European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Behavioural pharmacology

Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice Md. Mamun Al-Amin a,n, Hasan Mahmud Reza a, Hasan Mahmud Saadi a, Waich Mahmud a, Abdirahman Adam Ibrahim a, Musrura Mefta Alam a, Nadia Kabir a, A.R.M. Saifullah a, Sarjana Tarannum Tropa a, A.H.M. Ruhul Quddus b a b

Department of Pharmaceutical Sciences, North South University, Bashundhara, Dhaka, Bangladesh National University, Gazipur, Bangladesh

art ic l e i nf o

a b s t r a c t

Article history: Received 23 July 2015 Received in revised form 6 February 2016 Accepted 26 February 2016

Aluminum chloride induces neurodegenerative disease in animal model. Evidence suggests that aluminum intake results in the activation of glial cells and generation of reactive oxygen species. By contrast, astaxanthin is an antioxidant having potential neuroprotective activity. In this study, we investigate the effect of astaxanthin on aluminum chloride-exposed behavioral brain function and neuronal oxidative stress (OS). Male Swiss albino mice (4 months old) were divided into 4 groups: (i) control (distilled water), (ii) aluminum chloride, (iii) astaxanthin þaluminum chloride, and (iv) astaxanthin. Two behavioral tests; radial arm maze and open field test were conducted, and OS markers were assayed from the brain and liver tissues following 42 days of treatment. Aluminum exposed group showed a significant reduction in spatial memory performance and anxiety-like behavior. Moreover, aluminum group exhibited a marked deterioration of oxidative markers; lipid peroxidation (MDA), nitric oxide (NO), glutathione (GSH) and advanced oxidation of protein products (AOPP) in the brain. To the contrary, coadministration of astaxanthin and aluminum has shown improved spatial memory, locomotor activity, and OS. These results indicate that astaxanthin improves aluminum-induced impaired memory performances presumably by the reduction of OS in the distinct brain regions. We suggest a future study to determine the underlying mechanism of astaxanthin in improving aluminum-exposed behavioral deficits. & 2016 Elsevier B.V. All rights reserved.

Keywords: Memory Behavior Glutathione Nitric oxide Superoxide dismutase

1. Introduction Aluminum is a widely used household metal, which is associated with bone, blood and brain disease (Han et al., 2013). Aluminum accumulates in all brain regions following chronic exposure, especially in the mouse hippocampus (Yu et al., 2014). Growing evidence suggests that the deposition of aluminum is associated with the pathophysiology of neurodegenerative disease including alzheimer’s disease, parkinsonism, dementia, amyotrophic lateral sclerosis (Kawahara, 2005). In animal study, aluminum exposure has shown to induce deficits in memory formation, similar to alzheimer’s disease (Justin Thenmozhi et al., 2015b) and dementia (Rani et al., 2015). A previous study has reported that aluminum alters cholinergic and noradrenergic neurotransmission; via neuronal damage, disruption of glucose n

Correspondence to: Plot-15, Block-B, Bashundhara, Dhaka 1229, Bangladesh. E-mail addresses: [email protected], [email protected] (M.M. Al-Amin).

metabolism, interruption of signal transduction and production of reactive oxygen species (ROS) (Erasmus et al., 1993). ROS interacts with cell membrane lipids resulting in lipid peroxidation. In addition, ROS alters the level of antioxidant enzymes: (i) catalase (CAT) and (ii) superoxide dismutase (SOD). Aluminum toxicity generates oxidative damage (Nehru et al., 2007) by the accelerated production of ROS (Han et al., 2013; Wu et al., 2012). Aluminum-exposed ROS activates glial cells (Akinrinade et al., 2015) including astrocytes (Han et al., 2013). Aluminum has shown to induce neurodegeneration through iron accumulation and ROS formation (Wu et al., 2012). The exposure of 10 mg/kg aluminum for 12 weeks leads to region specific oxidative DNA-damage in the rat (Kumar et al., 2009), whereas the exposure of aluminum maltolate (0.1 mg/ml) enhances oxidative stress (OS) in mouse brain (Kaneko et al., 2004). A large body of research showed that aluminum-exposure leads to the increment of lipid peroxidation (Akinrinade et al., 2015; John et al., 2015; Kaur and Sodhi, 2015; Lakshmi et al., 2015; Pan et al., 2015; Qiu et al., 2015). Oral aluminum also increases peroxidized lipid-induced DNA damage (Rui and Yongjian, 2010). A previous study showed an inconsistent

http://dx.doi.org/10.1016/j.ejphar.2016.02.062 0014-2999/& 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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effect of aluminum on SOD activity. Several studies suggest a decreased activity of SOD in the hippocampus and cerebral cortex (Rui and Yongjian, 2010) and in the brain (Akinrinade et al., 2015; Lakshmi et al., 2015; Pan et al., 2015), whereas only one study showed elevated SOD activity (Wu et al., 2012). Aluminum also diminishes the formation of nitric oxide (NO) (John et al., 2015) as well as reduces the level of reduced glutathione (John et al., 2015; Lakshmi et al., 2015; Orihuela et al., 2005) and catalase enzyme (Akinrinade et al., 2015; Augusti et al., 2009; John et al., 2015; Lakshmi et al., 2015). Besides oxidative stress, aluminum diminishes mitochondrial activity, ATP synthesis, and promotes the metabolic deregulation in the astrocytes (Han et al., 2013). Chronic aluminum exposure results in intraneuronal neurofilamentous aggregation of proteins in the hippocampus, cerebral cortex, brain stem and spinal cord (Bharathi et al., 2006). Astaxanthin, a naturally occurring compound showed a strong antioxidant property as evidenced from previous studies (Belviranli and Okudan, 2015; Hussein et al., 2006; Zhang et al., 2014). Astaxanthin could be given to combat against the aluminum induced oxidative damage which is more potent than many other antioxidants like beta-carotene, vitamin C and lutein (Kurashige et al., 1990; Naguib, 2000). Our and others’ recent findings have shown that astaxanthin reduces neuronal OS in the rodent model (Al-Amin et al., 2015a; Lu et al., 2015; Wu et al., 2014). 1.1. Aim of the study Our present study aims to determine the effect of astaxanthin on spatial memory impairment, locomotor activity and oxidative markers in distinct brain regions and liver induced by aluminum.

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These animals had free access to food and water. 2.2. Experimental groups Animals were divided into four following groups (Fig. 1) (1) CON (control): – 200 ml distilled water was given. (2) AlCl3: Aluminum chloride was given at a dose of 50 mg/kg body weight per day (Singh and Goel, 2015). (3) AST_AlCl3 – astaxanthin at a dose of 20 mg/kg body weight together with aluminum chloride at a dose 50 mg/kg body weight was given (Pei et al., 2008; Singh and Goel, 2015). (4) AST – only astaxanthin at a dose of 20 mg/kg body weight was given (Pei et al., 2008). Behavioral experiments were conducted after 42 days of the astaxanthin treatment and AlCl3 exposure. At first, locomotor activity was recorded; then radial arm maze test was conducted; and finally biochemical tests were performed after sacrificing the animals. The experimental procedure was approved by the Institutional Animal Ethics Committee at North South University. 2.3. Drugs and chemicals Aluminum chloride was purchased from Merck, India and astaxanthin powder was collected from Pharmaraw Bangladesh as a gift. Natural astaxanthin was dissolved in distilled water at a concentration of 20 mg/20 ml. Astaxanthin was given orally once daily for 6 weeks. The control animals received 200 ml of distilled water. All chemicals and reagents used for the biochemical tests were AR grade. These chemicals were freshly prepared each time for biochemical tests.

2. Materials and methods

2.4. Behavioral experiments

2.1. Animals

2.4.1. Radial arm maze test Eight arm radial maze test devices were developed according to the previous study (Al-Amin et al., 2014; Tarragon et al., 2012).

Our present study was conducted on Swiss albino male mice weighing 30–35 g (age 4 months). The animals were housed in groups of 6 mice/cage in a controlled room having temperature of 22–28 °C and humidity (50%) and under a 12/12 h dark/light cycle.

2.4.1.1. Placement of food and cues. Food was placed at the end of the arm. The end of each arm contains a small well that hides food

Fig. 1. Schematic diagram of the experimental procedure. All animals were habituated and trained in the radial arm maze device for first 7 days. On day 8, animals were grouped into 4. In group (i) Control group animal (CON, n ¼ 6) was given 200 ml distilled water; in group (ii) Aluminum chloride (AlCl3, n ¼ 6) was given at a dose of 50 mg/kg/ day; in group (iii) (AST_AlCl3, n¼ 6), astaxanthin þ AlCl3 treatment was administered; and in group (iv) (AST, n ¼ 6), only astaxanthin (AST) was orally administered. All treatments were given for 42 days period. On day-49, behavioral tests were carried out, and on day-51 biochemical tests were conducted.

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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from direct eye-sight. Animals were allowed to freely explore the maze. To find out the food, animals must visit until the end of the arm. During the experiment, food was placed only in 1 arm. The wells of remaining 7 arms were empty. In each trial food was placed randomly to any of the 8-arms. 2.4.1.2. Training in the radial arm maze. All animals were habituated to the radial arm maze environment. Mice were placed on the central platform and allowed to explore the maze for 15 min per day. Initially for first 2 days, all baits (8) contain food. On day 3, the number of food containing baits were reduced to half (4 arms contained food among 8 baited arms), and the session ends when all eight arms have been visited. On day 4 and 5, food was kept randomly to 4 arms out of 8 baited arms. On day 6, food was placed in only 2 arms out of 8 baited arms. On day 7 and 8, food was placed in only 1 arm out of 8 baited arms. The training session ends when all eight arms have been visited once. 2.4.1.3. Experimental procedure. On the day of the experiment, food was placed only in 1 arm and all the animals were kept overnight fasting (except water). Mice were released in the center of the maze and allowed to explore the maze. Each trial was conducted for 10 min and behavior of mice was recorded using a webcam. After each run, apparatus was cleaned by 70% ethanol. Three trials were run for every mouse. 2.4.1.4. Parameter of radial arm maze test. Parameters such as “working memory correct”, “working memory incorrect” and “percentage entry into the food placed arm” were considered for data collection (Tarragon et al., 2012). “Working memory correct” was counted when mouse visited into the baited (target) arm that contains food, whereas "working memory incorrect" was counted when mice entered into the baited arm that had no food. Percentage entry to the food placed arm was counted as: (number of entry to the food placed arm/total number of entry to the rest of the arms)  100. Fewer number of mistakes indicate better spatial navigation with improved spatial memory performance. Mice were frequently allowed to move. The entrance of non-food containing arm for more than once was considered as “working memory incorrect”. For example; the result of “working memory incorrect” is 11. This value indicates that the animal have visited to the non-food containing arm for 12 times in 10 min long trial; whereas, “working memory correct” is considered as 4, when an animal has visited to the baited arm for 5 times. 2.4.2. Open field test Plastic wood was used to prepare open field test apparatus. The specification of open field test apparatus was followed according to Walsh and Cummins (1976). 35-W bulb was suspended approximately 1 m above the apparatus for background lighting. Home cage was used to transfer animals to the rest of the room. Animals were allowed to explore the field for 20 min. Apparatus was cleaned using 70% ethanol and allowed to dry in between the trials. Behavioral exploration was video recorded for analysis using a Logitech 4mp webcam. Video was analyzed by Smart (free trial version 3.0) video tracking software developed by Panlab. Open field parameters were analyzed as described in our previous study (Al-Amin et al., 2014). 2.5. Biochemical estimations and tissue processing Mice were anesthetized with ketamine (100 mg/kg body weight, 0.1 ml) and perfused through the heart with cold 0.9% sodium chloride to wash blood from the brain tissue. The entire brain was rapidly removed cautiously and kept in a petridish placed over ice. Six brain regions were dissected in the following

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order; prefrontal cortex (PFC), striatum (ST), hypothalamus (HT), parietal cortex (PCX), cerebellum (CB), and hippocampus (HC). Brain tissue was dissected according to the previous study (AlAmin et al., 2015b). At first, for PFC dissection, the olfactory bulb was removed, and cut into coronal slices of 4 mm below the olfactory bulb. Then striatum was isolated by removing the cortex; CB was dissected from the hindbrain region. Remaining brain part was turned upside down to dissect HT. For HIP dissection: HT was removed, sagittal incision was done to separate the two hemispheres, non-cortical forebrain and meningeal tissues were removed to isolate hippocampus. Ten percent (10%) (w/v) homogenates of various brain regions were prepared in phosphate buffer saline (PBS) (10 mmol/l, pH 7.0) using Ultra-Turrax T25 (USA) homogenizer. Homogenized tissue samples were sonicated at 5 s cycle for 150 s using an ultrasonic processor and centrifuged at 10,000 RPM (7960g) for 10 min. Then, the upper clear supernatants were collected for the biochemical analysis. 2.5.1. Lipid peroxidation (MDA) Lipid peroxidation was evaluated colorimetrically by measuring thiobarbituric acid reactive substances (TBARS) as described previously (Niehaus and Samuelsson, 1968). Briefly, 0.1 ml of sample (PBS buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCAHCl reagent (2-thiobarbituric acid 0.37%, 0.25 N HCl and 15% TCA) and placed in water bath for 15 min and cooled. The absorbance of clear supernatant was measured against reference blank at a wavelength of 535 nm using a 96-well plate. The level of MDA was measured by using standard curve and expressed as nmol/mg of tissue. 2.5.2. Advanced oxidation of protein product (AOPP) Determination of AOPPs was based on spectrophotometric detection according to the method described (Witko-Sarsat et al., 1996). Briefly 50 ml of sample (diluted 1:2 with phosphate-buffered saline (PBS), chloramine T (0–100 mmol/l) was used for the preparation of calibration curve and PBS was used as blank. One hundred microliter of 1.16 M potassium iodide and 50 ml of acetic acid were added to each well and absorbance at 340 nm was measured immediately. Concentration of APOP was expressed as μmol/mg. 2.5.3. Nitric oxide (NO) Nitric Oxide (NO) was assayed according to the method described (Tracey et al., 1995). In this study, Griess-Ilosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1% w/v) instead of 1-naphthylamine (5%). The reaction mixture (3 ml) containing brain homogenates (2 ml) and phosphate buffer saline (0.5 ml) was incubated at 25 °C for 15 min. Rest of the process was followed as described in previous experiment (AlAmin et al., 2015b). A pink colored chromophore was formed in diffused light and the absorbance was measured at a wavelength of 540 nm against the corresponding blank solutions. NO level was measured by using standard curve and expressed as μmol/mg of tissue. 2.5.4. Superoxide dismutase (SOD) The activity of SOD was measured according to the procedure (Ma et al., 2010). Each 300 μl reaction mixture contained sodium phosphate (pH 7.8, 50 mM), methionine (13 mM), nitroblue tetrazolium (NBT) (75 mM), riboflavin (2 mM), EDTA (100 mM), and 2 ml of tissue homogenate. Absorbance was taken at 560 nm followed by the production of blue formazan. 2.5.5. Glutathione (GSH) Glutathione in the brain was evaluated based on the method described (Ellman, 1959). Briefly, 1 ml of sample, 2.7 ml of PBS

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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(0.1 mol/l, pH 8) and 0.2 ml of 5, 5-dithio-bis (2-nitrobenzoic acid) were added and the color developed was determined instantly at 412 nm. Results of glutathione tests were expressed as mmol/mg protein. 2.5.6. Catalase (CAT) The activity of catalase enzyme was assayed colorimetrically at a wavelength of 240 nm (Sinha, 1972). The reaction mixture (1.5 ml) contained 1.0 ml of 0.01 M phosphate buffer (pH 7.0), 0.1 ml of tissue homogenate (supernatant) and 0.4 ml of 2 M H2O2. The reaction was stopped by the addition of 2.0 ml of dichromateacetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). 2.6. Statistical analysis One-way ANOVA analysis was carried out to compare the effect of various treatments and AlCl3 exposure on distinct brain regions and liver. Post-hoc test namely, “Newman-Keuls” was used to compare the difference between groups. All analyses were carried out in Graph Pad prism (version 6.0, Graph Pad Software, Inc.). The difference was considered significant when P o0.05. Data were represented as mean 7S.E.M.

3. Results 3.1. Effect of various treatment on working memory performance in radial arm maze test One-way analysis of variance (ANOVA) showed a significant main effect of various treatments on “working memory correct” [F(3,66) ¼5.32, Po0.01] (Fig. 2A), “working memory incorrect” [F(3,30) ¼ 6.17, P o0.01] (Fig. 2B) and “percentage of entry to the target arm” [F(3,30) ¼4.72, P o0.01] (Fig. 2C). The number of working memory correct was higher in astaxanthin plus aluminum (M ¼3.00, SD¼ 1.15) group than that in the AlCl3 group (M ¼1.4, SD¼ 1.07). Even, astaxanthin plus aluminum (M ¼ 7.00, SD ¼3.22) group made less working memory error than aluminum (M ¼11.00, SD¼ 2.61) group.

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3.3. Biochemical estimations 3.3.1. Brain oxidative stress 3.3.1.1. Glutathione (GSH). A significant main effect of various treatments on the FCX [F(3,19) ¼16.40, Po0.001] (Fig. 4A), ST [F(3,21) ¼37.44, P o0.001] (Fig. 4B), HT [F(3,20) ¼107.70, Po 0.001] (Fig. 4C), HIP [F(3,20) ¼10.03, Po0.001](Fig. 4D) and CB [F(3,19) ¼ 6.18, P o0.01] (Fig.4E). Post-hoc test showed that the levels of GSH in the FCX, HT, CB, HIP, ST were significantly (P o0.05) higher in the aluminum exposed group than the controls. Interestingly, combined aluminum and astaxanthin exposure reversed the levels of GSH in all brain regions except hippocampus. 3.3.1.2. Nitric oxide (NO). We found a significant main effect of treatment on the FCX [F(3,23) ¼24.61, P o0.001] (Fig. 5A), ST [F(3,21) ¼37.44, Po0.001] (Fig. 5B), HT [F(3,20) ¼ 24.07, Po 0.001] (Fig. 5C), HIP [F(3,20) ¼26.78, P o0.001] (Fig. 5D) and CB [F(3,21) ¼86.79, Po0.001] (Fig. 5E). Post-hoc test showed that the levels of NO in the FCX, HT, CB, HIP, ST were markedly (P o0.05) raised only in the aluminum while combined exposure with aluminum and astaxanthin significantly reduced NO levels in the cortex cerebellum and hippocampus. 3.3.1.3. Advanced oxidation of protein product (AOPP). Our ANOVA analysis revealed a significant main effect of various treatment in the FCX [F(3,20) ¼ 24.86, P o0.001] (Fig. 6A), ST [F(3,21) ¼10.34, Po0.01] (Fig. 6B), HT [F(3,20) ¼4.54, Po 0.01] (Fig. 6C), HIP [F(3,20) ¼21.69, Po0.001] (Fig. 6D) and insignificant main effect of treatment on CB [F(3,20) ¼1.0, P o0.25] (Fig. 6E). Post-hoc analysis revealed that the levels of AOPP in the FCX, HT, HIP, ST were markedly (Po 0.05) increased in the aluminum exposed group in comparison to the control group. On the contrary, exposure with aluminum and astaxanthin improved the levels of AOPP in most of the brain regions except CB.

3.2. Effect of different treatment on locomotor activity in open field test

3.3.1.4. Lipid peroxidation (MDA). Our results showed a significant main effect of various treatment on ST [F(3,21) ¼16.49, Po 0.001] (Fig. 7B), HT [F(3,21) ¼ 12.78, Po0.001] (Fig. 7C), HIP [F(3,21) ¼ 71.72, Po0.001] (Fig. 7D), CB [F(3,11) ¼9.32, Po 0.01] (Fig. 7E), and an insignificant main affect on FCX [F(3,12) ¼3.41, P 40.05] (Fig. 7A). Post-hoc test indicated that the levels of MDA in the PCX, FCX, HT, CB, HIP, ST were noticeably (P o0.05) raised in the aluminum exposed group. Moreover, exposure with aluminum plus astaxanthin improved the levels of MDA in all brain regions except FCX.

ANOVA analysis showed a significant main effect of treatment on “time spent in the border” [F(3,18) ¼27.36, Po0.001] (Fig. 3A), “total distance traveled”[F(3,20) ¼3.48, P o0.03] (Fig. 3B) and “zone transition” [F(3,24) ¼7.74, Po 0.001] (Fig. 3C).

3.3.1.5. Catalase (CAT). ANOVA analysis showed a significant main effect of various treatment on ST [F(3,20) ¼ 3.62, Po0.05] (Fig. 8B), CB [F(3,20) ¼16.90, Po0.001] (Fig. 8E), and HT [F(3,20) ¼5.97, Po0.01] (Fig. 8C). Post-hoc test showed that the levels of CAT in

Fig. 2. Effect of various treatments on working memory performance in radial arm maze test. Parameters including “working memory correct”, “working memory incorrect”, and “percentage of entry to the target arm” are used to measure working memory performances. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, ** indicate P values at the levels of 0.05, and 0.01 respectively.

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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Fig. 3. Effect of different treatments on open field test parameters. The parameters were “time spent in border region”, “total distance traveled” and “zone transition”. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, *** indicate P values at the levels of 0.05, and 0.001 respectively.

Fig. 4. Effect of various treatments on the level of Glutathione (GSH) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of GSH in millimole per mg (mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

the HT, CB, ST were significantly (P o0.05) lower in the aluminum group than that in the control group. Interestingly, exposure with aluminum plus astaxanthin reversed the levels of CAT in ST, and CB regions. 3.3.1.6. Superoxide dismutase (SOD). Results of one-way ANOVA analysis showed a significant main effect of treatments on ST [F(3,20) ¼ 7.58, Po 0.001] (Fig. 9B), HIP [F(3,22) ¼5.88, Po0.01] (Fig. 9D), HT [F(3,20) ¼34.88, Po0.001] (Fig. 9C), and CB [F(3,24) ¼ 4.97, P o0.01] (Fig. 9E), and FCX [F(3,20) ¼2.99, P 40.05] (Fig. 9A). Post-hoc test showed that the activities of SOD in the PCX, HT, CB, HIP, ST and liver were significantly (Po0.05) lower in the aluminum group than that in the control group. Interestingly, combined exposure aluminum and astaxanthin reversed the level of SOD in most of the brain regions. 3.3.1.7. Liver oxidative stress. One-way ANOVA analysis showed a significant main effect of various treatment on the level of GSH [F(3,21) ¼48.08, P o0.001] (Fig. 4F), NO [F(3,20) ¼38.87, Po0.001] (Fig. 5F), AOPP [F(3,19) ¼8.39, P o0.05] (Fig. 6F), MDA

[F(3,21) ¼28.44, Po 0.001] (Fig. 7F), CAT [F(3,20) ¼7.63, P o0.001] (Fig. 8F), and SOD [F(3,21) ¼ 26.79, Po0.001] (Fig. 9F). Post hoc analysis result showed that combined aluminum and astaxanthin exposure reduced the levels of GSH, MDA while increased the activity of CAT and SOD in the liver.

4. Discussion The present study revealed that exposure to aluminum causes a deficit of spatial memory performance and enhanced locomotion. Aluminum also enhanced the level of oxidative stress marker in the brain and liver tissues. Moreover, this study discovered that astaxanthin and aluminum co-administration causes less deficits in spatial memory performance and locomotor activity. Additionally the combination treatment group has shown a reduced oxidative damage. Radial-arm maze (RAM) experiment is a hippocampus dependent behavioral experiment to examine learning performance of spatial memory task in rodents which has been used in many

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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Fig. 5. Effect of treatment types on the level of nitric oxide (NO) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of NO in milli mole per mg (mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean 7S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

Fig. 6. Effect of numerous treatments on the level of advanced oxidation of protein product (AOPP) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of (AOPP) in milli mole per mg(mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

previous studies (Levin, 2015; Hritcu et al., 2015; Shanmugasundaram et al., 2015; Yamada et al., 2015). In this study, aluminum-exposed animals showed higher number of errors in maze task and thus aluminum exposure reduces the ability to solve maze task. This suggests that aluminum interrupts in retrieving information that was acquired during the training session. Regarding spatial memory task, a study on rabbit has shown that

intracerebral administration of aluminum causes deficits in learning in the morris water maze task (Rabe et al., 1982). This phenomenon could be attributed to the ability of aluminum to interfere with the downstream signaling, such as, cyclic GMP (cGMP). cGMP is involved in long-term potentiation (LTP) during learning and memory formation (Canales et al., 2001), whereas interference of cGMP could reduce learning ability of spatial

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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Fig. 7. Effect of diverse treatments on the level of malondialdehyde (MDA) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of (MDA) in millimole per mg (mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

Fig.8. Effect of various treatments on the activity of catalase (CAT) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of (CAT) in millimole per mg (mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was used to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

memory task. It is important to note that, all groups of animals have completed sufficient training to learn the task. Various treatments were given after this learning period. Remarkably, the combination of astaxanthin and aluminum treatment has shown less number of errors in spatial memory task when compared with the aluminum group. Animals that had received the combined treatment spent extended time in the target arm. Taken together,

our results demonstrate that the administration of astaxanthin might counteracts aluminum-induced toxicity resulting in improved performance in hippocampus dependent task. Open field test is an important tool to examine anxiety and exploratory behavior of rodents. In the open field test, aluminum treated mice displayed anxious behavior. Our findings on aluminum exposed behavioral alterations are consistent with the

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

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Fig. 9. Effect of different treatments on the activity of superoxide dismutase (SOD) in the frontal cortex, striatum, hypothalamus, hippocampus, cerebellum and liver. Horizontal axis represents the concentration of (SOD) in millimole per mg (mmol/mg) and vertical axis represents groups such as control, AlCl3, AlCl3 þ AST, AST. Values are represented as mean 7 S.E.M. Newman-keuls multiple comparisons test was followed to compare between the groups. *, **, *** indicate P values at the levels of 0.05, 0.01, and 0.001 respectively.

previous studies. Aluminum exposure resulted in behavioral changes such as spatial memory deficits in morris water maze (John et al., 2015; Justin Thenmozhi et al., 2015b; Kaur and Sodhi, 2015; Lakshmi et al., 2015; Ribes et al., 2008; Sharma et al., 2013) and locomotor activity (Justin Thenmozhi et al., 2015a; Lakshmi et al., 2015). By contrast, we demonstrate that aluminum-exposed animals were anxious. This anxiousness drives the animals to travel longer distance and frequent changes of the position in the open field test arena. We showed that astaxanthin plus aluminum group showed improved locomotor activity in the open field parameters. These results are consistent with our previous study where astaxanthin was used to treat valproic acid-induced mice model of autism (AlAmin et al., 2015b). We have observed that astaxanthin reduces the level of anxiety in mice model of autism in an open field test (Al-Amin et al., 2015b). We have observed that an association between the exposure to aluminum and OS exists in the brain. These changes could have been due to the reduced axonal mitochondrial turn over, disruption of the golgi and the release of oxidative products like malondialdehyde, carbonyls and peroxynitrites within the neurons (Bharathi et al., 2006). Malondialdehyde (MDA), a lipid peroxidation parameter, is considered as one of the key intermediates of free radical damages. In our study, MDA production was raised in aluminum treated group, and dropped when astaxanthin plus aluminum was given together. Previous reports are in line with our current result (Chan et al., 2009). Aluminum enhanced the level of MDA in the cortex (Qiu et al., 2015), hippocampus, cerebellum (Qiu et al., 2015) and in the whole brain (John et al., 2015; Kaur and Sodhi, 2015; Lakshmi et al., 2015; Pan et al., 2015; Sharma et al., 2013; Yu et al., 2014). This lipid peroxidation product interferes with the brain homeostasis between inhibitory and excitatory neurons (Ritter et al., 2015) and impairs mitochondrial function in the brain (Vaishnav et al., 2010). Reactive oxygen species also oxidizes protein products; such as,

oxidized albumin. We found that aluminum enhanced the levels of AOPP and NO in the brain and liver. Our findings are consistent with the previous reports regarding AOPP (Sharma et al., 2013) and NO (John et al., 2015). Nevertheless, combined treatment with astaxanthin has shown that this antioxidant reduces the protein oxidation and nitric oxide formation. These results are consistent with our recent findings regarding the effect of astaxanthin on autism (Al-Amin et al., 2015b) and age-dependent study (Al-Amin et al., 2015a). Accumulating evidence suggests that aluminum altered the level of enzymatic antioxidants in the brain, such as superoxide dismutase (SOD) catalase (CAT) and glutathione (GSH). SOD presents the first line of defense against superoxide. Superoxide dismutase converts superoxide anion to hydrogen peroxide and oxygen (Kumar et al., 2011). CAT inhibits oxidative damage by catalyzing the decomposition of hydrogen peroxide to water and oxygen (Chtourou et al., 2015). Our results showed that exposure to aluminum inhibited the activity of CAT and SOD enzymes. To the contrary, combined treatment enhanced this enzymatic activity. The effect of aluminum exposure is consistent with the previous studies that decreased levels of CAT (Akinrinade et al., 2015; John et al., 2015; Lakshmi et al., 2015) and SOD (Lakshmi et al., 2015; Pan et al., 2015; Yu et al., 2014) in the brain. Besides SOD and CAT, aluminum reduces the level of Glutathione (GSH), however, combined treatment enhanced the concentration of GSH. GSH in its reduced form is the most abundant intracellular antioxidant. GSH is involved in direct scavenging of free radicals or serving as a substrate for the glutathione peroxidase enzyme that catalyzes the detoxification of hydrogen peroxide (Kumar et al., 2011). Regarding GSH, our results are consistent with the previous reports (Al-Amin et al., 2015b; John et al., 2015; Lakshmi et al., 2015; Orihuela et al., 2005). Taken together, our study demonstrates that astaxanthin improves OS markers in most of the brain regions by improving antioxidant enzymes. This result implied that the improvement of antioxidant enzyme may reduce neural stress, and enhance synaptic communication.

Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062

M.M. Al-Amin et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Specific signaling pathway or underpinning molecular mechanism of astaxanthin on the improvement of OS has not been reported in the previous literature. Here, we propose that astaxanthin treatment improves behavioral brain function via counteracting aluminum-induced free radicals in the brain. Astaxanthin could considerably inhibit intracellular free radical and ROS generation specifically in the mitochondria (Liu et al., 2009). Diminished level of ROS would help the body to retain the level of cellular antioxidant enzymes. Consequently, unused antioxidant enzymes spare in the cell and are accumulated in the appropriate site for future use. Recent studies suggest that astaxanthin stimulates gene expression of antioxidant enzymes in shrimp (Wang et al., 2015). Astaxanthin has been shown to increase the level of antioxidant enzymes in various disease model as well (Kamath et al., 2008; Leite et al., 2010; Otton et al., 2012; Rao et al., 2013). We know that effect of aluminum on the level of neurotoxicity could be dose-dependent. Aluminum lactate (1 mg/g diet for 120 days) increased the number of proliferating cells in the dentate gyrus (Ribes et al., 2008), whereas aluminum lactate (11 mg/g of food for 180 days) unaffected neurogenesis in the hippocampus. In addition, aluminum (10 mg/kg/day for 56 days) reduced the total number of neurons and glial cells in the rat cortex. In our study, we used comparatively higher dose (50 mg/kg/day) which might induce loss of neuronal and glial cells in the brain. However, we were unable to count the neuron or glial cells in the brain, therefore, future study may address the effect of aluminum and astaxanthin on the number of immature neurons and mature neurons and glial cells in the brain. To be noted that our study has several limitations as we were unable to measure the level of neurotransmitters. Measurement of specific neurotransmitter may indicate neuroprotective and neurotoxic effect of various treatments on the level of neurotransmitter production. Moreover, the measurement of acetylcholinesterase enzyme and histological study could provide important insight on aluminum toxicity. Only one dose of astaxanthin was used in this study, whereas, higher dose could produce different aluminum induced toxicity in a greater extent. Future study may include a range of doses, which will provide a comparable picture of dose dependent effects of astaxanthin in aluminum-exposed neurotoxic model. Despite of the limitations, the present study is novel in several respects – this study first addresses the effect of astaxanthin on aluminum exposed oxidative damage. Moreover, our attention was given on the various brain tissues that are associated with vital brain functions.

5. Conclusions We used two behavioral tests and six OS markers to measure the contribution of astaxanthin in counteracting aluminum induced toxicity in brain. Our study specially focuses on spatial learning and memory task, locomotor activity and OS in major brain regions that are relevant with the behavioral task. We found that combined treatment with astaxanthin and aluminum in mice has shown better behavioral performance and OS than aluminumexposed animals. In conclusion, we suggest that treatment with astaxanthin might be able to rescue aluminum-exposed behavioral disorder presumably by the reduction of OS markers in the brain.

Conflict of interest There are no conflicts of interests with other organizations. No fund was received or no funding organization was involved in this study.

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Please cite this article as: Al-Amin, M.M., et al., Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.02.062