Oral administration of lutein attenuates ethanol-induced memory deficit in rats by restoration of acetylcholinesterase activity

Physiology & Behavior 204 (2019) 121–128

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Oral administration of lutein attenuates ethanol-induced memory deficit in rats by restoration of acetylcholinesterase activity

T

Júlia Maria Tonin Geissa, Sara Cristina Sagaeb, Edson Duarte Ribeiro Pazb, Mayara Lutchemeyer de Freitasc, Naiéli Schiefelbein Soutod, Ana Flavia Furianc,d, ⁎ Mauro Schneider Oliveirac, Gustavo Petri Guerraa, a

Programa de Pós-graduação em Tecnologia de Alimentos, Universidade Tecnológica Federal do Paraná, Medianeira, PR 85884-000, Brazil Departamento de Fisiologia, Universidade Estadual do Oeste do Paraná, Cascavel, PR 85819-110, Brazil c Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil d Programa de Pós-graduação em Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Carotenoids Object recognition Acetylcholinesterase Oxidative stress Hippocampus Cerebral cortex

The excessive consumption of alcohol affects the central nervous system, resulting in memory and learning deficits. Lutein is a carotenoid known for its antioxidant properties, which can be able to prevent neurodegenerative diseases and cognitive deficits. In the present study, we evaluated the effect of lutein on ethanolinduced memory deficits in the object recognition task in adult rats, as well as the possible involvement of oxidative stress and cholinergic system. Wistar rats were randomly divided into two groups receiving lutein (50 mg/kg) or olive oil (1 mL/kg) by oral gavage once daily for 14 days. On day 8 each group was divided again into two groups receiving either ethanol (3 g/kg) or saline by oral gavage once daily for 7 days. After the last administration, the animals were submitted on the object recognition task 24 h later (on days 15, 16 and 17). After the behavioral test, the hippocampus and cerebral cortex were removed for the determination of oxidative stress indicators (superoxide dismutase, thiobarbituric acid reactive substances, and non-protein thiol) and acetylcholinesterase activity. Ethanol administration induced a memory deficit and increased acetylcholinesterase activity, however, it did not alter the parameters of oxidative stress, evaluated in the cortex and hippocampus. Oral administration of lutein (50 mg/kg during 14 days) attenuated memory deficit and the increase of acetylcholinesterase activity induced by ethanol. These results provide evidence that lutein is an alternative treatment for ethanol-induced memory deficit, and suggest the involvement of cholinergic system.

1. Introduction

reduction of antioxidant enzyme activity, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [11–14]. Furthermore, ethanol increases acetylcholinesterase (AChE) activity [15] and decreases acetylcholine (ACh) released in the hippocampus [16], which correlates with impairments in the spatial memory [17,18]. Considering the prevalence of alcohol consumption over the last few years, it is important to prevent alcohol-related disorders that can impair daily activities as well as productivity at work and studies. Therefore, studies that contribute to the development of treatments and new therapeutical options able to prevent ethanol-induced memory deficits, that are also effective, safe, inexpensive, and with no side effects, are of extreme importance. In this sense, the evidence to support the important role of bioactive compounds and extra nutritionals constituents present mainly in plant origin food, that act in health maintenance and reduce the risk of diseases, without side effects, is growing

Alcohol is one of the most used drugs in the world, with 90% of people consume it at some point in their lives, and approximately 30% of these develop some type of disorder [1]. Excessive alcohol consumption affects many body systems, including the brain and the central nervous system, [2,3] which may cause cognitive, learning, and short- or long-term memory deficits [4,5]. In fact, it has been shown that brain damages and memory deficits are related to alcohol consumption in humans [6,7] and in animals [8,9]. A recent study shows there is significant spatial and non-spatial memory impairment in rats treated with intermittent ethanol exposure [10]. In this sense, ethanol exposure increases the production of reactive species and lipid peroxidation [11,12] and induces a significant

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Corresponding author at: Department of Food, Universidade Tecnológica Federal do Paraná, Medianeira, PR 85884-000, Brazil. E-mail address: [email protected] (G.P. Guerra).

https://doi.org/10.1016/j.physbeh.2019.02.020 Received 13 November 2018; Received in revised form 6 February 2019; Accepted 13 February 2019 Available online 15 February 2019 0031-9384/ © 2019 Elsevier Inc. All rights reserved.

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[19–21]. Bioactive compounds such as carotenoids, vitamins B, C, D and E, flavonoids, polyunsaturated fatty acids, curcumin, and piperine have been studied as alternatives treatments for cancer, neurodegenerative, and cardiovascular diseases [22–27]. Lutein is a xanthophyll carotenoid known for its antioxidant properties, with the ability to remove free radicals and, consequently, prevent damage to lipoproteins, membrane lipids, proteins, and DNA, thereby it prevents oxidative stress [28–30]. There are lines of evidence supporting the role of lutein in cognitive processes and neurodegenerative diseases. Firstly, supplementation or a diet rich in lutein sources are associated with low rates of cognitive decline due to aging [31–33]. In accordance with this view, oral administration of lutein (50 or 100 mg/kg) has been described to improve the cognitive deficits caused by Huntington's disease [34]. Secondly, lutein is the main carotenoid found in the human brain, forming approximately 30% of the total carotenoids concentration. It displays an ability to cross the blood–brain barrier, with preferential uptake compared to most carotenoids, showing a positive correlation between the serum and brain levels of lutein [35–37]. Moreover, lutein accumulation in the macula is associated with the levels present in some brain regions such as the cerebellum, frontal, and occipital cortex [37–39]. Thirdly, an improvement in cognitive function has been associated with increased levels of lutein in the serum and the brain [36,40]. Furthermore, the increase in macular pigment optical density is positively correlated to cognitive function in the elderly [38,41]. Fourthly, individuals with mild cognitive deficit [36,42] or Alzheimer's disease [42–45] show reduced levels of lutein in the plasma and the brain compared to normal individuals. Furthermore, the low macular pigment optical density is associated to a lower cognitive performance in the elderly [46,47]. Therefore, considering the confluent results that support the role of lutein in cognitive functions and the prevention of degenerative diseases [31–33,36,42,44,45,48], in the present study, we investigated whether lutein prevents ethanol-induced memory deficit as well as possible mechanisms of action, involving oxidative stress and cholinergic system.

Fig. 1. A) Schematic representation of the experimental protocol for dose–response curve of lutein. B) Administration of lutein (15–100 mg/kg) for 14 days on the discrimination index in the object recognition task. Data are mean ± SEM, for n = 9–10 animals in each group. * Indicates a significant difference (p < .05) compared to the control group (Olive Oil).

administration, the animals were returned to their home cages and submitted on the object recognition task 24 h later (on days 15, 16 and 17). The dose range and administration time were selected based on a previous study which shows that lutein (50 mg/kg) improves deficits induced by Huntington's disease [34]. The treatment schedule is depicted at Fig. 1A. 2.2.2. Experiment 2: effect of ethanol administration on object recognition task performance A dose–response curve for ethanol was performed to define the dose that induces memory deficit for the subsequent experiments. The rats were weighed and randomly divided into two groups receiving ethanol (0.3, 1 or 3 g/kg) or saline (0.9% NaCl, 10 mL/kg) by oral gavage, once daily for 7 days. After the last administration, the animals were returned to their home cages and submitted on the object recognition task 24 h later (on days 8, 9 and 10). The dose range and administration time were selected based on a previous study which shows that ethanol administration (2 or 3.4 g/kg) causes memory deficit [49,50]. The treatment schedule is depicted at Fig. 2A.

2. Materials and methods 2.1. Animals and reagents All experiments reported in this study were conducted in accordance to the National and International legislation [guidelines of the Brazilian Council of Animal Experimentation (CONCEA) and of the U.S. Public Health Service's Policy on Humane Care and Use of Laboratory Animals (PHS Policy)], and with the approval of the Ethics Committee for Animal Research of the Universidade Estadual do Oeste do Paraná (01/2016). Male Wistar rats (weight: 250–300 g; n = 142) were bred in the Animal House of the Universidade Estadual do Oeste do Paraná, housed 5 to a cage, and maintained on a natural day/night cycle, at 21 °C with access to water and rodent laboratory chow (Algomix, Ouro Verde do Oeste, PR, Brazil) ad libitum. Behavioral tests were conducted during the light phase of the cycle (from 9:00 a.m. to 5:00 p.m.). Lutein (Fagron, Brazil) was dissolved in olive oil, immediately before administration. Ethanol was purchased from Merck (Darmstadt, Germany) and diluted in saline (0.9% NaCl) to achieve a concentration of 20% v/v ethanol. All the other reagents used were of analytical grade.

2.2.3. Experiment 3: effect of lutein on memory deficit induced by ethanol administration and oxidative stress indicators and acetylcholinesterase (AChE) activity Once it had been determined that lutein at the dose of 50 mg/kg did not alter memory per se and ethanol (3 g/kg) induced a memory deficit, we tested whether this dose of lutein prevented the memory deficits induced by ethanol administration. The rats were randomly divided into two groups receiving lutein (50 mg/kg) or olive oil (1 mL/kg) by oral gavage once daily for 14 days. On day 8 each group was randomly divided again into two groups receiving either ethanol (3 g/kg) or saline (0.9% NaCl, 10 mL/kg) by oral gavage once daily for 7 days. After the last administration, the animals were returned to their home cages and submitted on the object recognition task 24 h later (on days 15, 16 and 17). After the behavioral test, the hippocampus and cerebral cortex were removed for the determination of oxidative stress indicators (SOD, TBARS and NPSH) and AChE activity. The treatment

2.2. Experimental design 2.2.1. Experiment 1: effect of lutein on object recognition task performance A dose–response curve for lutein was performed to evaluate the effect on object recognition task performance and to define the dose for the subsequent experiments. The rats were weighed and randomly divided into two groups receiving lutein (15, 50 and 100 mg/kg) or olive oil (1 mL/kg) by oral gavage, once daily for 14 days. After the last 122

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experiments, the objects were used in a counterbalanced manner, and the rats did not previously display a preference for any of the objects. Chambers and objects were cleaned after each subject was tested with 30% ethanol. The task consisted on habituation, training, and testing sessions; each of them with the duration of 10 min. In the first session, rats were habituated to the behavioral apparatus and then returned to their home cage. In this session, the number of crossing and rearing responses were recorded to identify any motor disabilities, which might influence the performance of the task. For this evaluation, the wooden chamber had the floor divided into 12 squares. Twenty-four hours later, the training session occurred, where rats were exposed to two of the same objects (Object A), and the exploration time was recorded using two stopwatches. Exploration was defined as sniffing or touching the object with the nose and/or forepaws, at a distance of < 2 cm. Climbing or sitting on the object was not considered as exploration. The test session was carried out 24 h after the training session. Each rat was placed back in the behavioral chamber and one of the already known objects (Object A) was replaced by a novel object (Object B). The times spent exploring the already known and novel objects were recorded. The discrimination index was then calculated, taking into account the difference of time spent exploring the novel (B) and already known (A) object x 100 divided by the sum of time spent exploring the novel (B) and already known (A), and used as a cognitive parameter {[T novel (B) - T already known (A)/T novel (B) + T already known (A)] × 100}.

Fig. 2. A) Schematic representation of the experimental protocol for dose–response curve of ethanol. B) Administration of ethanol (0.3–3 g/kg) for 7 days on the discrimination index in the object recognition task. Data are mean ± SEM, for n = 10–11 animals in each group. * Indicates a significant difference (p < .05) compared to the control group (Saline).

2.4. Measurement of oxidative stress indicators and acetylcholinesterase (AChE) activity 2.4.1. Preparation of tissues Immediately after the test session of the novel object recognition task, the rats were decapitated, the hippocampus and cerebral cortex were removed, weighed, and homogenized in Tris–HCl (50 mM, pH 7.4) buffer. The resulting homogenate was then centrifuged at 10,000 rpm × 10 min at 4 °C and supernatant fraction (S1) was used for the determination of enzymatic and non-enzymatic indicators of oxidative stress and AChE activity. The protein content was measured colorimetrically using the Bradford method [52], and bovine serum albumin (1 mg/mL) was used as the standard.

schedule is depicted at Fig. 3A. 2.3. Object recognition task The object recognition task was performed to evaluate the declarative memory, according to Bevins and Besheer [51], with minor modifications. The task was performed in a wooden chamber (60 × 40 × 30 cm), with black painted walls, the front wall consisting of clear Plexiglas, and the floor covered with an ethyl vinyl acetate sheet. A light bulb, hanging 60 cm above the behavioral apparatus, provided constant illumination of approximately 40 lx, and an airconditioner provided constant background sound isolation. The objects used were pairs of plastic mounting bricks, with each pair composed of different shapes (rectangular, pyramid, and stair-like shapes), and colors (white, red, and blue), but all of the same size. Throughout the

2.4.2. Superoxide dismutase (SOD) activity The activity of SOD was determined according to the method described by Misra and Fridovich [53]. This method is based on the ability of SOD to inhibit the autoxidation of adrenaline to adrenochrome. In Fig. 3. A) Schematic representation of the experimental protocol for administration of lutein and ethanol. B) Administration of lutein (50 mg/kg), ethanol (3 g/kg), and the co-administration of ethanol and lutein on the discrimination index in the object recognition task. Data are mean ± SEM, for n = 10–11 animals in each group. * Indicates a significant difference (p < .05) compared to the control group (Olive Oil/Saline).

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summary, the supernatant fraction (20–60 μL) was added to a medium containing glycine buffer (50 mM, pH 11) and adrenaline (1 mM). The kinetic analysis of SOD was started after adrenaline addition, at 38 °C, and the color reaction was measured at 480 nm. One unit of enzyme was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50% at 38 °C, and the results were expressed as units (U)/mg of protein.

Table 1 Effect of administration of lutein (15–100 mg/kg) for 14 days, on exploratory behavior of animals (number of crossing and rearing responses), during the habituation session in object recognition task.

2.4.3. Thiobarbituric acid reactive substance (TBARS) determination Lipid peroxidation was estimated by measuring TBARS and was expressed in terms of the malondialdehyde (MDA) content, according to the method described by Ohkawa et al. [54]. In this method, MDA, a final product of fatty acid peroxidation, reacts with thiobarbituric acid (TBA) to form a colored complex. The content of TBARS was measured in a medium containing 100 μL of tissue homogenate of the hippocampus or of the cerebral cortex; 15 μL of 8.1% sodium dodecyl sulfate (SDS), 60 μL of acetic acid buffer (2.5 M, pH 3.4), and 115 μL of 0.81% TBA. The mixture was then heated at 95 °C for 120 min in a water bath. After cooling to room temperature, the absorbance was measured in the supernatant at 532 nm. The results were calculated as μmol MDA/mg of protein.

Groups

Crossing

Rearing

N

Olive oil Lutein 15 mg/kg Lutein 50 mg/kg Lutein 100 mg/kg Statistical analysis

81.6 ± 3.6 71.2 ± 4.7 75.8 ± 6.7 70.1 ± 8.7 F(3,34) = 0.7552 p > .05

46.6 ± 2.7 42.5 ± 3.3 42.7 ± 4.0 37.1 ± 5.1 F(3,34) = 1.020 p > .05

10 10 9 9

Data are means ± SEM; N, number of animals in each group.

lutein on the exploratory behavior of rats, during the habituation session in the object recognition task. Statistical analysis (one-way ANOVA) revealed that lutein did not alter the number of crossing or rearing responses (F values shown in Table 1), suggesting that its administration did not cause gross motor disabilities during training and testing sessions. The dose of lutein used in the subsequent experiments (50 mg/kg) was selected based on the lack of effect on memory shown in this experiment.

2.4.4. Non protein thiol (NPSH) NPSH levels were determined according to the method described by Ellman et al. [55] with some modifications. Samples were precipitated with trichloroacetic acid (TCA, 10%) and subsequently centrifuged at 3000 x g for 10 min. After the centrifugation, the supernatant fraction (60 μL) was added to a reaction medium containing potassium phosphate buffer (1 M, pH 7.4) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, 10 mM). The NPSH levels were measured spectrophotometrically at 412 nm. The results were calculated using a standard curve constructed with reduced glutathione (GSH) and corrected based on the protein content. The results were calculated as nmol NPSH/mg of protein.

3.2. Experiment 2 Fig. 2B shows the effect of oral administration of ethanol (0.3–3 g/ kg) for 7 days on the discrimination index in the test session of the object recognition task. Statistical analysis (one-way ANOVA) revealed that ethanol significantly decreased the discrimination index [F(3,37) = 3.06; p < .05]. Post hoc comparisons demonstrated that ethanol administration, at the dose of 3 g/kg, impaired memory in the object recognition task, compared to the control group. There was no significant difference between the groups for the time spent exploring both objects in the training session, indicating no biased exploration of objects (data not shown). Table 2 shows the effect of ethanol on the exploratory behavior of rats, during the habituation session of the object recognition task. Statistical analysis (one-way ANOVA) revealed that ethanol did not alter the number of crossing or rearing responses (F values shown in Table 2), suggesting that its administration did not cause gross motor disabilities during training and testing sessions. No signs of spontaneous ethanol withdrawal was observed in rats treated with ethanol. The dose of ethanol used in the subsequent experiments (3 g/kg) was selected due to induce memory deficit in this experiment.

2.4.5. Determination of acetylcholinesterase activity The AChE activity was measured by the method described by Ellman et al. [56], using acetylthiocholine iodide as a substrate in homogenates of the hippocampus. Each sample was assayed in triplicate. The rate of hydrolysis of acetylthiocholine iodide was measured at 412 nm through the release of thiol compounds, which reacted with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), producing the colored product, thionitrobenzoic acid.

3.3. Experiment 3

2.5. Statistical analysis

Figs. 3B, 4 and 5 show the effect of the oral administration of lutein (50 mg/kg), ethanol (3 g/kg), and the co-administration of ethanol and lutein on the discrimination index in the test session of the object recognition task, oxidative stress indicators (SOD, TBARS and NPSH) and AChE activity in the hippocampus and cerebral cortex. Statistical analysis (two-way ANOVA) revealed a significant effect for interaction factor (lutein or olive oil versus ethanol or saline) [F(1,39) = 5.62; p < .05] and ethanol [F(1,39) = 12.11; p < .05] on the discrimination

GraphPad Prism 7 Software was used for statistical analysis and plotting graphs. Statistical analyses were performed by one-way or twoway analysis of variance (ANOVA), followed by Bonferroni's post hoc test, depending on the experiment. Values of p < .05 were considered statistically significant. All data are expressed as the mean and S.E.M. 3. Results 3.1. Experiment 1

Table 2 Effect of administration of ethanol (0.3–3 g/kg) for 7 days on exploratory behavior of animals (number of crossing and rearing responses), during the habituation session in object recognition task.

Fig. 1B shows the effect of the oral administration of lutein (15–100 mg/kg) for 14 days, on the discrimination index in the test session of the object recognition task. Statistical analysis (one-way ANOVA) revealed that lutein, significantly increased the discrimination index [F(3,34) = 7.13; p < .05]. Post hoc comparisons demonstrated that lutein administration, at the dose of 100 mg/kg, improved memory in the object recognition task, compared to the control group. There was no significant difference between the groups for the time spent exploring both objects in the training session, thus indicating no biased exploration of objects (data not shown). Table 1 shows the effect of

Groups

Crossing

Rearing

N

Saline 0.9% Ethanol 0.3 g/kg Ethanol 1 g/kg Ethanol 3 g/kg Statistical analysis

78.8 ± 9.4 81.1 ± 8.2 94.9 ± 6.1 76.2 ± 8.2 F(3,37) = 1.049 p > .05

43.5 ± 6.9 46.6 ± 3.1 47.6 ± 3.5 40.0 ± 2.6 F(3,37) = 0.6262 p > .05

10 10 10 11

Data are means ± SEM; N, number of animals in each group. 124

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Fig. 4. Administration of lutein (50 mg/kg), ethanol (3 g/kg), and the co-administration of ethanol and lutein on oxidative stress indicators A) Superoxide Dismutase, B) MDA and C) Non-protein thiol in the hippocampus and D) Superoxide Dismutase, E) MDA and F) Non-protein thiol in the cerebral cortex of rats. Data are mean ± SEM, for n = 5–7 animals in each group.

increase of AChE activity induced by ethanol in the hippocampus (Fig. 5A) and cerebral cortex (Fig. 5B). Table 3 shows the effect of the oral administration of lutein, ethanol and co-administration of ethanol and lutein on exploratory behavior of the rats, during the habituation session of the object recognition task. Statistical analysis (two-way ANOVA) revealed that pharmacological treatments did not alter the number of crossing or rearing responses (F values shown in Table 3), suggesting that none of the compounds tested caused gross motor disabilities during training and testing sessions. No significant difference in weight gain between the treated rats (four groups) was evident (data not shown).

index. Post hoc comparisons demonstrated that lutein co-administration attenuated the ethanol-induce memory deficit in the object recognition task (Fig. 3B). There was no significant difference between groups for the time spent exploring both objects in the training session, indicating no biased exploration of objects (data not shown). Statistical analysis also revealed that the administration of lutein, ethanol and the co-administration of ethanol and lutein did not alter the SOD activity and levels of TBARS and NPSH in the hippocampus (Fig. 4 A, B and C) and the cerebral cortex of rats (Fig. 4D, E and F). Two-way ANOVA revealed a significant effect for lutein or olive oil versus ethanol or saline interaction on AChE activity in the hippocampus [F(1,20) = 5.28; p < .05] and cerebral cortex [F(1,20) = 26.76; p < .0001]. Post hoc comparisons demonstrated that lutein co-administration prevented the 125

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in the cognitive deficits in elderly or caused by age-related diseases, regardless of the effects per se [31–33,47,57]. However, little is known about the effects of lutein on the prevention or reversal of memory deficit caused by factors others than aging [34]. In support of this view, the protective effects of lutein on ethanol-induced memory deficits has not been demonstrated. We showed that lutein attenuated the ethanolinduced memory deficit in the object recognition task. Aiming at providing information related to memory impairment and lutein, the AChE activity was assessed. AChE is an enzyme that catalyzes the hydrolyse of ACh that functions as neurotransmitter, which is responsible for the termination of cholinergic responses [58]. The ACh released in the synaptic cleft is directly involved in the process of learning and memory [59]. Therefore, cholinergic system dysfunction reduces ACh levels in the hippocampus and cerebral cortex leading to memory deficits [60]. In this sense, systemic administration of ethanol (1.0–2.4 g/kg) reduces ACh released in the hippocampus and prefrontal cortex [16,61,62]. Considering that the cholinergic system is important for memory processing, it can be suggested that an increase in AChE activity might be responsible for memory deficits induced by ethanol administration. Interestingly, the administration of lutein reduced AChE activity and prevented ethanol-induced memory deficit. Another hypothesis to explain the protective effect of lutein on ethanol-induced memory deficit is that it would be through prevention of oxidative stress induced by ethanol. Carotenoids can be used to treat of pathologies caused by oxidative stress [30]. In this context, lutein antioxidant action and the ability to combat oxidative stress are responsible for the protective effect on dysfunctions or deficits caused by different factors, such as diabetes [63,64], 3-nitropropionic acid [34], ischemic stroke [65] or alcohol [66]. Furthermore, ethanol administration leads to increased oxidative stress, due to a decrease in the antioxidant enzymes activity [11,12,67]. Surprisingly, however, our results indicated that ethanol administration did not show effect on oxidative stress indicators. Regarding this point, there are also some conflicting results [68–70]. The administration of ethanol (2.5 g/kg) for 4 days increases the level of TBARS and does not alter the activity of SOD and GPx in the hippocampus, whereas in the cortex, the activity of antioxidant enzymes and level of TBARS are not modified [69]. In addition, the administration of ethanol (4 g/kg; single dose) does not alter the antioxidant enzyme activity, such as SOD, CAT and GPx in the rat brain [68]. Similarly, the chronic consumption of ethanol (5% w/v) for 2 months, does not alter the activity of SOD in the cerebellum [71]. Furthermore, ethanol (1.6 g/kg) increases the MDA levels and SOD activity in the cortex, as well as the CAT and GPx activity in the striatum [70]. Moreover, the administration of ethanol (4.5 g/kg) for 14 days prenatal and 21 days postnatal does not alter the SOD and CAT activity in the hippocampus and cerebellum, respectively [67]. It is difficult to explain why the oxidative stress indicators remained unchanged upon ethanol exposure. It is suggested that the differences in the routes of administration, doses of ethanol, and periods of exposure can explain the differences in the results for these parameters. In summary, our results support the fact that lutein attenuated ethanol-induced memory deficit but such an effect does not involves oxidative stress or antioxidant systems in the hippocampus and cerebral cortex of rats. The protective effect of carotenoids seems to be not only related to antioxidant action, whereas α-tocopherol (vitamin E), a potent antioxidant, is less related to the improvement of cognitive deficits than carotenoids [36]. In addition, cortical carotenoids seem to play other synaptic functions, like increase of communication among neurons and gap junction [72,73]. Although we do not have experimental evidence, it is interesting to consider that the effect of lutein on cognitive deficit may involve an additional mechanism, beyond those investigated in this study. Evidence shows that lutein, the major carotenoid found in the human brain, prevents decreases in BDNF (brain-derived neurotrophic factor) levels in the retina of diabetic mice [29,74]. BDNF, the most abundant neurotrophic factor in the brain, found mainly in the hippocampus and

Fig. 5. Administration of lutein (50 mg/kg), ethanol (3 g/kg), and the co-administration of ethanol and lutein on AChE activity in the A) hippocampus B) cerebral cortex of rats. Data are mean ± SEM, for n = 6 animals in each group. * Indicates a significant difference (p < .05) compared to the control group (Olive Oil/Saline). Table 3 Effect administration of lutein (50 mg/kg), ethanol (3 g/kg), and the co-administration of ethanol and lutein, on exploratory behavior of animals (number of crossing and rearing responses), during the habituation session in object recognition task. Groups

Crossing

Rearing

N

Olive oil/Saline Olive oil/Ethanol Lutein/Saline Lutein/Ethanol Statistical analysis

87.0 ± 3.8 92.1 ± 7.1 87.2 ± 9.4 80.2 ± 8.4 F(1,39) = 0.6610 p > .05

42.3 ± 2.9 48.9 ± 1.4 39.9 ± 2.7 41.6 ± 3.8 F(1,39) = 0.8508 p > .05

11 11 11 10

Data are means ± SEM; N, number of animals in each group.

4. Discussion In the present study, we evaluated the effect of lutein on ethanolinduced memory deficits and the possible mechanism involved. Our results showed that ethanol administration impaired memory (Fig. 2) and increased AChE activity (Fig. 5), but did not alter oxidative stress indicators, such as SOD, TBARS, and NPSH (Fig. 4) in the hippocampus and the cerebral cortex. Lutein, a xanthophyll with antioxidant properties, showed to improve memory per se (Fig. 1). However, the most important finding is that this study provides evidence for the protective potential of oral lutein treatment (50 mg/kg during 14 days) on ethanol-induced memory deficit in the object recognition task (Fig. 3) and restoration of AChE activity in the hippocampus and the cerebral cortex (Fig. 5). Numerous studies have shown the protective effect of lutein, mainly 126

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frontal cortex [75], plays an important role in neurogenesis, neuronal development and regeneration, synaptic plasticity and cognition, formation and storage of memories [76,77]. These effects occur mainly due to the activation of the tyrosine kinase B receptor (TrkB) by BDNF, triggering several signaling cascades [78]. In addition, the expression of both BDNF [79] and TrkB receptor [80] are decreased in the hippocampus and cortex, after alcohol exposure. Interestingly, some studies suggest that BDNF enhances ACh release [81–83]. In this sense, a possible mechanism of action for the lutein effects on attenuation of ethanol-induced memory deficits can involve an increase in BDNF levels and activation of TrkB receptors, beyond the effects on the cholinergic system balance in the hippocampus and cortex, demonstrated in study. In summary, our results support the fact that lutein attenuated ethanol-induced memory deficit and restored AChE activity, suggesting that neuroprotective effect on memory involves cholinergic neurotransmission in the hippocampus and cerebral cortex. These currently reported results support lutein as a therapeutic target for the development of drugs able to prevent memory deficits, thus allowing the identification of effective pharmacological strategies for treatments. However, further investigation are needed to clarify the precise mechanisms involved in the neuroprotective effects of lutein on memory deficits.

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