown to have neuroprotective, antioxidant and anti-inflammatory properties making that a potential candidate for the treatment of central nervous system (CNS) disorders. Here we assessed the effect of Ator on the D-galactose (D-gal)-induced aging in mice. For this purpose, Ator (0.1 and 1 mg/kg/p.o.), was administrated daily in D-gal-received (500 mg/kg/p.o.) mice model of aging for six weeks. Anxiety-like behaviors and cognitive functions were evaluated by the elevated plus-maze and novel object recognition tasks, respectively. Physical power was assessed by forced swimming capacity test. Animals brains were analyzed for the superoxide dismutase (SOD) and brain-derived neurotrophic factor (BDNF). We found that Ator decreases the anxiety-like behaviors in D-gal-treated mice. Also, our behavioral tests showed that Ator reverses the D-gal induced learning and memory impairment. Furthermore, we found that Ator increases the physical power of D-gal-treated mice. Our results indicated that the neuroprotective effect of Ator on D-gal induced neurotoxicity is mediated, at least in part, by an increase in the SOD and BDNF levels. The results of present study suggest that Ator could be used as a novel therapeutic strategy for the treatment of age-related conditions. Key words: Atorvastatin; Aging; D-galactose
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Introduction During the last century, human lifespan has increased and it is expected that the elderly (over 65 year) population increase over 800 million by the year 2025 [1]. Aging is an extremely complex and slow biological phenomenon leading to the molecular, cellular and organic dysfunction [2]. These structural and functional alterations increase the incidence and severity of various pathological conditions such as cognitive decline, anxiety and sarcopenia [3]. One of the most established theories of aging is the oxidative stress theory [4]. It suggests that the overproduction of reactive oxygen species (ROS) can lead to mitochondrial dysfunction which results in cellular senescence and aging [5]. The oxidative stress is determined by the balance between the ROS production and clearance by various antioxidant enzymes such as superoxide dismutase (SOD) [6]. Moreover, it has been demonstrated that the level of brain-derived neurotrophic factor (BDNF) decreases in senescence [7]. BDNF is an important growth factor for neuronal proliferation and integrity [8]. Thus, the study of agedependent pathophysiology and age-related diseases is a significant challenge for medical gerontology. Previous studies demonstrated that long-term administration of D-galactose (D-gal) induces a mimetic natural aging effect in various tissues of rodents [9]. At high levels, D-gal could produce reactive oxygen species (ROS) and inflammatory reactions. Oxidative damage and inflammation are thought to play a critical role in age-related changes in tissues such as liver, brain, muscle and kidney [10]. Therefore, D-galactose-induced aging serves as a good for anti-aging research. Statins such as atorvastatin (Ator) are competitive inhibitors of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase that convert HMG-CoA to mevalonate and reduce the biosynthesis as well as the plasma level of cholesterol [11]. Statins are among the most
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widely prescribed drugs and for patients with atherosclerotic disease and hyperlipidemia [12]. In addition to this well-established property, statins exert a number of pleiotropic effects which are cholesterol-independent such as anti-inflammatory, antioxidant, analgesic and antineoplastic which may extend their clinical indications [13]. Moreover, the neuroprotective effect of statins has been demonstrated in various neurological conditions such as Alzheimer’s disease [14], Parkinson’s disease [15], multiple sclerosis [16], traumatic brain injury (Lu etal.,2004), seizures [17] and acute stroke [18]. Furthermore, it has also been reported that statins have protective effect against running wheel activity-induced fatigue and anxiety-like behavior in mice [19]. The current investigation aimed to evaluate the effect of Ator on D-gal-induced aging in mice by behavioral tests (Elevated plus-maze test, Novel object recognition task and the forced swimming capacity test). Furthermore, the level of SOD and BDNF in the brain were measured by ELISA. Materials and methods Animals The experiments were performed on 40 male NMRI (Naval Medical Research Institute) mice weighing 18–22 g. Animals were kept at 25±1°C on 12-h light/dark cycle with free access to food (pellet chow) and water. 10 mice were housed in standard polypropylene cages with wired-net top in a controlled room. All efforts were made to minimize the number of animals used and their suffering. It should be considered that the animal housing, surgery and testing rooms (22 ± 2°C) were close together and the animals were transported between the rooms in their home cages. Behavioural tests and animal care were conducted in accordance with the standard ethical guidelines (NIH, publication no. 85-23, revised 1985; European Communities Directive 86/609/EEC) and approved by the local ethical committee.
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Drugs Ator (Lipitor™) was purchased from Pfizer Pharmaceuticals (New York, NY, USA). D-gal was purchased from Sigma Aldrich (Germany). Ator was dissolved in standard drinking water. Daily doses were based on daily body weight measurements and this solution was freshly dissolved and administered by oral route (p.o.) in a volume of 10 ml/kg body weight using the gavage technique [20]. D-gal was dissolved in a measured quantity of mice drinking water. D-gal was given to three out of four groups of animals at the dose of 500 mg/kg per 10 ml drinking water for 6 weeks [21-23]. Experimental procedures and treatment After 2 weeks of acclimatization, mice were randomly divided into four groups as follows: control, model (D-gal group), Ator 0.1 and Ator 1. Control group served as healthy normal animals without any intervention. D-gal group received D-gal at the dose of 500 mg/kg per 10 ml drinking water for 6 weeks. D-gal + Ator 0.1 group received D-gal at the dose of 500 mg/kg per 10 ml drinking water plus Ator 0.1 mg/kg/day intragastrically for 6 weeks. D-gal + Ator 1 group received D-gal at the dose of 500 mg/kg per 10 ml drinking water plus Ator 1 mg/kg/day intragastrically for 6 weeks. The body weights of mice were recorded once a week. 24 h after the last administration, mice were subjected to behavioral tests and all experiments were carried out at the same time of the day. The elevated plus-maze test The elevated plus-maze (EPM) test is one of the most widely used methods to assess the anxiety-like behaviors in rodents [24]. This maze consists of two open and two closed arms as a plus sign. The percent of entries into the open arms (%OAE) and the percent of time spent on the open arms (%OAT) were recorded for 5 min. Significant increases in these
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indices represent an anxiolytic-like response. We also recorded the total arm entries as a measure of spontaneous locomotor activity. The novel object recognition task Exploratory behavior and preference for novelty are the important components of learning [25]. The novel object recognition task (NORT) is a standard method to assess this behavior [26]. The test was carried out in a 50 × 50 × 50 cm Plexiglas white box. The day before the test, mice were placed in the chamber to get familiar with the environment for 5 min. During the training phases, two identical objects (object A) were placed in the chamber, and the mouse was allowed to explore the objects freely for 5 min. Exploration was considered when the head of the mouse was oriented toward the objects with its nose within 2 cm of the object. The test phase was carried out 4 h later. An object A was replaced by an object B. The time spent with the two objects (novel and familiar) was recorded. Results were expressed as discrimination ratio (the difference between exploration time of novel and familiar divided by the total time spent exploring the objects in the test phase) [27]. All mice were placed in the chamber at the same point, and they were facing the same direction. At the end of each session, the mouse was removed from the chamber and the experimental chamber was thoroughly cleaned with 50% ethanol and dried. The forced swimming capacity test Mice were taken for forced swimming capacity test to assess the animals’ physical power and endurance. It is commonly accepted that swimming is an experimental exercise model [28]. Briefly, the mice were dropped separately into a columnar swimming pool (45 cm tall and radius 20 cm) filled with fresh water to a depth of 35 cm so that mice could not support themselves by touching the bottom with their tails. The temperature of the water was maintained at 34±1°C. A (steel ring) weighting equivalent to 5% of body weight attached to
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the tail root of each mouse. The animal exhaustion time was recorded when they failed to rise to the surface of the water to breathe within 7 s. The swimming time to exhaustion was used as the index of the forced swimming capacity [29]. the tests were done in the order of EPM (43th day of experiment), NORT (44th and 45th day of experiment) and forced swimming capacity test (46th day of experiment), respectively. Evaluation of BDNF and SOD levels 24 h after forced swimming capacity test, mice were sacrificed by decapitation and the brains were immediately removed from the skull within 30 s under aseptic condition. Brains were homogenized (1:10 w/v) in sterile phosphate-buffered saline (PBS). The tissue homogenates were centrifuged (Eppendorf, Germany) at 16,000 × g, at 4°C for 20 min and the supernatants were obtained and stored at −80 °C for measurements of the biochemical analyses. BDNF and SOD levels were measured with ELISA kit (Zellbio, Germany). Briefly, the samples were put in the kit wells. In the next stage, a type of antibody labeled with enzyme named conjugated was added and they were incubated for 45 minutes on the shaker in the room temperature. After incubation, free antibodies or antigens were washed by using a washer buffer. Then the substrate was added and the incubation was done again in a 15-minute time interval. Then, an interrupter solution was used for stopping the enzyme-substrate reaction. At the end, the light absorption was read by the ELISA Reader device (RT-2100C, Rayto, China) at the wavelength of 450 mm. Results were expressed as pg/mg protein. Statistical analysis Statistical analysis was performed via GraphPad Prism version 6.01 for Windows (GraphPad Software, USA). Data are expressed as mean ± SEM and the differences between the groups were tested with analysis of variance (one-way ANOVA) followed by the Tukey post-hoc test. Repeated measurement ANOVA (RMA) followed by the Tukey post hoc test were used
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for comparison of weight changes in groups. Differences between means were considered statistically significant if p<0.05. Results The effect of atorvastatin on body weight As shown in figure 1, at the beginning of the experiment, we did not find any difference in mean body weight of different groups. Results from RMA showed a significant difference between groups (p < 0.001). Tukey post-hoc analysis showed that in comparison to control group, administration of D-gal and Ator (0.1 and 1 mg/kg), decreased (p < 0.001) and increased (all p < 0.001) the body weight respectively. These results suggested that Ator at the doses of 0.1 and 1 mg/kg could improve body weight of D-gal-treated mice. The effect of atorvastatin on anxiety-like behaviors The results of the EPM test showed that D-gal decreases %OAT and %OAE compared to the control group (all p<0.05) and Ator at the doses of 0.1 and 1 mg/kg increases %OAT (all p<0.01) and %OAE (p<0.01 and p<0.001, respectively) compared to the D-gal group (Figure 2A and B). Statistical analysis revealed that the locomotor activity of different groups did not change significantly (Figure 2C). The effect of atorvastatin on novel object recognition task The D-gal group mice showed impaired working memory and preference for novelty on the NORT. Ator at the doses of 0.1 and 1 mg/kg reversed the D-gal-induced working memory and preference for novelty deficiency, as evidenced by higher discrimination ratio as compared to D-gal model (p<0.05 and p<0.001, respectively) (Figure 3). The effect of atorvastatin on exhaustion swimming time
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As shown in figure 4, the exhaustion swimming time of mice treated with D-gal showed a significant decrease compared to the control group (p<0.05). The mice treated with Ator at the doses of 0.1 and 1 mg/kg exhibited an increased exhaustion swimming time compared to the D-gal group (all p<0.001). The effect of atorvastatin on BDNF level As shown in figure 5, the BDNF level, was significantly decreased in D-gal group compared to the control group (P< 0.01). In contrast, Ator at the doses of 0.1 and 1 mg/kg increased the BDNF level in D-gal treated mice (P< 0.05 and P< 0.01, respectively). The effect of atorvastatin on brain level of SOD Our results (Figure 6) demonstrated that the level of SOD in the brain of D-gal treated mice was significantly lower than that of the control mice (P<0.05). Interestingly, Ator at the doses of 1 mg/kg attenuated D-gal induced decrease in SOD level (P<0.001). Discussion The results of the present study imply that administration of D-gal at dose 500 mg/kg per 10 ml drinking water for 6 weeks, causes severe aging-related changes, including significant decrease in body weight, physical power, BDNF level, SOD level and discrimination ratio as well as increase in anxiety-like behaviors. However, Ator, could partially (at the dose 0.1 mg/kg) and completely (at the dose 1 mg/kg) reverse these deteriorating effects. D-gal aging model has been widely used for anti-aging pharmacology research [9, 23, 30-32]. In addition, it is well established in previous studies that chronic administration of D-gal impairs cognitive performance in rodents [33, 34]. It has been shown that during ageing the cognitive functions such as memory are adversely affected which may due to the loss of neurons in specific brain regions. [32]. Consistent with these studies, we observed a
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significant difference in NORT results between control and D-gal treated mice. This suggests that chronic administration of D-gal for 6 weeks causes impairments of novelty-induced exploratory behaviors. Our results also showed that atorvastatin (0.1 and 1 mg/kg) improves working memory and preference for novelty in D-gal treated mice. It has been reported that statins could improve cognitive functions. In human subjects, Rej et al., have shown that high-dose statin use in patients with coronary artery disease is associated with higher visuospatial memory and executive functioning [35]. Moreover, there was low-strength evidence for deterioration effects of statins on memory function in the elderly [36]. Furthermore, there are some reports that suggest statin therapy might be protective against Alzheimer's disease [37]. In animal models, beneficial effects of statin on learning and memory have been reported in t various conditions such as nicotine treated rats [38], ischemic stroke [39], focal cerebral ischemia [40], postoperative cognitive decline [41] and Alzheimer's disease [42]. It seems that these effects on cognitive function are due to the neuroprotective effects of Ator via different mechanisms such as antioxidant signaling [38], downregulating the activation of the caspase-3 [39] and inhibiting inflammatory process [41, 42]. Accordingly, the Ator may possibly decrease the cognitive impairments and improve object recognition performance in mice through the neuroprotective effects. Consistent with improving the cognitive function, our data indicated that administration of Ator at the doses of 0.1 and 1 mg/kg significantly increases the BDNF level compared to Dgal treated mice. We also found that level of BDNF is decreased in D-gal treated mice in comparison with control mice. BDNF is a powerful neurotrophic factor that has been demonstrated to promote the survival of all types of neurons [8]. Moreover, BDNF is known to be involved in regulation of neurocognitive functions like learning, memory, synaptic transmission and plasticity [43]. Furthermore, the level of BDNF is reduced in neurodegenerative disorders such as Alzheimer as well as in senescence which is correlated 10
with cognitive impairment [44, 45]. Previous studies showed that BDNF level and expression decreases in both in vitro and in vivo models of accelerated aging [46, 47]. On the other hand, various studies have consistently demonstrated that Ator elevates the BDNF level. Zhang et al., reported that Ator treatment for 6 weeks increases the BDNF level and improves functional recovery in stroke patients [48]. In another study, Chen et al., showed that Ator promotes brain plasticity and enhances functional recovery following stroke in mice through increasing the expression of BDNF [49]. So, there is the possibility that increased BDNF level contributes to Atro restorative effects. In this study, we observed that senescence induction using D-gal attenuates the level of brain SOD in mice. Consistent to this finding, ROS production increases with age and this may account for the occurrence of age-related degenerative processes [4]. ROS promote neurological diseases and aging via oxidative damage to DNA, lipids and proteins. On the other hand, it has been reported that antioxidant enzymes such as SOD are decreased in Dgal-treated animals [33]. In our study, the SOD activity, as an intracellular antioxidative enzyme, was measured in the brains of mice. SOD rapidly and specifically reduces superoxide to hydrogen peroxide, then another endogenous antioxidants detoxify hydrogen peroxide to water [50]. We also found that, Ator at dose of 1 mg/kg significantly increases the level of brain SOD compared to D-gal treated mice. Many studies have indicated that Ator has a potent antioxidant activity. Mehrzadi et al., investigated the protective effect of atorvastatin against gentamicin-induced nephrotoxicity in rat kidney. They found that Ator enhances the activity of renal antioxidants such as SOD and decreases renal ROS and MDA levels [51]. In another study, Haendeler et al., showed that statins reduce ROS in endothelial cells via enhancing the enzymatic activity of thioredoxin [52]. Moreover, Prajapati et al., demonstrated that Ator decreases lipid peroxidases and increases SOD in 6-
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hydroxydopamine-induced dopaminergic toxicity in rats [53]. So, this implies that the effect of Ator in aging might be possibly mediated by increasing the antioxidative defense. The prevalence of anxiety disorders in older adults is high with estimates ranging from 15% to 28% [54]. Late-life anxiety has been associated with reduced life satisfaction and functional impairment [55]. It is well established that administration of D-gal leads to increased anxiety-like behavior which probably could result in ROS overproduction [56]. In addition, it has been shown that administration of an antioxidant can attenuate the anxietylike behavior in humans and animals [6, 57]. Consistent with previous studies, the results of our study showed that anxiety-like behavior increases in D-gal group compared to the control mice. We also found that Ator at the doses of 0.1 and 1 mg/kg decreases this behavior in Dgal treated animals. The antioxidant activity of statin was demonstrated in several studies. Girona et al., demonstrated that simvastatin selectively blocks the production of ROS in stimulated macrophages [58]. In another study, Herbet et al., showed that Ator at the dose of 10 mg/kg for 28 days, increases the total antioxidant level (representing improved serum potential to quench free radical production) [59]. Moreover, Alizadeh-Tabrizi et al., showed the antioxidant effect of Ator on paraquat-induced oxidative stress, pulmonary fibrosis and inflammation in rodent model [60]. Furthermore, Kumar et al., demonstrated that treatment with Ator (10 and 20 mg/kg) for 21 days significantly decreases anxiety-like behavior (decreased number of entries and time spent in open arm) and increases oxidative defense [19]. Hence, it seems that treatment with Ator may reduce anxiety-like behavior in D-gal treated mice via the antioxidant effect. Our results indicate that D-gal reduced the physical power in forced swimming capacity test compared to the control group. We also demonstrated that the treatment of aged mice with Ator (0.1 and 1 mg/kg) restores the physical power to a normal level. It was confirmed that sarcopenia (a progressive loss of muscle mass and strength) is a common characteristic of 12
aging. Sarcopenia reduces the quality of life and increases disability and mortality in aging people [31]. It has been shown that aging have deleterious effects on mitochondrial function that have a critical role in the onset and progression of sarcopenia via enhancing the ROS production [61]. Previous studies demonstrated that D-gal model induces aging in skeletal muscle like other organs [10]. Chang et al., suggested that poor skeletal muscle strength induced by D-gal might be due to the mitochondrial dysfunction caused by complex I deficiency [31]. Ator plays an important role in the regulation of lysosomes and mitochondria stability [62]. Song et al., demonstrated that Ator has protective effects in cerebral ischemia-reperfusion injury by blocking the mitochondrial permeability transition pore [63]. In another study, Kumar et al., demonstrated that treatment with Ator (10 and 20 mg/kg) for 21 days improves the fatigue induced by rotating/running wheel activity test session (increased number of wheel rotations and locomotor activity) and mitochondrial complex enzyme dysfunction [19]. Accordingly, Ator may possibly reduce the sarcopenia and increase the skeletal muscle strength through improving the mitochondrial functions and the suppression of ROS production. Although one of the most serious side effect of statins is myotoxicity but large evidence base for this side effect does not exist for the secondgeneration statins such as Ator [64]. On the other hand, Camerino et al., found that Atorinduced myotoxicity occurs at the dose 10 mg/kg in aged rat, however, no histological features were found in these animals [65]. It is probable that the low dose of Ator (0.1 and 1 mg/kg), as used in our, does not cause myotoxicity. In conclusion, the results of this study demonstrated that Ator improves aging-related disorders such as cognitive impairments, anxiety and sarcopenia. Based on our data, we concluded that mechanism/s underlying Ator effects on d-gal-induced neurotoxicity might be mediated by: (i) increasing the activity of antioxidant enzymes such as SOD and (ii)
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increasing the level of neurotrophins such as BDNF. However, further investigations are required to unveil the precise cellular mechanisms. Conflict of interest statement The authors declare no conflict of interest relevant to this study. Acknowledgments This work was supported by a grant from Research Deputy of Rafsanjan University of Medical Sciences (grant number 9/296).
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Figures
Figure 1. The effect of atorvastatin (0.1 and 1 mg/kg) on the body weight. Values are expressed as mean±SEM. In each group n=10. *** P< 0.001 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 2. (A, B) The effect of atorvastatin (0.1 and 1 mg/kg) on anxiety-like behaviors and (C) locomotor activity of d-galactose-induced aging mice. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 3. The effect of atorvastatin (0.1 and 1 mg/kg) on working memory and preference for novelty. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 4. The effect of atorvastatin (0.1 and 1 mg/kg) on swimming time to exhaustion. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ### P< 0.001 compared to the D-gal group.
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Figure 5. The effect of atorvastatin (0.1 and 1 mg/kg) on BDNF level. Values are expressed as mean±SEM. In each group n=10. ** P< 0.01 compared to the control group. # P< 0.05 and ## P< 0.01 compared to the D-gal group.
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Figure 6. The effect of atorvastatin (0.1 and 1 mg/kg) on brain level of SOD. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ### P< 0.001 compared to the D-gal group.
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S0166-4328(17)30662-9 http://dx.doi.org/doi:10.1016/j.bbr.2017.07.029 BBR 11001
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
22-4-2017 17-7-2017 22-7-2017
Please cite this article as: Kaviani Elham, Rahmani Mohammadreza, Kaeidi Ayat, Shamsizadeh Ali, Allahtavakoli Mohamad, Mozafari Nazanin, Fatemi Iman.Protective Effect of Atorvastatin on D-galactose-Induced Aging Model in Mice.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective Effect of Atorvastatin on D-galactose-Induced Aging Model in Mice Elham Kaviani1, Mohammadreza Rahmani1,2, Ayat Kaeidi1, 2, Ali Shamsizadeh1, 2, Mohamad Allahtavakoli1, 2, Nazanin Mozafari1, Iman Fatemi1, 2* 1
Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences,
Rafsanjan, Iran 2
Department of Physiology and Pharmacology, School of Medicine, Rafsanjan University of
Medical Sciences, Rafsanjan, Iran Running title: Atorvastatin and Aging * Corresponding author: Iman Fatemi, Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Postal code: 7717684884 Tel: +9834-31315083 Mobile: +989133431737 Fax: +983431315003 Email: [email protected], Alternate Email: [email protected] Highlights
Chronic exposure to d-galactose induces oxidative damage and cognitive impairment. Atorvastatin exerted substantial benefit in preventing neurodegeneration. Atorvastatin improves aging-related disorders such as cognitive impairments, anxiety and sarcopenia.
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Abstract Atorvastatin (Ator), competitive inhibitors of 3-hydroxymethyl-3-glutaryl-coenzyme-A reductase, is a cholesterol lowering drug. Ator has been shown to have neuroprotective, antioxidant and anti-inflammatory properties making that a potential candidate for the treatment of central nervous system (CNS) disorders. Here we assessed the effect of Ator on the D-galactose (D-gal)-induced aging in mice. For this purpose, Ator (0.1 and 1 mg/kg/p.o.), was administrated daily in D-gal-received (500 mg/kg/p.o.) mice model of aging for six weeks. Anxiety-like behaviors and cognitive functions were evaluated by the elevated plus-maze and novel object recognition tasks, respectively. Physical power was assessed by forced swimming capacity test. Animals brains were analyzed for the superoxide dismutase (SOD) and brain-derived neurotrophic factor (BDNF). We found that Ator decreases the anxiety-like behaviors in D-gal-treated mice. Also, our behavioral tests showed that Ator reverses the D-gal induced learning and memory impairment. Furthermore, we found that Ator increases the physical power of D-gal-treated mice. Our results indicated that the neuroprotective effect of Ator on D-gal induced neurotoxicity is mediated, at least in part, by an increase in the SOD and BDNF levels. The results of present study suggest that Ator could be used as a novel therapeutic strategy for the treatment of age-related conditions. Key words: Atorvastatin; Aging; D-galactose
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Introduction During the last century, human lifespan has increased and it is expected that the elderly (over 65 year) population increase over 800 million by the year 2025 [1]. Aging is an extremely complex and slow biological phenomenon leading to the molecular, cellular and organic dysfunction [2]. These structural and functional alterations increase the incidence and severity of various pathological conditions such as cognitive decline, anxiety and sarcopenia [3]. One of the most established theories of aging is the oxidative stress theory [4]. It suggests that the overproduction of reactive oxygen species (ROS) can lead to mitochondrial dysfunction which results in cellular senescence and aging [5]. The oxidative stress is determined by the balance between the ROS production and clearance by various antioxidant enzymes such as superoxide dismutase (SOD) [6]. Moreover, it has been demonstrated that the level of brain-derived neurotrophic factor (BDNF) decreases in senescence [7]. BDNF is an important growth factor for neuronal proliferation and integrity [8]. Thus, the study of agedependent pathophysiology and age-related diseases is a significant challenge for medical gerontology. Previous studies demonstrated that long-term administration of D-galactose (D-gal) induces a mimetic natural aging effect in various tissues of rodents [9]. At high levels, D-gal could produce reactive oxygen species (ROS) and inflammatory reactions. Oxidative damage and inflammation are thought to play a critical role in age-related changes in tissues such as liver, brain, muscle and kidney [10]. Therefore, D-galactose-induced aging serves as a good for anti-aging research. Statins such as atorvastatin (Ator) are competitive inhibitors of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase that convert HMG-CoA to mevalonate and reduce the biosynthesis as well as the plasma level of cholesterol [11]. Statins are among the most
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widely prescribed drugs and for patients with atherosclerotic disease and hyperlipidemia [12]. In addition to this well-established property, statins exert a number of pleiotropic effects which are cholesterol-independent such as anti-inflammatory, antioxidant, analgesic and antineoplastic which may extend their clinical indications [13]. Moreover, the neuroprotective effect of statins has been demonstrated in various neurological conditions such as Alzheimer’s disease [14], Parkinson’s disease [15], multiple sclerosis [16], traumatic brain injury (Lu etal.,2004), seizures [17] and acute stroke [18]. Furthermore, it has also been reported that statins have protective effect against running wheel activity-induced fatigue and anxiety-like behavior in mice [19]. The current investigation aimed to evaluate the effect of Ator on D-gal-induced aging in mice by behavioral tests (Elevated plus-maze test, Novel object recognition task and the forced swimming capacity test). Furthermore, the level of SOD and BDNF in the brain were measured by ELISA. Materials and methods Animals The experiments were performed on 40 male NMRI (Naval Medical Research Institute) mice weighing 18–22 g. Animals were kept at 25±1°C on 12-h light/dark cycle with free access to food (pellet chow) and water. 10 mice were housed in standard polypropylene cages with wired-net top in a controlled room. All efforts were made to minimize the number of animals used and their suffering. It should be considered that the animal housing, surgery and testing rooms (22 ± 2°C) were close together and the animals were transported between the rooms in their home cages. Behavioural tests and animal care were conducted in accordance with the standard ethical guidelines (NIH, publication no. 85-23, revised 1985; European Communities Directive 86/609/EEC) and approved by the local ethical committee.
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Drugs Ator (Lipitor™) was purchased from Pfizer Pharmaceuticals (New York, NY, USA). D-gal was purchased from Sigma Aldrich (Germany). Ator was dissolved in standard drinking water. Daily doses were based on daily body weight measurements and this solution was freshly dissolved and administered by oral route (p.o.) in a volume of 10 ml/kg body weight using the gavage technique [20]. D-gal was dissolved in a measured quantity of mice drinking water. D-gal was given to three out of four groups of animals at the dose of 500 mg/kg per 10 ml drinking water for 6 weeks [21-23]. Experimental procedures and treatment After 2 weeks of acclimatization, mice were randomly divided into four groups as follows: control, model (D-gal group), Ator 0.1 and Ator 1. Control group served as healthy normal animals without any intervention. D-gal group received D-gal at the dose of 500 mg/kg per 10 ml drinking water for 6 weeks. D-gal + Ator 0.1 group received D-gal at the dose of 500 mg/kg per 10 ml drinking water plus Ator 0.1 mg/kg/day intragastrically for 6 weeks. D-gal + Ator 1 group received D-gal at the dose of 500 mg/kg per 10 ml drinking water plus Ator 1 mg/kg/day intragastrically for 6 weeks. The body weights of mice were recorded once a week. 24 h after the last administration, mice were subjected to behavioral tests and all experiments were carried out at the same time of the day. The elevated plus-maze test The elevated plus-maze (EPM) test is one of the most widely used methods to assess the anxiety-like behaviors in rodents [24]. This maze consists of two open and two closed arms as a plus sign. The percent of entries into the open arms (%OAE) and the percent of time spent on the open arms (%OAT) were recorded for 5 min. Significant increases in these
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indices represent an anxiolytic-like response. We also recorded the total arm entries as a measure of spontaneous locomotor activity. The novel object recognition task Exploratory behavior and preference for novelty are the important components of learning [25]. The novel object recognition task (NORT) is a standard method to assess this behavior [26]. The test was carried out in a 50 × 50 × 50 cm Plexiglas white box. The day before the test, mice were placed in the chamber to get familiar with the environment for 5 min. During the training phases, two identical objects (object A) were placed in the chamber, and the mouse was allowed to explore the objects freely for 5 min. Exploration was considered when the head of the mouse was oriented toward the objects with its nose within 2 cm of the object. The test phase was carried out 4 h later. An object A was replaced by an object B. The time spent with the two objects (novel and familiar) was recorded. Results were expressed as discrimination ratio (the difference between exploration time of novel and familiar divided by the total time spent exploring the objects in the test phase) [27]. All mice were placed in the chamber at the same point, and they were facing the same direction. At the end of each session, the mouse was removed from the chamber and the experimental chamber was thoroughly cleaned with 50% ethanol and dried. The forced swimming capacity test Mice were taken for forced swimming capacity test to assess the animals’ physical power and endurance. It is commonly accepted that swimming is an experimental exercise model [28]. Briefly, the mice were dropped separately into a columnar swimming pool (45 cm tall and radius 20 cm) filled with fresh water to a depth of 35 cm so that mice could not support themselves by touching the bottom with their tails. The temperature of the water was maintained at 34±1°C. A (steel ring) weighting equivalent to 5% of body weight attached to
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the tail root of each mouse. The animal exhaustion time was recorded when they failed to rise to the surface of the water to breathe within 7 s. The swimming time to exhaustion was used as the index of the forced swimming capacity [29]. the tests were done in the order of EPM (43th day of experiment), NORT (44th and 45th day of experiment) and forced swimming capacity test (46th day of experiment), respectively. Evaluation of BDNF and SOD levels 24 h after forced swimming capacity test, mice were sacrificed by decapitation and the brains were immediately removed from the skull within 30 s under aseptic condition. Brains were homogenized (1:10 w/v) in sterile phosphate-buffered saline (PBS). The tissue homogenates were centrifuged (Eppendorf, Germany) at 16,000 × g, at 4°C for 20 min and the supernatants were obtained and stored at −80 °C for measurements of the biochemical analyses. BDNF and SOD levels were measured with ELISA kit (Zellbio, Germany). Briefly, the samples were put in the kit wells. In the next stage, a type of antibody labeled with enzyme named conjugated was added and they were incubated for 45 minutes on the shaker in the room temperature. After incubation, free antibodies or antigens were washed by using a washer buffer. Then the substrate was added and the incubation was done again in a 15-minute time interval. Then, an interrupter solution was used for stopping the enzyme-substrate reaction. At the end, the light absorption was read by the ELISA Reader device (RT-2100C, Rayto, China) at the wavelength of 450 mm. Results were expressed as pg/mg protein. Statistical analysis Statistical analysis was performed via GraphPad Prism version 6.01 for Windows (GraphPad Software, USA). Data are expressed as mean ± SEM and the differences between the groups were tested with analysis of variance (one-way ANOVA) followed by the Tukey post-hoc test. Repeated measurement ANOVA (RMA) followed by the Tukey post hoc test were used
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for comparison of weight changes in groups. Differences between means were considered statistically significant if p<0.05. Results The effect of atorvastatin on body weight As shown in figure 1, at the beginning of the experiment, we did not find any difference in mean body weight of different groups. Results from RMA showed a significant difference between groups (p < 0.001). Tukey post-hoc analysis showed that in comparison to control group, administration of D-gal and Ator (0.1 and 1 mg/kg), decreased (p < 0.001) and increased (all p < 0.001) the body weight respectively. These results suggested that Ator at the doses of 0.1 and 1 mg/kg could improve body weight of D-gal-treated mice. The effect of atorvastatin on anxiety-like behaviors The results of the EPM test showed that D-gal decreases %OAT and %OAE compared to the control group (all p<0.05) and Ator at the doses of 0.1 and 1 mg/kg increases %OAT (all p<0.01) and %OAE (p<0.01 and p<0.001, respectively) compared to the D-gal group (Figure 2A and B). Statistical analysis revealed that the locomotor activity of different groups did not change significantly (Figure 2C). The effect of atorvastatin on novel object recognition task The D-gal group mice showed impaired working memory and preference for novelty on the NORT. Ator at the doses of 0.1 and 1 mg/kg reversed the D-gal-induced working memory and preference for novelty deficiency, as evidenced by higher discrimination ratio as compared to D-gal model (p<0.05 and p<0.001, respectively) (Figure 3). The effect of atorvastatin on exhaustion swimming time
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As shown in figure 4, the exhaustion swimming time of mice treated with D-gal showed a significant decrease compared to the control group (p<0.05). The mice treated with Ator at the doses of 0.1 and 1 mg/kg exhibited an increased exhaustion swimming time compared to the D-gal group (all p<0.001). The effect of atorvastatin on BDNF level As shown in figure 5, the BDNF level, was significantly decreased in D-gal group compared to the control group (P< 0.01). In contrast, Ator at the doses of 0.1 and 1 mg/kg increased the BDNF level in D-gal treated mice (P< 0.05 and P< 0.01, respectively). The effect of atorvastatin on brain level of SOD Our results (Figure 6) demonstrated that the level of SOD in the brain of D-gal treated mice was significantly lower than that of the control mice (P<0.05). Interestingly, Ator at the doses of 1 mg/kg attenuated D-gal induced decrease in SOD level (P<0.001). Discussion The results of the present study imply that administration of D-gal at dose 500 mg/kg per 10 ml drinking water for 6 weeks, causes severe aging-related changes, including significant decrease in body weight, physical power, BDNF level, SOD level and discrimination ratio as well as increase in anxiety-like behaviors. However, Ator, could partially (at the dose 0.1 mg/kg) and completely (at the dose 1 mg/kg) reverse these deteriorating effects. D-gal aging model has been widely used for anti-aging pharmacology research [9, 23, 30-32]. In addition, it is well established in previous studies that chronic administration of D-gal impairs cognitive performance in rodents [33, 34]. It has been shown that during ageing the cognitive functions such as memory are adversely affected which may due to the loss of neurons in specific brain regions. [32]. Consistent with these studies, we observed a
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significant difference in NORT results between control and D-gal treated mice. This suggests that chronic administration of D-gal for 6 weeks causes impairments of novelty-induced exploratory behaviors. Our results also showed that atorvastatin (0.1 and 1 mg/kg) improves working memory and preference for novelty in D-gal treated mice. It has been reported that statins could improve cognitive functions. In human subjects, Rej et al., have shown that high-dose statin use in patients with coronary artery disease is associated with higher visuospatial memory and executive functioning [35]. Moreover, there was low-strength evidence for deterioration effects of statins on memory function in the elderly [36]. Furthermore, there are some reports that suggest statin therapy might be protective against Alzheimer's disease [37]. In animal models, beneficial effects of statin on learning and memory have been reported in t various conditions such as nicotine treated rats [38], ischemic stroke [39], focal cerebral ischemia [40], postoperative cognitive decline [41] and Alzheimer's disease [42]. It seems that these effects on cognitive function are due to the neuroprotective effects of Ator via different mechanisms such as antioxidant signaling [38], downregulating the activation of the caspase-3 [39] and inhibiting inflammatory process [41, 42]. Accordingly, the Ator may possibly decrease the cognitive impairments and improve object recognition performance in mice through the neuroprotective effects. Consistent with improving the cognitive function, our data indicated that administration of Ator at the doses of 0.1 and 1 mg/kg significantly increases the BDNF level compared to Dgal treated mice. We also found that level of BDNF is decreased in D-gal treated mice in comparison with control mice. BDNF is a powerful neurotrophic factor that has been demonstrated to promote the survival of all types of neurons [8]. Moreover, BDNF is known to be involved in regulation of neurocognitive functions like learning, memory, synaptic transmission and plasticity [43]. Furthermore, the level of BDNF is reduced in neurodegenerative disorders such as Alzheimer as well as in senescence which is correlated 10
with cognitive impairment [44, 45]. Previous studies showed that BDNF level and expression decreases in both in vitro and in vivo models of accelerated aging [46, 47]. On the other hand, various studies have consistently demonstrated that Ator elevates the BDNF level. Zhang et al., reported that Ator treatment for 6 weeks increases the BDNF level and improves functional recovery in stroke patients [48]. In another study, Chen et al., showed that Ator promotes brain plasticity and enhances functional recovery following stroke in mice through increasing the expression of BDNF [49]. So, there is the possibility that increased BDNF level contributes to Atro restorative effects. In this study, we observed that senescence induction using D-gal attenuates the level of brain SOD in mice. Consistent to this finding, ROS production increases with age and this may account for the occurrence of age-related degenerative processes [4]. ROS promote neurological diseases and aging via oxidative damage to DNA, lipids and proteins. On the other hand, it has been reported that antioxidant enzymes such as SOD are decreased in Dgal-treated animals [33]. In our study, the SOD activity, as an intracellular antioxidative enzyme, was measured in the brains of mice. SOD rapidly and specifically reduces superoxide to hydrogen peroxide, then another endogenous antioxidants detoxify hydrogen peroxide to water [50]. We also found that, Ator at dose of 1 mg/kg significantly increases the level of brain SOD compared to D-gal treated mice. Many studies have indicated that Ator has a potent antioxidant activity. Mehrzadi et al., investigated the protective effect of atorvastatin against gentamicin-induced nephrotoxicity in rat kidney. They found that Ator enhances the activity of renal antioxidants such as SOD and decreases renal ROS and MDA levels [51]. In another study, Haendeler et al., showed that statins reduce ROS in endothelial cells via enhancing the enzymatic activity of thioredoxin [52]. Moreover, Prajapati et al., demonstrated that Ator decreases lipid peroxidases and increases SOD in 6-
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hydroxydopamine-induced dopaminergic toxicity in rats [53]. So, this implies that the effect of Ator in aging might be possibly mediated by increasing the antioxidative defense. The prevalence of anxiety disorders in older adults is high with estimates ranging from 15% to 28% [54]. Late-life anxiety has been associated with reduced life satisfaction and functional impairment [55]. It is well established that administration of D-gal leads to increased anxiety-like behavior which probably could result in ROS overproduction [56]. In addition, it has been shown that administration of an antioxidant can attenuate the anxietylike behavior in humans and animals [6, 57]. Consistent with previous studies, the results of our study showed that anxiety-like behavior increases in D-gal group compared to the control mice. We also found that Ator at the doses of 0.1 and 1 mg/kg decreases this behavior in Dgal treated animals. The antioxidant activity of statin was demonstrated in several studies. Girona et al., demonstrated that simvastatin selectively blocks the production of ROS in stimulated macrophages [58]. In another study, Herbet et al., showed that Ator at the dose of 10 mg/kg for 28 days, increases the total antioxidant level (representing improved serum potential to quench free radical production) [59]. Moreover, Alizadeh-Tabrizi et al., showed the antioxidant effect of Ator on paraquat-induced oxidative stress, pulmonary fibrosis and inflammation in rodent model [60]. Furthermore, Kumar et al., demonstrated that treatment with Ator (10 and 20 mg/kg) for 21 days significantly decreases anxiety-like behavior (decreased number of entries and time spent in open arm) and increases oxidative defense [19]. Hence, it seems that treatment with Ator may reduce anxiety-like behavior in D-gal treated mice via the antioxidant effect. Our results indicate that D-gal reduced the physical power in forced swimming capacity test compared to the control group. We also demonstrated that the treatment of aged mice with Ator (0.1 and 1 mg/kg) restores the physical power to a normal level. It was confirmed that sarcopenia (a progressive loss of muscle mass and strength) is a common characteristic of 12
aging. Sarcopenia reduces the quality of life and increases disability and mortality in aging people [31]. It has been shown that aging have deleterious effects on mitochondrial function that have a critical role in the onset and progression of sarcopenia via enhancing the ROS production [61]. Previous studies demonstrated that D-gal model induces aging in skeletal muscle like other organs [10]. Chang et al., suggested that poor skeletal muscle strength induced by D-gal might be due to the mitochondrial dysfunction caused by complex I deficiency [31]. Ator plays an important role in the regulation of lysosomes and mitochondria stability [62]. Song et al., demonstrated that Ator has protective effects in cerebral ischemia-reperfusion injury by blocking the mitochondrial permeability transition pore [63]. In another study, Kumar et al., demonstrated that treatment with Ator (10 and 20 mg/kg) for 21 days improves the fatigue induced by rotating/running wheel activity test session (increased number of wheel rotations and locomotor activity) and mitochondrial complex enzyme dysfunction [19]. Accordingly, Ator may possibly reduce the sarcopenia and increase the skeletal muscle strength through improving the mitochondrial functions and the suppression of ROS production. Although one of the most serious side effect of statins is myotoxicity but large evidence base for this side effect does not exist for the secondgeneration statins such as Ator [64]. On the other hand, Camerino et al., found that Atorinduced myotoxicity occurs at the dose 10 mg/kg in aged rat, however, no histological features were found in these animals [65]. It is probable that the low dose of Ator (0.1 and 1 mg/kg), as used in our, does not cause myotoxicity. In conclusion, the results of this study demonstrated that Ator improves aging-related disorders such as cognitive impairments, anxiety and sarcopenia. Based on our data, we concluded that mechanism/s underlying Ator effects on d-gal-induced neurotoxicity might be mediated by: (i) increasing the activity of antioxidant enzymes such as SOD and (ii)
13
increasing the level of neurotrophins such as BDNF. However, further investigations are required to unveil the precise cellular mechanisms. Conflict of interest statement The authors declare no conflict of interest relevant to this study. Acknowledgments This work was supported by a grant from Research Deputy of Rafsanjan University of Medical Sciences (grant number 9/296).
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Figures
Figure 1. The effect of atorvastatin (0.1 and 1 mg/kg) on the body weight. Values are expressed as mean±SEM. In each group n=10. *** P< 0.001 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 2. (A, B) The effect of atorvastatin (0.1 and 1 mg/kg) on anxiety-like behaviors and (C) locomotor activity of d-galactose-induced aging mice. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 3. The effect of atorvastatin (0.1 and 1 mg/kg) on working memory and preference for novelty. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ## P< 0.01 and ### P< 0.001 compared to the D-gal group.
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Figure 4. The effect of atorvastatin (0.1 and 1 mg/kg) on swimming time to exhaustion. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ### P< 0.001 compared to the D-gal group.
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Figure 5. The effect of atorvastatin (0.1 and 1 mg/kg) on BDNF level. Values are expressed as mean±SEM. In each group n=10. ** P< 0.01 compared to the control group. # P< 0.05 and ## P< 0.01 compared to the D-gal group.
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Figure 6. The effect of atorvastatin (0.1 and 1 mg/kg) on brain level of SOD. Values are expressed as mean±SEM. In each group n=10. * P< 0.05 compared to the control group. ### P< 0.001 compared to the D-gal group.
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