Blackberry extract improves behavioral and neurochemical dysfunctions in a ketamine-induced rat model of mania

Journal Pre-proof BLACKBERRY EXTRACT IMPROVES BEHAVIORAL AND NEUROCHEMICAL DYSFUNCTIONS IN A KETAMINE-INDUCED RAT MODEL OF MANIA Vitor C. Chaves, Mayara S.P. Soares, Luiza Spohr, Fernanda Teixeira, Andriele Vieira, Larissa S. Constantino, Felipe Dal Pizzol, Claiton L. Lencina, Roselia M. Spanevello, Matheus P. Freitas, ´ ´ Claudia M.O. Simo˜ es, Flavio H. Reginatto, Francieli M. Stefanello

PII:

S0304-3940(19)30669-X

DOI:

https://doi.org/10.1016/j.neulet.2019.134566

Reference:

NSL 134566

To appear in:

Neuroscience Letters

Received Date:

13 September 2018

Revised Date:

20 September 2019

Accepted Date:

15 October 2019

Please cite this article as: Chaves VC, Soares MSP, Spohr L, Teixeira F, Vieira A, Constantino LS, Pizzol FD, Lencina CL, Spanevello RM, Freitas MP, Simo˜ es CMO, Reginatto FH, Stefanello FM, BLACKBERRY EXTRACT IMPROVES BEHAVIORAL AND NEUROCHEMICAL DYSFUNCTIONS IN A KETAMINE-INDUCED RAT MODEL OF MANIA, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134566

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BLACKBERRY EXTRACT IMPROVES BEHAVIORAL AND NEUROCHEMICAL DYSFUNCTIONS IN A KETAMINE-INDUCED RAT MODEL OF MANIA

Vitor C. Chavesa#, Mayara S. P. Soaresb#, Luiza Spohrb, Fernanda Teixeirab, Andriele Vieirad, Larissa S. Constantinoe, Felipe Dal Pizzold, Claiton L. Lencinac, Roselia M. Spanevellob, Matheus P. Freitasg, Cláudia M.O. Simõesa, Flávio H. Reginattoe, Francieli M. Stefanello*f a

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Laboratório de Virologia Aplicada, Programa de Pós-Graduação em Biotecnologia & Biociências Universidade Federal de Santa Catarina, SC, Brasil b Laboratório de Neuroquímica, Inflamação e Câncer. Programa de Pós-Graduação em Bioquímica Bioprospecção – Universidade Federal de Pelotas, RS, Brasil c Curso de Farmácia, Universidade Federal de Pelotas, RS, Brasil d Laboratório de Fisiopatologia Experimental, Programa de Pós-Graduação em Ciências da Saúde Universidade do Extremo Sul Catarinense, SC, Brasil e Programa de Pós-Graduação em Farmácia – Universidade Federal de Santa Catarina, SC, Brasil f Laboratório de Biomarcadores, Programa de Pós-Graduação em Bioquímica e Bioprospecção Universidade Federal de Pelotas, RS, Brasil g Departamento de Educação Física, Faculdade Anhanguera de Pelotas, RS, Brasil

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These authors equally contributed to this work * Author to whom correspondence should be addressed, e-mail: [email protected]

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HIGHLIGHTS

Blackberry extract prevents the maniac episodes in rat model of mania.

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Blackberry extract exhibits neuroprotection in rat model of mania.

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Blackberry extract has antioxidant and anti-inflammatory effects in rat model of

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mania.

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Abstract

Bipolar disorder is a chronic mood disorder characterized by episodes of mania and depression. The aim of this study was to investigate the effects of blackberry extract on behavioral parameters, oxidative stress and inflammatory markers in a ketamine-induced model of mania. Animals were pretreated with extract (200 mg/kg, once a day for 14 days), lithium chloride (45 mg/kg, twice a day for 14 days), or vehicle. Between the 8th and 14th days, the animals received an injection of ketamine (25 mg/kg) or vehicle. On the 15th day, thirty minutes after ketamine administration,

the animals’ locomotion was assessed using open-field apparatus. After the experiments, the animals were euthanized and cerebral structures were removed for neurochemical analyses. The results showed that ketamine treatment induced hyperlocomotion and oxidative damage in the cerebral cortex, hippocampus and striatum. In contrast, pretreatment with the extract or lithium was able to prevent hyperlocomotion and oxidative damage in the cerebral cortex, hippocampus, and striatum. In addition, IL-6 and IL-10 levels were increased by ketamine, while the extract prevented these effects in the cerebral cortex. Pretreatment with the extract was also effective in decreasing IL-6 and increasing the level of IL-10 in the striatum. In summary, our findings suggest that blackberry consumption could help prevent or reduce manic episodes, since this extract have demonstrated neuroprotective properties as well as antioxidant and anti-inflammatory effects in

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the ketamine-induced mania model.

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Keywords: Rubus sp.; Blackberry; Anthocyanins; Mania; Antioxidant; Anti-inflammatory.

1. Introduction

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Bipolar disorder (BD) is a mood disorder associated with cyclical episodes of mania and depression. Its estimated prevalence is about 1 to 3% in the population worldwide [1]. Patients with BD have increased pro-inflammatory cytokines such as TNF-α and IL-6, which is related to

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changes in cholinergic receptors and in the metabolism of monoamines [1-3]. Furthermore, the degradation of the neurotransmitters tryptophan and serotonin induced by these cytokines may cause a decrease in mitochondrial energy metabolism, free radical generation and lipid

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peroxidation leading to neurodegeneration [3-6]. Alterations in antioxidant enzymes are also found in BD [7]. Moreover, increased TNF-α and IL-6 as well as activation of the NMDA receptor through glutamate, may also increase nitric oxide production which may lead to nitrosative

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damage to the biomolecules [2-6]. Neuroimaging and neuropathological studies have shown that the neural circuits involving several brain structures are significantly decreased in volume in BD.

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Studies of neurocognitive function in this psychiatric disorder indicate deficits in attention, executive function, and emotional processing. It should be noted that these are important functions are performed by the cerebral cortex, hippocampus and striatum [8-11]. The drugs used for the treatment of manic episodes are mood stabilizers, such as lithium,

which presents several side effects, such that many BD patients do not respond positively to the treatment [1-3]. Therefore, the search for therapeutic alternatives for BD treatment remains relevant. In this context, the research and development of new drugs including those derived from natural products might be expanded significantly [12-13].

The consumption of berries, a huge group of functional foods, has demonstrated neuroprotective effects against psychiatric [12] and neurodegenerative disorders [14]. These effects have been related to specific phytochemicals, mainly phenolic compounds, such as phenolic acids, tannins, and anthocyanins [15]. In this context, blackberry fruits belong to the Rubus genus, classified in the Rosaceae family [16]. The chemical composition of its fruits is characterized by the presence of anthocyanins as major compounds, which are responsible for their typical color. Many studies have associated blackberries consumption with several pharmacological activities such as antioxidant and anti-inflammatory properties [17-18]. Considering the limited options for the prevention and treatment of BD, this study evaluates the neuroprotective effects of the blackberry methanolic extract (Rubus sp.) on oxidative

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stress and inflammatory markers in rats submitted to a model of manic-like behavior induced by ketamine.

2. Materials and methods 2.1 Animals

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Male Wistar rats (60 days old) were obtained from the Central Animal House of the

Federal University of Pelotas, Brazil. The animals were maintained on a 12/12 h light/dark cycle

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in an air-conditioned environment at constant temperature (22 ± 1ºC). The rats had free access to a commercial chow and water. The animals were divided in five experimental groups (n = 8-10) and the experiments were conducted in accordance with the guidelines of the Committee of Ethics

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and Animal Experimentation of the institution, under protocol number 4609.

2.2 Plant material and extraction

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Rubus sp. fruits, of the Tupy cultivar were cultivated and harvested at Empresa Brasileira de Pesquisa Agropecuária de Clima Temperado (EMBRAPA) in Pelotas, RS, Brazil (31°40’50.6”S; 52°26’23.1”W). The extract was prepared according to Chaves et al. [19]. Briefly,

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unprocessed frozen blackberry fruits were sonicated for 60 min at room temperature in methanol. Next, the crude extract was filtered, the methanol was evaporated under reduced pressure and the

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residue was lyophilized, yielding the extract to be tested. These procedures were performed in triplicate and protected from light.

2.3 Total phenolic and anthocyanin contents The total phenolic content was determined by the Folin-Ciocalteu assay [20]. A gallic acid standard curve was prepared with concentrations ranging from 60 to 300 µg mL-1 (r2 = 0.998). The readings were performed at 760 nm, and the results were expressed as milligrams of gallic acid equivalent per gram of fresh fruit (mg GAE g-1 FF) ± SD values. The anthocyanin content, was performed using the pH differential method [21]. The molar absorptivity coefficient (Ɛ) for

cyanidin-3-O-glucoside in a methanol solution used was 26600 L/M/cm. The results were expressed as mg of cyanidin-3-O-glucoside equivalents in 100 g of fresh fruits (mg CGE 100 g-1 FF) ± SD values. All analyses were performed in triplicate.

2.4 Anthocyanin identification Anthocyanins were individually identified by high performance liquid chromatography (Acquity-UPLCTM) coupled to a photodiode array detector (PDA) and a high-resolution mass spectrometer (Xevo® G2 QTof model – Waters®) equipped with an electrospray ionization source (ESI) operating in positive mode as previously described by Chaves et al. [22]. All samples were analyzed in triplicate. The methodology was validated, and the specificity, linearity, accuracy,

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precision, and detection and quantitation limits were measured.

2.5 Experimental protocol of mania state and treatment with blackberry extract

The animals were divided into six groups: saline, saline+blackberry extract,

saline+ketamine, ketamine+blackberry, ketamine+lithium chloride. Blackberry extract was

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administrated at 200 mg/kg (once a day) and lithium chloride 45 mg/kg (twice a day) both for 14 days by oral administration. From the 8th to the 14th days the animals also received saline or

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ketamine 25 mg/kg (once a day) by intraperitoneal injection. On the 15th day of treatment, the animals received a single injection of ketamine or saline, and the locomotor activity was assessed

2.6 Open-field test

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in the open-field apparatus after 30 min (Figure 1) [12-13].

The open-field apparatus consisted of a wooden box measuring 72 × 72 × 33 cm. The

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floor of the arena was divided into 16 equal squares (18 × 18 cm) and the box was placed in a sound free room. The animals were placed in the rear left square and left to explore the box freely for 5 min. The total number of squares crossed with all paws (crossing) was manually measured,

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to evaluate ambulatory behavior [12-13].

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2.7 Biochemical parameters

Rats were submitted to euthanasia immediately after the open-field test. The cerebral

cortex, hippocampus and striatum were dissected, and homogenized for the biochemical measurements.

2.7.1 Thiobarbituric acid reactive species (TBARS) levels Homogenates were mixed with trichloroacetic acid and thiobarbituric acid, and then heated in a boiling water bath for 25 min. The results were expressed as nmol of TBARS/mg protein [23].

2.7.2

Total sulfhydryl content Homogenates were added to PBS buffer pH 7.4 containing EDTA. The reaction was

started by adding of 5,5’-dithio-bis(2-nitrobenzoicacid). The results were reported as nmol TNB/mg protein [24].

2.7.3

Reactive oxygen species (ROS) measurements The oxidation of dichloro-dihydro-fluorescein diacetate (DCFH-DA) to fluorescent 2’,7’-

dichlorofluorescein (DCF) was measured to detect intracellular ROS. The results were expressed

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as µmol DCF/mg protein [25].

2.7.4 Superoxide dismutase (SOD) activity

This protocol was based on the inhibition of superoxide dependent adrenaline autooxidation to adenochrome. SOD activity was expressed as units/mg protein [26].

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2.7.5 Catalase (CAT) activity

The disappearance of H2O2 was monitored for 180s. CAT activity was expressed as

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units/mg protein [27]. Glutathione peroxidase (GPx) activity

GPx activity was measured using a commercial kit (RANSEL®; Randox Lab, Antrim,

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UK). GPx activity was expressed as units/mg protein.

Acetylcholinesterase (AChE) activity

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System DTNB, phosphate buffer (pH 7.5) and homogenate were incubated. Next, acetylthiocholine were added. AChE activity was expressed as μmolAcSCh/h/mg protein [28].

Nitrite levels

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2.7.8

Sulfanilamide in 5% phosphoric acid was added to supernatants and incubated for 10 min.

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Subsequently, samples were mixed with Griess reagent and incubated for 10 min. Nitrite levels were expressed as μmol of nitrite/mg protein [29].

2.7.9

Cytokine analysis

Levels of IL-6 and IL-10 were measured using immunoassay kits (DuoSet ELISA Development, R&D Systems, Inc., USA) according to the manufacturer’s instructions.

2.8 Statistical analysis

Statistical analyses were performed with Stata 12.0. The normality of the variables was tested by the Shapiro-Wilk test and Bartlett’s test to verify the homogeneity of variances. The 2x5 two-way ANOVA was used to evaluate the interaction between the "group" (saline; ketamine) and the "treatment" (saline; blackberry; ketamine; ketamine+blackberry; ketamine+lithium chloride) with the different outcomes studied. For non-parametrical data, Tukey's "ladder of powers" method was used to discern the necessary transformation for normality, then the variable was normalized. As there were no significant interactions, the difference between the outcomes was evaluated through one-way ANOVA, followed by Bonferroni’s post-hoc and the KruskalWallis test, with the Dunn’s post-hoc test for non-parametric data. The variables were expressed

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as mean ± SEM and the results were considered significant at p<0.05.

3. Results Chemical characterization

The total phenolic content of the methanolic extract of Rubus sp. fruit was111.53± 0.62 mg GAE/g of dried extract. Total anthocyanin content was 10.56 ± 0.29 mg CGE/g of dried

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extract.

The LC/DAD/MS of blackberry extract was also performed. The chromatographic

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analyses were carried out on a reverse phase column, which provided an efficient separation for the identification of detected anthocyanin compounds. The methodology was validated according to the ICH guidelines [30] using cyanidin-3-O-glucoside as analytical standard. The parameters

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evaluated were accuracy, precision, specificity, linearity, and limits of detection and quantification (data not shown). Since the anthocyanins exhibited a characteristic absorption band at wavelengths ranging from 500 to 550 nm in the visible region of the electromagnetic spectra,the

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identification of anthocyanins was previously performed by the PDA detector [31]. The anthocyanins identified were cyanidin-O-galactoside, as the major anthocyanin component, plus

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cyanidin-O-rutinoside, pelargonidin-O-glucoside and cyanidin-O-malonylhexoside (Table 1).

Table 1 - Anthocyanins identified in the blackberry methanolic extract.

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Anthocyanin

Cyanidin-O-galactoside Cyanidin-O-glucoside Cyanidin-O-rutinoside Pelargonidin-O-glucoside Cyanidin-O-malonylhexoside

Behavioral evaluation

Retentionti ʎmax me (min) (nm) 7.70 8.49 9.14 10.29 12.87

515 515 517 500 518

Molecular formula

[M]+m/z (Error - ppm)

C21H21O11+ C21H21O11+ C27H31O15+ C21H21O10+ C24H23O14+

449.1065 (-4.2) 449.1065 (-4.2) 595.1705 (7.1) 433.1130 (1.2) 535.1062 (-4.9)

Main fragments m/z 287 287 449 271 449; 287

Ketamine increased animal locomotion, which was observed by an increase in the number of crossing in the open-field test (Figure 2). This was expected and is reported in the literature as a feature of hyperlocomotion as commonly found in BD patients [32]. Furthermore, pretreatment with lithium or the blackberry extract prevented this behavior [F(4-37)=9.75, p<0.001].

Neurochemical parameters in cerebral cortex Ketamine administration decreased SH concentration, and pretreatment with lithium or blackberry extract prevented this effect [F(4-18)=9.28, p<0.001]. ROS levels were increased by ketamine, while pretreatment with lithium or blackberry extract was able to prevent this alteration [H(2)=10.900, p<0.05]. Additionally, TBARS levels were increased by ketamine and ketamine

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plus blackberry when compared to the saline group. Meanwhile, pretreatment with lithium prevented this increase [F(4-17)=10.06, p<0.001]. No changes were observed in nitrite content (p>0.05) (Figure 3).

Both SOD and CAT activities were decreased by ketamine treatment when compared to the saline group [F(4-17)= 4.02, p<0.05]; [F(4-17)=5.51, p<0.01]. No changes were observed in

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the GPx activity (p>0.05).

Finally, AChE activity was increased by ketamine administration and ketamine plus

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lithium.

Neurochemical parameters in the hippocampus

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The results of the oxidative stress markers evaluation in the rats’ hippocampus are shown in Figure 4. ROS [H(2)=13.668, p<0.01] and TBARS [F(4-17)=14.85, p<0.001] levels were significantly increased by ketamine administration, and the pretreatment with lithium or

levels (p>0.05).

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blackberry extract were able to prevent these effects. No changes were found in SH and nitrite

Ketamine treatment decreased SOD activity [F(4-17)=5.74, p<0.01] and blackberry

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extract treatment was able to prevent this change. CAT activity was reduced by ketamine administration, and lithium and blackberry treatments restored the enzyme activity [F(4-16)=4.61,

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p<0.05]. As regards GPx, ketamine plus blackberry increased this enzyme activity when compared to blackberry, ketamine and ketamine+lithium groups [H(2)=9.844, p<0.05]. No changes were observed in AChE activity (p>0.05).

Neurochemical parameters in striatum Ketamine increased ROS [H(2)=9.988, p<0.05], nitrite [H(2)=9.937, p<0.05] and TBARS [F(4-16)=12.88, p<0.001] levels, while pretreatments with the lithium and blackberry extract prevented these alterations. No changes were found in SH levels (p>0.05) (Figure 5).

No changes were observed in SOD and CAT (p>0.05) activities, while GPx activity was decreased only after ketamine treatment [F(4-17)=4.68, p<0.01]. Finally, AChE activity was increased by ketamine administration and the pretreatment with lithium prevented this increase [H(2)=15.309, p<0.01].

Cytokine analysis IL-6 was increased in the cerebral cortex [F(4-16)= 8.087, p<0.001] in ketamine group, while pretreatments with lithium or blackberry extract prevented this change. This cytokine was increased in striatum [H(2)=11.655, p<0.05] in the ketamine+lithium group compared to the saline group. However, ketamine+blackberry reduced IL-6 levels when compared to other

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ketamine groups. No changes were observed in the hippocampus (p>0.05) (Figure 6). In the cerebral cortex, IL-10 levels were increased in the blackberry and ketamine groups, while pretreatment with lithium or blackberry extract reduced IL-10 levels [H(2)=16.35, p<0.01]. In the striatum, IL-10 was enhanced in the ketamine+blackberry group compared to the saline

group [F(4-22)=6.57, p<0.01]. In contrast, these cytokine levels were higher in the blackberry

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group when compared to the ketamine group [H(2)=20.577, p<0.001]. In addition, in the ketamine+blackberry group, these levels were decreased in comparison to the other ketamine

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groups and the saline group (Figure 6).

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4. Discussion The chemical profile and preventive effects of the blackberry methanolic extract on behavioral and neurochemical dysfunctions in rats submitted to a ketamine-induced model of mania were evaluated.

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Concerning the chemical composition, phenolic compounds are the main set of secondary metabolites present in blackberry fruits. Anthocyanins belong to the most abundant subgroup of phenolics and are responsible for the antioxidant potential of these fruits [12,15,33]. The high-

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resolution mass spectrometry of the bioactive compounds presents in blackberry methanolic extract allowed the identification of five anthocyanins, four cyanidin aglycone derivatives and one from pelargonidin derivative. All these compounds have been previously described for these

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fruits [12,34-35].

In this work the effects of a blackberry extract were determined in an experimental model

of manic-like behavior induced by ketamine in rats [12-13]. Ketamine is an anesthetic drug that presents different actions in sub doses on mood and behavior. At lower doses it has antidepressant effect and at higher doses it induces hyperlocomotion and a manic-like behavior [12-13]. Moreover, ketamine acts as a potent antagonist of N-methyl-aspartate (NMDA) glutamate receptors, which are commonly found in brain areas involved in behavioral functions. Although ketamine induces behavioral and biochemical alterations, such as hyperlocomotion, oxidative

stress, and changes in levels of brain-derived neurotrophic factor similar to those found in bipolar disorder [12-13,36], we should acknowledge that a limitation of this model is its lack of specificity for mania since ketamine-induced hyperactivity is also observed in other animal model for psychiatric diseases such as schizophrenia [37]. The primary production of superoxide anion (O2-) and secondary free radical reactions in the mitochondria have been indicated as causes for several CNS disorders [37]. Moreover, Kunz et al. [38] have shown an increase in serum TBARS in manic bipolar patients, as well as an increased in oxidative stress in the peripheral tissues and brains in these patients was also observed. [38]. Our results showed that ketamine treatment induced hyperlocomotion and oxidative brain damage, since alterations in some oxidative stress markers were observed, such

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as an increase in TBARS and ROS levels in the cerebral cortex, hippocampus and striatum of rats. Moreover, ketamine administration decreased the total thiol content in the cerebral cortex, as well as the antioxidant enzyme activity in the cerebral structures. The reduction of SOD and CAT

activities is also an indicative of oxidative damage, as it can lead to an increase of O2˙-and hydrogen peroxide (H2O2) levels, which may generate hydroxyl radicals unleashing lipid

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peroxidation [39].

Pretreatment with lithium or blackberry extract prevented hyperlocomotion as well as

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decreased oxidative damage. These promising properties could be explained by the presence of anthocyanins in the tested extract. Anthocyanins have a high antioxidant effect when compared to other conventional antioxidants such as α-tocopherol and catechin. These effects derive from

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the chemical structure of the anthocyanin molecule and, especially, the number of hydroxyl groups, the catechol moiety in the B ring, and the oxonium ion in the C ring [12-13,15,33]. These findings are in agreement with other studies that use the same experimental model testing different

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samples to prevent manic episodes, such as a blueberry extract, and other medicinal plant extracts rich in phenolic compounds [12-13].

Since several studies have demonstrated the involvement of different neurotransmitter

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pathways in mood disorders [40-41], the role of the cholinergic route in the mania model mediated by AChE activity was also evaluated. Our results showed an increase in AChE activity in the

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cerebral cortex and striatum of the animals submitted to the mania model. On the other hand, treatment with lithium and blackberry extract were effective in reducing enzyme activity in the striatum. It is important to highlight that in another study using the cell line HEK293T, lithium treatment demonstrated a rapid proteasomal degradation of overexpressed AChE-S [40]. In contrast, in a model of schizophrenia mediated by ketamine, using the same protocol, the AChE activity did not show any differences in the cortex, hippocampus or striatum [41]. Our results suggest that the inhibition of AChE activity detected herein could represent advantages for its use in the treatment of mood disorders [40-41].

Inflammation is another important component in psychiatric disturbances, since an increase in plasma levels of cytokines and inflammatory mediators were described in models of depression and mood disorders [2-3]. Moreover, since the relationship between inflammation and oxidative stress is close, the relationship between ROS production and stimulation of synthesis and release of IL-6 has also been described [2-3]. In the same way, an increase in proinflammatory cytokines levels, such as IL-6, was found in different phases of BD and in manic episodes [3-42]. In this work, an increase in IL-6 levels was detected in the cerebral cortex after administration of ketamine. In contrast, the pretreatment with lithium or blackberry extract prevented these alterations. These results are in agreement with those of previous studies, which found highest levels of IL-6 in manic episodes [3,12-13,42]. Furthermore, ROS may be

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responsible for stimulating the synthesis of IL-6 since this association can occur in physiological and pathological conditions [42]. As previously described, the increase of IL-10 in the cerebral cortex was observed in animals that received ketamine [43]. In our study, pretreatment with the blackberry extract was effective in decreasing IL-6 and enhanced IL-10 levels in the striatum,

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suggesting an anti-inflammatory effect of blackberry on this cerebral structure.

Conclusion

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Our findings suggest that blackberry consumption may help the prevention or reduce manic episodes, since this extract showed neuroprotective properties as well as antioxidant and

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anti-inflammatory effects in the ketamine-induced mania model.

Figures

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Fig 1 Treatment protocol.

Vehicle Lithium (45 mg/kg) Blackberry (200 mg/kg)

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Number of crossings

Fig 2 Effect of blackberry methanolic extract (200 mg/kg, p.o.) and lithium chloride (45 mg/kg, p.o.) pretreatment on hyperlocomotion in rats submitted to ketamine-induced manic-like. Data are reported as mean±S.E.M. #p<0.05compared to vehicle/saline group. *p<0.05 compared to vehicle/ketamine group (n=8-10).

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Ketamine

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Fig 3 Effect of blackberry methanolic extract (200 mg/kg, p.o.) and lithium chloride (45 mg/kg, p.o.) pretreatment on TBARS (A), total thiol content (SH) (B), ROS (C) and nitrite (D) levels; superoxide dismutase (SOD) (E), catalase (CAT) (F), glutathione peroxidase (GPx) (G) and acetylcholinesterase (AChE) (H) activities in cerebral cortex of rats submitted to ketamineinduced manic-like. Data are reported as mean±S.E.M. #p<0.05 compared to vehicle/saline group. * p<0.05 compared to vehicle/ketamine group (n=4–5).

Cerebral cortex A SH (nmol TNB/mg protein)

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Fig 4 Effect of blackberry methanolic extract (200 mg/kg, p.o.) and lithium chloride (45 mg/kg, p.o.) pretreatment on TBARS (A), total thiol content (SH) (B), ROS (C) and nitrite (D) levels; superoxide dismutase (SOD) (E), catalase (CAT) (F), glutathione peroxidase (GPx) (G) and acetylcholinesterase (AChE) (H) activities in hippocampus of rats submitted to ketamine-induced manic-like. Data are reported as mean±S.E.M. #p<0.05 compared to vehicle/saline group. *p<0.05 compared to vehicle/ketamine group (n=4–5).

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Fig 5 Effect of blackberry methanolic extract (200 mg/kg, p.o.) and lithium chloride (45 mg/kg, p.o.) pretreatment on TBARS (A), total thiol content (SH) (B), ROS (C) and nitrite (D) levels; superoxide dismutase (SOD) (E), catalase (CAT) (F), glutathione peroxidase (GPx) (G) and acetylcholinesterase (AChE) (H) activities in striatum of rats submitted to ketamine-induced manic-like. Data are reported as mean±S.E.M. #p<0.05 compared to vehicle/saline group. *p<0.05 compared to vehicle/ketamine group (n=4–5).

Striatum #

4

*

*

*

2 1 0

300

#

*

*

100

0

50

Ketamine

D #

40 30 20

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10 0

Ketamine

*

Ketamine

-p

Saline

E

CAT (Units/mg of protein)

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F

3

re

40

20

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SOD (Units/mg of protein)

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#

Saline

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60

#

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40 20

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Vehicle

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Ketamine

Lithium (45 mg/kg)

#

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GPx (Units/mg of protein)

20

C

*

80

40

Ketamine

200

60

B

60

Nitrite (M nitrite/mg of protein)

ROS (mol DCF/mg of protein)

Saline

80

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3

SH (nmol TNB/mg protein)

A

AChE (mol AcScH/h/mg of protein)

TBARS (nmol/mg of protein)

5

Saline

10

Ketamine

H #

8

#

6

*

4

#

*

2 0

Saline

Ketamine

Blackberry (200 mg/kg)

Fig 6 Effect of blackberry methanolic extract (200 mg/kg, p.o.) and lithium chloride (45 mg/kg, p.o.) pretreatment in brain evels of IL-6 and IL-10 of animals submitted to ketamine-induced manic-like behavior. Data are reported as mean±S.E.M. #p<0.05 compared to vehicle/saline group. *p<0.05 compared to vehicle/ketamine group (n = 4–8).

Cerebral Cortex IL 6

IL 10 4

#

0.8 0.6

*

0.4

#

*

IL 10 levels (pg/mg of protein)

IL 6 levels (pg/mg of protein)

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0.2 0.0

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# 2

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1

* 0

Saline

Ketamine

Saline

Ketamine

IL 6

IL 10

20

IL 10 levels (pg/mg of protein)

2.0 1.5 1.0 0.5

15 10

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*

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#*

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IL 6 levels (pgmg of protein)

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Hippocampus

0.0

0

Ketamine

Saline

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Saline

Ketamine

IL 6

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ur

Saline

Ketamine

Lithium (45 mg/kg)

*

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Ketamine

Blackberry (200 mg/kg)

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Vehicle

*

IL 10 levels (pg/mg of protein)

1.0

IL 10

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#

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IL 6 levels (pg/mg of protein)

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Striatum

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V.C.C. was responsible by preparation of Rubus sp. extract, the chemical analysis as well as part of in vivo experiments. V.C.C. and M.S.P. was also responsible by the article drafting. M.S.P., L.S., and F.T., was involved in the in vivo experiments as well as acquisition, and interpretation of in vitro biochemical assays. A.V. and L.C. were involved in cytokine analysis. C.L., R.S., and F.S. was involved in concept and design in vivo experiments, interpretation of data and critical revision of the article. M.P.F was responsible by statistical analysis. F.R. was involved in concept and design of all experiments, drafting and critical revision of the manuscript. R.S., F.S., F.D-P., and F.R. were responsible from the grants supports of this research.