Doxycycline prevents and reverses schizophrenic-like behaviors induced by ketamine in mice via modulation of oxidative, nitrergic and cholinergic pathways

Accepted Manuscript Title: Doxycycline prevents and reverses schizophrenic-like behaviors induced by ketamine in mice via modulation of oxidative, nitrergic and cholinergic pathways Authors: Benneth Ben-Azu, Itivere Adrian Omogbiya, Adegbuyi Oladele Aderibigbe, Solomon Umukoro, Abayomi Mayowa Ajayi, Ezekiel O. Iwalewa PII: DOI: Reference:

S0361-9230(17)30602-0 https://doi.org/10.1016/j.brainresbull.2018.02.007 BRB 9374

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

14-10-2017 21-1-2018 2-2-2018

Please cite this article as: Benneth Ben-Azu, Itivere Adrian Omogbiya, Adegbuyi Oladele Aderibigbe, Solomon Umukoro, Abayomi Mayowa Ajayi, Ezekiel O.Iwalewa, Doxycycline prevents and reverses schizophrenic-like behaviors induced by ketamine in mice via modulation of oxidative, nitrergic and cholinergic pathways, Brain Research Bulletin https://doi.org/10.1016/j.brainresbull.2018.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Doxycycline prevents and reverses schizophrenic-like behaviors induced by ketamine in mice via modulation of oxidative, nitrergic and cholinergic pathways Benneth Ben-Azu a,*, Itivere Adrian Omogbiya a,b, Adegbuyi Oladele Aderibigbe a, Solomon Umukoro a, Abayomi Mayowa Ajayi a and Ezekiel O. Iwalewa a Neuropharmacology Unit, Department of Pharmacology and Therapeutics, College of

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Medicine, University of Ibadan, Ibadan, PMB 5017, Oyo State, Nigeria; b

Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, Delta State University, Abraka, PMB 1, Delta State, Nigeria.

*Corresponding Author

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Neuropharmacology Unit, Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria

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Email address: [email protected]; Tel. Number: +2348030881152

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Graphical abstract

Fig. 1. Treatment protocols: VEH = Vehicle, KET = ketamine, DOX = Doxycycline, RIS = Risperidone

Highlights Doxycycline (DOX) prevented and reversed ketamine (KET)-induced hyperlocomotion

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DOX prevented and reversed ketamine-induced social interaction impairment

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DOX prevented and reversed ketamine-induced cognitive impairment

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DOX ameliorated ketamine-induced neurochemical alterations in mice brains

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DOX enhanced the antipsychotic effects of risperidone in KET-induced schizophrenia

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Abstract

The involvement of oxidative/nitrosative stress, cholinergic and inflammatory pathways have

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been reported to contribute to the pathophysiology of schizophrenia, a debilitating neuropsychiatric disorder. Our previous studies have shown that doxycycline (DOX), a notable

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member of tetracyclines with proven antioxidant and anti-inflammatory properties, attenuated

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psychotic-like behaviors induced by apomophine and ketamine (KET) in mice. This present study was designed to further evaluate in detail the ability of DOX and its combination with

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risperidone (RIS) to prevent and reverse KET-induced schizophrenic-like behaviors and the role of oxidative/nitrosative and cholinergic pathways in mice. In the prevention protocol, mice were treated orally with DOX (25, 50 or 100 mg/kg), RIS (0.5 mg/kg), DOX (50 mg/kg) in

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combination with RIS, or vehicle for 14 consecutive days. In addition, the animals received intraperitoneal injection of KET (20 mg/kg/day) from the 8th to the 14th day. In the reversal

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protocol, the animals received KET or vehicle for 14 days prior to DOX, RIS, DOX incombination with RIS or vehicle treatments. Schizophrenic-like behaviors consisting of positive, negative and cognitive symptoms were evaluated using open field, social interaction, Y-maze and novel object recognition tests. Thereafter, the brain levels of biomarkers of oxidative stress, nitrite and acetylcholinesterase activity were determined. DOX given alone or in combination with RIS attenuated schizophrenic-like behaviors induced by chronic injection

of KET in both preventive and reversal treatment protocols. DOX significantly increased glutathione, superoxide dismutase and catalase levels in the brain of chronic KET-treated mice. However, it decreased malonyladehyde, nitrite levels and acetylcholinesterase activity when given alone or in-combination with RIS in both protocols. Taken together, these findings showed that doxycycline ameliorated schizophrenic-like behaviors induced by ketamine in both

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preventive and reversal treatment protocols in mice via inhibition of oxidative stress, nitrergic stress and acetylcholinesterase activity. Our data further suggests that doxycycline supplementation may augment the therapeutic efficacy of resperidone particularly for the treatment of negative and cognitive symptoms associated with schizophrenia.

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Keywords: schizophrenia; antipsychotics; oxidative stress; antioxidant; antibiotics

1. Introduction

Schizophrenia is a chronic debilitating neuropsychiatric disease that is characterized by positive (hallucinations, delusions), negative (social withdrawal, depression) and cognitive (executive and working memory deficits) symptoms (Lewis and Lieberman, 2000; Monte et al., 2013). Schizophreni is a complex disease of multiple pathologies including dysfunction in central dopaminergic, serotonergic, glutaminergic and cholinergic pathways (Chatterjee et al., 2012a).

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However, converging lines of evidence have implicated increased oxidative and nitrosative stress in the pathogenesis of schizophrenia (Ng et al., 2008; Zhang et al., 2010). Moreover, increased reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as altered antioxidant molecules in the brain of schizophrenic patients have been reported; which further support the role of oxidative and nitrosative stress in the pathology of the disease (Zhang et al., 2010).

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Oxidative and nitrosative stress may be a common pathogenic mechanisms underlying the

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pathophysiology of the disease; as the brain has greater susceptibility factors (including low antioxidant defense system, comparatively high oxygen utilization and high levels of readily

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oxidizable membrane polyunsaturated fatty acid, as well as redox regulated activity at dopamine

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and glutamate receptors) (Ng et al., 2008; Bókkon and Antal, 2011). Thus, it has been suggested that these culprits might be targets for the treatments of schizophrenia especially negative and

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cognitive symptoms associated with the disease (Monte et al., 2013). This suggestion is based on

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the fact that depression and memory deteriorations, which represent the negative and cognitive symptoms associated with schizophrenia, are known to be closely connected with increased oxidative stress (Meltzer, 2010). Moreover, studies have also shown that ketamine (KET)

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induces cognitive impairment through the induction of acetylcholinesterase (AChE) enzyme that leads to decreased brain concentrations of acetylcholine (Ach), a neurotransmitter relevant in

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memory (Chatterjee et al., 2012a, 2012b). However, the positive symptoms produced by chronic KET administration is due to antagonism of NMDA receptors located on gamma aminobutyric

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acid (GABA) interneurons, resulting in decreased firing of GABAergic inhibitory neurons (disinhibition); thus, increasing the release of dopamine in the limbic circuits (Moghaddam et al., 1997; Monte et al., 2013). The current available antipsychotic drugs have several limitations thus necessitating the search for newer agents that could be used for the treatment of patients with schizophrenic disorders (Melzer, 2010). Doxycycline (DOX), a prominent member of tetracycline antibiotic has been

reported to exhibit antioxidant, neuroprotective, anti-neuroinflammatory and antidepressant properties (Leite et al., 2011; Mello et al., 2013) Moreover, Nogueira et al. (2011) have demonstrated the anticonvulsant effect of DOX in pilocarpine-induced convulsion through decrease in glutamatergic transmission, increase in GABAergic neurotransmission, as well as antioxidant mechanisms. However, in our recent study, we have reported that DOX attenuated

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psychotic-like behaviors induced by apomorphine and KET injection in mice (Ben-Azu et al., 2016a). This present study was designed to further evaluate the ability of DOX and its combination with risperidone (RIS) to prevent and reverse ketamine-induced schizophrenic-like behaviors and the role of oxidative/nitrosative and cholinergic pathways in mice.

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2. Materials and Methods

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2.1. Laboratory Animals

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Male Swiss mice (20–25 g; 6 weeks old) were obtained from the Central Animal House, Delta

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State University, Abraka, Nigeria. The animals were housed in a well-ventilated animal house: five per plastic cage (42 × 30 × 27 cm) at a room temperature (25 ± 1 °C) and relative humidity of 60 ± 5% with a 12-h light/dark cycle. They were fed with standard rodent pellet food and

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water ad libitum throughout the experimental period. They were acclimatized for at least 1 week

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prior to commencement of the experiments. The experiments were performed according to the National institutes of Health Guide for Care and Use of Laboratory Animals (Publication No.

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85–23, revised 1985). Also, efforts were made to minimize the suffering of the animals by

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careful handling, treatments, behavioral tests of the animals, and euthanization.

2.2. Drugs and Chemicals

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Doxycycline (Hovid Pharmaceutical industry, Perak, Malaysia), ketamine hydrochloride (SigmaAldrich, St. Louis, MO, USA) and risperidone (Sigma-Aldrich, St. Louis, MO, USA), trichloroacetic acid (TCA) (Burgoyne Burbidges & Co., Mumbai, India), thiobarbituric acid (TBA) (Guanghua Chemical Factory Co. Ltd., Guangzhou, China), Ellman Reagent [5′,5′Dithiobis-(2-nitrobenzoate) DTNB] (Sigma-Aldrich, St. Louis, MO, USA), hydrogen peroxide

(H2O2) (BDH Chemicals Ltd., Poole, UK), adrenaline (Sigma-Aldrich, St. Louis, MO, USA) were used in this study. 2.3. Drug Preparation DOX and RIS were dissolved in distilled water immediately before use and administered orally

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(p.o.). KET was diluted with distilled water in appropriate concentrations and administered intraperitoneally (i.p.). The doses of DOX (25, 50 and 100 mg/kg) used in this study were selected based on the results from previous investigation (Ben-Azu et al., 2016a); while the doses of KET (20 mg/kg) and RIS

(0.5 mg/kg) were selected based on results from previous

investigation [2]. Vehicle (distilled water) was administered in a volume of 10 mL/kg per body

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weight.

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2.4. Experimental Protocol

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Schizophrenia-like behaviors and biochemical alterations were induced using the method

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previously described (Monte et al., 2013) with slight modifications. Briefly, in the prevention paradigm, animals were randomly grouped into 7 treatment groups (n = 7). Groups 1 and 2 were

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pretreated with VEH (10 mL/kg, p.o.), groups 3–5 received DOX (25, 50 and 100 mg/kg, p.o.), group 6 received RIS (0.5 mg/kg, p.o.), while group 7 received both DOX (50 mg/kg, p.o.) plus

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RIS (0.5 mg/kg, p.o.) for 14 days. Between the 8th and 14th day, these animals received a daily dose of KET (20 mg/kg, i.p.) or vehicle 30 min after DOX or RIS administration. In the reversal

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model, animals were grouped into 7 groups (n = 7). Groups 1–7 received KET (20 mg/kg) or vehicle (10 mL/kg) once daily for 14 days. From the 8th day of treatment onwards to the 14th

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day, group 2 was treated with vehicle (10 mL/kg, p.o.), groups 3–5 were treated with DOX (25, 50 and100 mg/kg, p.o.), group 6 received RIS (0.5 mg/kg, p.o.), and group 7 received both DOX

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(50 mg/kg, p.o.) and RIS (0.5 mg/kg, p.o.) once daily with a 30 min interval between treatments (Fig. 1).

2.5. Behavioral Studies

The behavioral studies were performed 24 h after the administration of DOX and KET respectively. Each animal was evaluated for schizophrenia-like behavioral phenotypes i.e., (a) hyperlocomotor activity (representing positive symptoms); (b) Social interaction test (representing negative symptoms); and (c) Y-maze and novel object recognition tests (representing spatial and non-spatial working memory, cognitive symptoms). All behavioral tests

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were carried out between 8:30 a.m. and 1:00 p.m.

2.5.1. Open-field test

Locomotor behavior was monitored using the open field test. The open-field test (OFT) apparatus consists of a wooden box measuring 35 × 30 × 23 cm with visible lines drawn to

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divide the floor into 36 (20 cm × 20 cm) squares with a frontal glass wall, and placed in a sound

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free room. The animals were placed in the rear left square and left to explore it. The observed

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parameters include number of line crossed for 5 min using a stopwatch (Omogbiya et al., 2013).

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2.5.2. Social Interaction Test

The testing apparatus consisted of a 60 × 40 cm Plexiglas box divided into three chambers (A, B

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and C). Mice moved between chambers through a small opening (6 × 6 cm) in the dividers. An iron restraining cage was placed in each of the two side chambers (A and C), with chamber A

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containing the probe mice. Test (experimental) mouse was placed in the center chamber (chamber B) and allowed 5 min of exploration time in all chambers. At the end of the 5 min

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exploration time, an unfamiliar, same-sex probe mouse from the same experimental group was placed in one of two restraining cages in chamber A, while chamber C was without mice

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(Radyushkin et al., 2009). Thereafter, the test mouse was placed back into chamber B and allowed to explore between chamber A (containing probe mouse) and chamber C (without

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mouse) in the social test box for another 5 min. The time spent in chamber A and C was measured with different stopwatch, and social preference was defined as follows: (% time spent in the social chamber) − (% time spent in the opposite chamber). 2.5.3. Y-Maze Test The effect of the DOX on spontaneous alternation performance was assessed using Y-maze test (YMT), which allows the evaluation of cognitive searching behavior, as an index of spatial

working memory dysfunction of schizophrenia. Animals were gently placed individually in the Y-maze apparatus, which consisted of three identical arms (33 × 11 × 12 cm each) in which the arms are symmetrically separated at 120°. Each mouse was placed at the end of arm A, and allowed to explore all the three arms (labeled A, B, C) freely for 5 min, taking the following parameters: the number of arm visits and sequence (alternation) of arm visits visually.

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Alternation behavior was defined as consecutive entries into all the three arms (i.e., ABC, CAB or BCA but not ABA, BAB or CAC). The percentage alternation, which is an index of spatial working memory, was calculated by dividing the total number of alternations by the total number of arm entries, minus two and multiplied by 100 (Casadesus et al., 2006). After each mouse session, the observation chamber was cleaned with 70% ethanol to remove residual odor.

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2.5.4. Novel Object Recognition Test

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The effect of DOX on preventive and reversal effects of ketamine-induced cognitive impairment, as a measure of non-spatial cognitive dysfunction associated with schizophrenia was also

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evaluated by the method described by Zhu et al. (2014) on the 14th and 15th days using the

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novel object recognition test (NORT). The NORT test consists of two sessions; the training session (day 14) and test session (day 15). NORT was conducted in an observation box (43 ×

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2643 × 2630 × 5 cm) with discriminated objects (A, B and C) identically sized (5 cm diameter

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and 12 cm height) plastic cylinder bottles. Objects A and B were red, whereas object C had a red and green pattern. A preceding 5 min habituation was done 24 h after treatment on the 14th day to reduce the contribution of anxiety and stress on the outcome. The training session was carried

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out by placing each mouse in the middle of the two identical objects (A and B) on the opposite sides, symmetrically fixed to the floor of the box with a distance of 10 cm from the walls and 35

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cm from each other. Each animal was allowed to explore in the box for 5 min. An animal was considered to be exploring the object when its head was facing the object (the distance between

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the head and object is an approximately 1 cm or less) or was touching or sniffing the object. After the training, the mouse was immediately returned to their home cages, and the box and objects was cleaned with 70% ethanol to avoid any possible instinctive odorant cues. Retention tests were carried out on the next day of the training (24 h after the training session on the 15th day). During the retention test, each mouse was placed back into the same box, with one of the objects used during training replaced with a novel object (object C). Each mouse was then

allowed to freely explore for 5 min, and the time spent exploring each object (object A and C) was recorded by the observer who was blind to the experiment design. Exploratory preference, the ratio of the amount of time spent exploring any one of the two objects A and C during the retention test session over the total time spent exploring both objects multiplied by one hundred,

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was used to measure the memory performance. 2.6. Biochemical Assays

3.1. Preparation of whole Brain Tissues homomgenates for Biochemical Assay

Immediately after the behavioral tests, the animals were decapitated under ether anesthesia and the brains were immediately removed. Thereafter, the whole brain was weighed and

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homogenized with 5 mL of 10% w/v phosphate buffer (0.1 M, pH 7.4). Each brain tissue homogenates were centrifuged (Anke TGL-16G, Nanjing, China) at 5400 g for 10 min at 4 °C,

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the pellet was discarded and the supernatant was immediately separated into various portions for

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the different biochemical assays.

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2.6.1. Determination of Superoxide Dismutase Activity

The level of superoxide dismutase (SOD) activity was measured by the method previously

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described (Misra and Fridovich, 1972). This method is based on the inhibition of superoxide

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dependent adrenaline auto-oxidation in a spectrophotometer adjusted at 480 nm. Brain supernatant of 1 mL was diluted in 9 mL of distilled water to make a 1 in 10 dilution. An aliquot

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of 0.2 mL of the diluted sample was added to 2.5 mL of 0.05 M carbonate buffer (pH 10.2) to equilibrate in the spectrophotometer and the reaction was started by the addition of 0.3 mL of

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freshly prepared 0.3 mM adrenaline to the mixture which was quickly mixed by inversion. The reference cuvette (Blank) contained 2.5 mL buffer, 0.3 mL of substrate (adrenaline) and 0.2 mL of distilled water. The increase in absorbance at 480 nm was monitored for 30 s for 150 s. The

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amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline was considered1 unit of SOD activity. 2.6.2. Determination of Catalase Activity Catalase (CAT) activity was assayed by the method previously described (Sinha, 1971), which was based on the disappearance of hydrogen peroxide (H2O2) in the presence of an enzyme

source (catalase). Brain supernatant of the sample homogenate (1 mL) was mixed with 19 mL of distilled water to give a 1:20 dilution. Then, 1 mL of this was added to 5 mL of phosphate buffer (pH 7.0) and 4 mL of H2O2 solution (800 µmoles). The reaction mixture was mixed by a gentle swirling motion at room temperature. Then, 1 mL of this portion of the reaction mixture was withdrawn and added into 2 mL dichromate/acetic acid reagent. The absorbance was measured

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using spectrophotometer at 570 nm and change in absorbance at 60 s interval. The catalase activity was expressed as µmoles of H2O2 decomposed per min per mg protein. 2.6.3. Determination of Glutathione Concentration

Glutathione (GSH) concentration was assayed by the method previously described (Jollow et al., 1974), which was based upon the development of a relatively stable (yellow) color when 5′,5′-

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dithio-bis-(2-nitrobenzoic acid) (DTNB) is added to sulfhydryl compounds. Brain homogenates

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of 0.4 mL were added to 0.4 mL of 20% trichloroacetic acid (TCA) and mixed by a gentle swirling motion and then centrifuged in a cold (4 °C) centrifuge at 5400 g for 20 min. Then, 0.25

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mL of the supernatant was added to 2 mL of 0.6 mM DTNB and the final volume of the solution

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was made up to 3 mL with phosphate buffer (0.2 M, pH 8.0). Absorbance was read at 412 nm against blank reagent (2 mL of 0.6 mM DTNB + 1 mL phosphate buffer (0.2 M, pH 8.0)) using a

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spectrophotometer. The concentration of reduced GSH in the brain tissues were expressed as

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nanomoles per gram tissue (nmol/mg protein). 2.6.4. Estimation of Brain Level of Malondialdehyde

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The brain level of malondialdehyde (MDA) was measured according to the method previously described (Okhawa et al., 1979). This assay principle is based on the fact that lipid peroxidation

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generates unstable lipid peroxides that decompose to form a complex series of compounds including reactive carbonyl compounds. The polyunsaturated fatty acid peroxides produced,

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generate MDA upon decomposition. MDA form a 1:2 adduct with thiobarbituric acid (TBA) that give rise to a pink color product when heated in acidic pH, with a maximum absorbance of 532 nm. In this context, an aliquot of 0.4 mL of the sample was mixed with 1.6 mL of Tris-potassium chloride (Tris-KCl) buffer to which 0.5 mL of 30% trichloroacetic acid (TCA) was added. Then, 0.5 mL of 0.75% TBA was added and placed in a water bath for 45 min at 80 °C. This was then cooled in ice and centrifuged at 1200 g for 15 min. The clear supernatant was collected and

absorbance measured against a reference blank of distilled water at 532 nm. The MDA concentration was calculated using a Molar extinction coefficient of 1.56 × 105 M−1 cm−1 and the value was expressed as nanomole of MDA per g tissue (nmole MDA/g tissue). 2.6.5. Determination of Brain Nitrite Level

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In order to assess the effects of treatments with respective drugs on nitric oxide (NO) production, nitrite levels were determined in the mice brains homogenates according to the method described by Green et al. (1981). After centrifugation (5400 g for 10 min), the homogenate supernatant was collected and the production of NO was determined based on Griess reaction. Briefly, 100 µL of supernatant was incubated with 100 µL of Griess reagent (sulfanilamine in 1% H3PO4/ 0.1%

N-(1-naphthyl)-ethylenediamine

dihydrochloride/1%H3PO4/distilled

water,

1:1:1:1)

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(Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 min. The absorbance was

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measured at 550 nm via a UV-spectrophotometer (752N INESIA, China). The standard curve was prepared with several concentrations of NaNO2 (ranging from 0.75 to 100 µM) and was

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expressed as nmol/g tissue.

2.6.6. Determination of Acetylcholinesterase Activity in Mice Brain

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Acetylcholinesterase (AChE) enzyme activity, a marker for cholinergic neurotransmission for cognitive effect of DOX was evaluated according to the method previously described (Ellman et

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al., 1961). Briefly, the acetylcholinesterase enzyme activity in the homogenates was measured by adding 2.6 mL of phosphate buffer (0.1 M, pH 7.4), 0.1 mL of DTNB and 0.4 mL of the

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homogenate. Then, 0.1 mL of acetylthiocholine iodide solution was added to the reaction mixture. The absorbance was read using a spectrophotometer at a wavelength of 412 nm and

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change in absorbance for 10 minutes at two-minute intervals was recorded. The rate of acetylcholinesterase activity was measured by following the increase of color produced from

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thiocholine when it reacts with DTNB. The change in absorbance per minute was determined and the rate of acetylcholinesterase activity was calculated and expressed as µmoles/min/g tissue. 2.6.7. Protein Content Estimation This assay was done according to method described by Gornall et al. (1949), using the Biuret method. The diluted sample (1 mL) was taken and added to 3 mL of Biuret reagent in triplicate. The mixture was incubated at room temperature for 30 min after which the absorbance was read

at 540 nm using distilled water as blank. Bovine serum albumin (1 mg/mL) was used as standard and was measured in the range of 0.01–0.1 mg/mL.

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2.7. Statistical Analysis Data were expressed as mean ± S.E.M. (standard error of the mean). The data was analyzed using two-way analysis of variance (ANOVA) followed by post-hoc test (Bonferroni test) for multiple comparisons where appropriate using Graph Pad Prism software version 5 (GraphPad Software, Inc. La Jolla, CA 92037 USA). A level of p < 0.05 was considered as statistically

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significant for all tests.

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3. Results

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3.1. Effect of Doxycycline on Ketamine-Induced Hyperlocomotion Fig. 2 showed the effect of DOX on KET–induced hyperlocomotion as assessed by the number

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of line crossings in the OFT in the preventive (A) and reversal (B) treatment protocols. Two-way ANOVA revealed that there were significant differences between treatment groups: preventive

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protocol (F (6, 42) = 27.90, p < 0.0001), reversal treatment protocol (p < 0.05) (F (6, 42) = 32.12, p < 0.0001). As shown in Fig. 2, KET (20 mg/kg, i.p.) significantly increased the number

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of line crossings in the OFT and this was significantly (p < 0.05) reduced by DOX (50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) in the preventive and reversal treatment protocols (Fig.

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2A). In the interaction studies, pretreatment with DOX (50 mg/kg) significantly enhanced the ability of RIS (0.5 mg/kg, p.o.) to reduce the hyper-motility effect of KET in both preventive [F (3, 24) = 43.69, P < 0.0001] and reversal [F (3, 24) = 59.49, P < 0.0001] treatment protocols

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(Fig. 2A,B).

3.2. Effect of Doxycycline on Ketamine-Induced Social Interaction Deficits Fig. 3 showed the effect of DOX and RIS on KET-induced impairment in social interaction in mice. Two-way ANOVA revealed that there were significant differences between treatment groups: preventive protocol (F (6, 42) = 56.15, p < 0.0001), reversal protocol (p < 0.001) (F (6,

42) = 95.41, p < 0.0001). As depicted in Fig. 3, intraperitoneal administration of KET (20 mg/kg) significantly (p < 0.05) impaired social interaction behavior in both treatment protocols when compared with vehicle. The impaired social interaction behavior was ameliorated by DOX (25, 50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) in comparison with KET in both protocols. Moreover, the positive effect of RIS (0.5 mg/kg) on KET-induced social interaction deficits was

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also enhanced by DOX (50 mg/kg) in a significant (p < 0.05) manner: preventive protocol [F (3, 24) = 61.95, P < 0.001] and reversal protocol [F (3, 24) = 103.4, P < 0.05] (Fig 3).

3.3. Effect of Doxycycline on Ketamine-Induced Impairment in Spatial Working Memory

The effect of DOX on chronic ketamine-induced deficit in spatial working memory based on alternation behavior in YMT is presented in Fig. 4. Two-way ANOVA showed that there were

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significant differences between treatment groups: preventive protocol (F (6, 42) = 13.90, p <

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0.0001) and reversal protocol (F (6, 42) = 14.06, p < 0.0001). As depicted in Fig. 4, KET (20 mg/kg, i.p.) in the both treatment protocols, significantly (p < 0.001) decreased percentage

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alternations in the YMT relative to vehicle, suggesting decrease in spatial working memory.

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However, DOX (50 and 100 mg/kg) produced a significant (p < 0.05) increase in alternation behaviors in the YMT relative to KET group. In the interaction studies, combination of DOX (50

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mg/kg, p.o.) with RIS (0.5 mg/kg, p.o.) produced greater positive effect on memory function in

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mice chronically treated with KET in both treatment protocols. 3.4. Effect of Doxycycline on Ketamine-Induced Impairment in Non-Spatial Recognition Memory

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The effect of DOX on ketamine-induced impairment in non-spatial memory in mice is depicted in Fig. 5. There were significant differences (two-way ANOVA) between treatment group:

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preventive protocol (p < 0.001) (F (6, 35) = 8.395, p < 0.0001) and reversal protocol (p < 0.001) (F (6, 42) = 15.73, p < 0.0001). In both treatment protocols, chronic KET (20 mg/kg, i.p.)

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administration produced significant (p < 0.001) decrease in the percentage preference for novel object relative to vehicle, which suggests impairment of memory function. As shown in Fig. 5, the decrease in preference for novel object was significantly (p < 0.05) increased by DOX (50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) when compared with KET group in the preventive and reversal treatments respectively. The memory promoting effect of RIS (0.5 mg/kg, p.o.) was also enhanced by DOX (50 mg/kg, p.o.) in the interaction studies (Fig. 5A,B).

3.5. Effects of Doxycycline on Superoxide Dismutase and Catalase Activities in KetamineTreated Mice Ketamine (20 mg/kg, i.p.) decreased the brain activities of SOD and CAT in both preventive and reversal treatment protocols when compared with vehicle-treated group (Tables 1 and 2). As

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presented in Tables 1 and 2, DOX (50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) significantly (p < 0.05) increased the brain activity of SOD in KET-treated mice in both preventive and reversal protocols. Moreover, pretreatment with DOX (50 mg/kg, p.o.) significantly (p < 0.05) increased the effect of RIS (0.5 mg/kg, p.o.) on the brain activity of SOD in the reversal protocol but not in the preventive protocol (Tables 1 and 2). The results of Tables 1 and 2 also showed that DOX (50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) significantly increased KET-induced decreased brain activity of CAT in both treatment protocols. Also, pretreatment with DOX (50

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mg/kg, p.o.) enhanced the ability of RIS (0.5 mg/kg, p.o.) to increase the brain activity of CAT in

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both treatment protocols (Tables 1 and 2).

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3.6. Effect of Doxycycline on Brain Concentrations of Glutathione, Malondialdehyde and Nitrite in Ketamine-Treated Mice

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The effects of DOX or RIS on KET-induced decreased brain concentrations of GSH are

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presented in Tables 3 and 4. Two-way ANOVA revealed that there were significant differences between treatment groups: preventive protocol (F (6, 42) = 38.82, p < 0.0001) and reversal

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protocol (F (6, 42) = 40.80, p < 0.0001). As shown in Tables 3 and 4, KET (20 mg/kg, i.p.)induced decreased brain GSH levels were significantly (p < 0.05) elevated by DOX (50 and 100 mg/kg, p.o.) or RIS (0.5 mg/kg, p.o.) in both treatment protocols (Tables 3 and 4). Tables 3 and 4

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also showed the effects of DOX and RIS on KET-induced increased MDA concentrations in both preventive and reversal treatment protocols. Two-way ANOVA revealed that there were

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significant differences between treatment groups: preventive protocol (F (6, 42) = 50.93, p < 0.0001) and reversal protocol (F (6, 42) = 105.4, p < 0.0001). The increased brain concentrations of MDA produced by KET (20 mg/kg, i.p.) was reduced by DOX (50 and 100 mg/kg, p.o.) in a similar manner to RIS (0.5 mg/kg, p.o.) in both treatment protocols. Furthermore, DOX (50 and 100 mg/kg) or RIS (0.5 mg/kg) reduced the increased brain levels of nitrite produced by KET (20 mg/kg, i.p.) in the both treatment protocols. Moreover, in the interaction studies,

pretreatment

with

DOX

(50

mg/kg,

p.o.)

enhanced

the

effect

of

RIS

(0.5 mg/kg, p.o.) to ameliorates the altered brain levels of GSH, MDA and nitrite in KET-treated mice in the preventive and reversal protocols respectively (Tables 3 and 4). 3.7. Effects Doxycycline on Ketamine-Induced Changes in Acetylcholinesterase Enzyme Activity

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The effects of DOX and RIS on KET-induced changes in AChE enzyme activity, as a measure of acetylcholine concentration in both preventive and reversal treatment protocols are shown in Tables 5 and 6. Two-way ANOVA revealed that there were significant differences between treatment groups: preventive protocol (F (6, 42) = 42.85, p < 0.0001) and reversal protocol (p < 0.001) (F (6, 42) = 35.32, p < 0.0001). As presented in Tables 5 and 6, the increased brain AChE activity produced by KET (20 mg/kg, i.p.) was reduced by DOX (50 and 100 mg/kg, p.o.) or RIS

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(0.5 mg/kg, p.o.) in a significant (p < 0.05) manner in both treatments. Moreover, pretreatment

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with DOX (50 mg/kg, p.o.) was found to enhance the effect of RIS (0.5 mg/kg, p.o.) in inhibiting AchE activity in KET-treated mice in the reversal protocol (p < 0.05) but not in the preventive

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protocol (p > 0.05).

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4. Discussion

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The results of this study showed that DOX prevented and reversed schizophrenic-like behaviors induced by chronic injection of KET in mice. Doxycycline significantly increased glutathione,

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superoxide dismutase and catalase levels in the brain of chronic ketamine-treated mice. However, it decreased malonyladehyde, nitrite levels and acetylcholinesterase activity in the

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brains of mice chronically treated with KET. Moreover, its anti-schizophrenic-like effect was enhanced by RIS in both protocols based on the behavioral assays. Risperidone also enhanced the antioxidant and anti-cholinesterase activities demonstrated by doxycycline in the both

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preventive and reversal protocols. Ketamine-induced hyperlocomotion has been used as a screening model for the evaluation of novel compounds for potential antipsychotic property in rodents and is known to be partly due to the blockade of NMDA receptors located on the inhibitory GABAergic neurons in the limbic and sub-cortical regions of the brain. This has been linked to increased neuronal activity in the limbic-striatal pathways and thus, the hyperlocomotion that characterized some of the positive

symptoms of the disease (Irifune et al., 1991; Chatterjee et al., 2012b). However, compounds with antipsychotic property have been shown to attenuate KET-induced hyperlocomotion. In this context, our data showed that DOX demonstrated significant inhibition of ketamine-induced hyperlocomotion in both protocols, which suggests antipsychotic-like activity in mice. Moreover, treatment with RIS enhanced the inhibitory effect of DOX (50 mg/kg) against KET-

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induced hyperlocomotion in the preventive protocol. This finding suggests that DOX may enhance the efficacy of RIS, an atypical antipsychotic in the treatment of schizophrenia.

In this present study, we also evaluated the effect of DOX on the negative symptoms associated with schizophrenia using the social preference test in mice. This test mimic social withdrawal symptom, which is one of the major negative symptoms seen in patients with schizophrenia (Monte et al., 2013). Ketamine-induced impaired social interaction is a well recognized animal

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model in psychopharmacology and antipsychotics are known to attenuate this effect of KET

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(Neill et al., 2010). The results of our study further support previous investigations, which showed that KET impaired social interactions in rodents (Becker et al., 2003; Monte et al., 2013;

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Vasconcelos et al., 2015). However, the ability of DOX to prevent and reverse the decrease in

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social interaction produced by KET, as evidenced by increased percentage social preference, further confirms its antipsychotic-like activity. Recently, our research group showed that DOX

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attenuated KET-enhanced immobility in forced swim test (behavioral despair test); another

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model for negative symptoms of schizophrenia (Ben-Azu et al., 2016a). Ketamine-enhanced immobility has been found to be mediated, at least in part, through 5-HT2A receptors (Becker et al., 2003; Chindo et al., 2012). Taken together, this result may have therapeutic implications for

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the treatment of negative symptoms of schizophrenia and other disorders associated with social withdrawal. However, additional studies are necessary before speculating on how DOX

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alleviates negative symptoms in KET-treated mice. In this study, we also evaluated the effect of DOX on cognitive symptoms commonly seen in

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patients with schizophrenic disorders using YMT and NORT. These tests are used for evaluation of both short-term and long-term memories, one of the cognitive domains that have been known to be impaired in schizophrenia (Meltzer, 2010). Impairment of learning and memory following chronic KET injection has been reported in several studies (Jentsch et al., 1981; Rujescu et al., 2006) via mechanisms related to inhibition of alpha-7 nicotinic acetylcholine receptor (Coates and Flood, 2001) and release of proinflammatory cytokines (Behrens et al., 2009; Chatterjee et

al., 2012a; Lykhmus et al., 2016). In this study, KET was found to cause a significant decrease in memory performance in both YMT and NORT, which is in line with previous investigations (Monte et al., 2013; Vasconcelos et al., 2015). Moreover, previous studies have also established that KET increases the activity of AChE enzyme in the brain resulting in reduced concentration of ACh and thus, memory deficit (Chatterjee et al., 2012b). However, DOX prevented and

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reversed the memory impairment produced by chronic KET treatment, suggesting that it might offer beneficial effect in psychotic patients with cognitive deficits. The memory promoting effect of DOX observed in this study may be related to enhancement of cholinergic neurotransmissions. This suggestion is based on the findings that DOX attenuated the increase in the activity of AChE, the enzyme responsible for the breakdown of ACh, a neurotransmitter implicated in memory function.

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Several studies have shown that oxidative stress and nitrosative stress may contribute to the

N

pathogenesis of several neuropsychiatric disorders including schizophrenia (Ng et al., 2008; Monte et al., 2013). Both preclinical and clinical studies have shown that the severity of the

A

disease may depend on the activity of free radicals generated during metabolism of

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neurotransmitters (Pavlovic et al., 2002; Pazvantoglu et al., 2009). For example, metabolism of neurotransmitters including dopamine and glutamate generates large amounts of ROS that are

D

known to affect synaptic plasticity and signal transduction pathways via redox-sensitive

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receptors, such as NMDA and D2 receptors (Janaky et al., 1981; Bókkon and Antal, 2011). Indeed, continuous generation of ROS and RNS further cause neuronal cell death via glutamatemediated oxidative stress processes (West and Grace, 2000; Braidy et al., 2009; Chatterjee et al.,

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2012a). Moreover, the increased levels of MDA and NO found in postmortem studies of schizophrenia brains further support the role of oxidative stress and nitrosative stress in the

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pathogenesis of the disease (Nogueira et al., 2011; Zhang et al., 2012). Thus, the inhibition of nitrosative and oxidative stress mechanisms is currently being targeted as novel therapeutic

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approaches for the reduction of neuronal damage in schizophrenic brains (Pazvantoglu et al., 2009; Zhang et al., 2012). Recent studies demonstrated that treatments with clozapine, risperidone and minocycline restored antioxidant levels, and to a greater extent reversed the increase in nitrite levels (Monte et al., 2013; Vasconcelos et al., 2015; Ben-Azu et al., 2016b). However, the increase in oxidative stress and nitrosative stress produced by KET was attenuated by DOX when given alone or with RIS. Thus, the ability of DOX to prevent or reverse

schizophrenic-like behaviors in KET-treated mice may be mediated, in part, through inhibition of oxidative stress and nitrosative stress. Indeed, literature data has also shown that doxycycline alone demonstrates antioxidant property (Riba et al., 2017), while risperidone alone shows little or no antioxidant activity and remains controversial (Vinay et al., 2003). Meanwhile, as to whether the antibacterial effect of DOX plays a role in its anti-schizophrenic-

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like activity or psychotropic effects warrants further investigations. However, both preclinical and clinical studies have implicated microbial infections in the pathogenesis of neuropsychiatric disorders (Cunningham et al., 2005; Nemeroff et al., 2005; Perry et al., 2007; Dantzer et al., 2008; Capuron et al., 2011). A constellation of behavioral symptoms referred to as sickness behavior (depressive-like behavior, anhedonia, fatigue, psychomotor slowing, decreased appetite, sleep alterations and increased sensitivity to pain) have been widely reported in humans

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and laboratory animals suffering from infectious diseases (Perry et al., 2007; Dantzer et al.,

N

2008). The activation of the immune system in response to infections triggers the release of proinflammatory cytokines (interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha)

A

from immune cells, which act as mediators of neuronal injury that underlie the neurobiological

M

basis of neuropsychiatric disorders (Cunningham et al., 2005; Perry et al., 2007; Dantzer et al., 2008). As a matter of fact, cytokines have been shown to act on several neurotransmitter systems

D

and neurocircuitry relevant to the development of psychopathology (Perry et al., 2007; Dantzer

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et al., 2008). Thus, pro-inflammatory cytokines are the signal molecules that communicate the impact of systemic infections to the brain and it is expected that the clearance of the infections with antibiotics should attenuate neuropsychiatric diseases. However, it has been reported that

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microbial disease is an adaptive response to infection by pathogens and fully reversible once the pathogen has been cleared (Nemeroff et al., 2005; Dantzer et al., 2008). Meanwhile, this is not

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the case for the neurological disorder, which represents a maladaptive version of cytokineinduced sickness, which occurs in response to activated immune cells (Nemeroff et al., 2005;

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Dantzer et al., 2008). Moreover, the findings that cytokine inhibitors attenuate the behavioral symptoms of cytokines further confirm their role in the mediation of neuropsychiatric diseases (Nemeroff et al., 2005; Perry et al., 2007; Dantzer et al., 2008; Capuron et al., 2011). Thus, it might be speculated that the positive effects of DOX in KET model of schizophrenia may be related to its antibiotic property since KET-induced schizophrenia-like behaviors has recently

been found to be, as least in part, due to neuroimmune alterations (Behrens et al., 2008; da Silva

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et al., 2017). However, more studies are needed to clarify this assertion.

5. Conclusions

The results of this present study showed that doxycycline ameliorated schizophrenic-like behaviors induced by ketamine in both preventive and reversal treatment protocols in mice via inhibition of oxidative stress, nitrergic stress and acetylcholinesterase activity. Our data further suggests that doxycycline supplementation may augment the therapeutic efficacy of conventional

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antipsychotic drugs especially for the treatment of negative and cognitive symptoms associated

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with schizophrenia.

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Author Contributions: “B.B., I.A.O. and A.M.A conceived and designed the experiments; B.B. and I.O.M. performed the experiments; B.B. and S.U. analyzed the data; A.O.A contributed reagents/materials/analysis tools; B.B, S.U and E.O.I. wrote the first draft of the manuscript;

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B.B. and S.U. completed the final draft of the paper.”

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Funding

This research did not receive any specific grant from funding agencies in the public, commercial,

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or not-for-profit sectors.

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Conflicts of interests: The authors declare no conflict of interest

Acknowledgment: We thank all technical staffs of the Department of Pharmacology and Therapeutics, Delta State University, Abraka for their assistance during the course of this study.

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Figure Captions

Fig. 1. Treatment protocols: VEH = Vehicle, KET = ketamine, DOX = Doxycycline,

A

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D

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A

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RIS = Risperidone

* * *,** *,#,**

75 50

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Number of line crossings

VEH (10 mL/kg) KET (20 mg/kg) DOX (25 mg/kg) + KET DOX (50 mg/kg) + KET DOX (100 mg/kg) + KET RIS (0.5 mg/kg) + KET DOX (50 mg/kg) + RIS + KET

**

100

25 0

Treatments

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**

100

*,**

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*

D

25 0

A

* 75 50

VEH (10 mL/kg) KET (20 mg/kg) KET + DOX (25 mg/kg) KET + DOX (50 mg/kg) KET + DOX (100 mg/kg) KET + RIS (0.5 mg/kg) KET + DOX (50 mg/kg) + RIS

N

125

Treatments

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Number of line crossings

(A)

(B)

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Fig. 2. Effect of DOX on ketamine-induced hyperlocomotion in the preventive (A) and reversal (B) treatment protocols in mice. Bars represents the mean ± S.E.M (standard error

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of the mean) (n = 7 animals/group). ** p < 0.05 as compared to vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET + RIS group according to two-way

A

analysis of variance (ANOVA) followed by Bonferroni test.

100

*# *

50

*

*

25

*

0 -25

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% Social Preference

75

VEH (10 mL/kg) KET (20 mg/kg) DOX (25 mg/kg) + KET DOX (50 mg/kg) + KET DOX (100 mg/kg) + KET RIS (0.5 mg/kg) + KET DOX (50 mg/kg) + RIS + KET

**

-50

Treatments

* 0

*

**

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*

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25

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50

*#

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% Social preference

75

-50

VEH (10 mL/kg) KET (20 mg/kg) KET + DOX (25 mg/kg) KET + DOX (50 mg/kg) KET + DOX (100 mg/kg) KET + RIS (0.5 mg/kg) KET + DOX (50 mg/kg) + RIS

N A

100

-25

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(A)

Treatments

(B)

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Fig. 3. Effect of DOX on ketamine-induced social interaction deficits in the preventive (A) and reversal (B) treatment protocols in mice. Bars represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET + RIS group according to two-way ANOVA followed by Bonferroni test.

*

**

40

DOX (50 mg/kg) + RIS + KET

20 0

Treatments

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% Correct Alternations

* 60

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*#

80

VEH (10 mL/kg) KET (20 mg/kg) DOX (25 mg/kg) + KET DOX (50 mg/kg) + KET DOX (100 mg/kg) + KET RIS (0.5 mg/kg) + KET

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(A)

A *#

* 40 20

*

Treatments

(B)

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0

**

*

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60

M

80

VEH (10 mL/kg) KET (20 mg/kg) KET + DOX (25 mg/kg) KET + DOX (50 mg/kg) KET + DOX (100 mg/kg) KET + RIS (0.5 mg/kg) KET + DOX (50 mg/kg) + RIS

TE

% Correct alternations

100

Fig. 4. Effect of DOX on ketamine-induced impairment in spatial working memory in the preventive (A) and reversal (B) treatment protocols. Bars represents the mean ± S.E.M (n =

A

7 animals/group). ** p < 0.05 as compared to vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET + RIS group according to two-way ANOVA followed by Bonferroni test.

* * *

60

40

*

**

VEH (10 mL/kg) KET (20 mg/kg) DOX (25 mg/kg) + KET DOX (50 mg/kg) + KET DOX (100 mg/kg) + KET RIS (0.5 mg/kg) + KET DOX (50 mg/kg) + RIS + KET

SC RI PT

% Exploraroty preference

80

20

0

Treatments

U

(A)

60

N

VEH (10 mL/kg) KET (20 mg/kg) KET + DOX (25 mg/kg) KET + DOX (50 mg/kg) KET + DOX (100 mg/kg) KET + RIS (0.5 mg/kg) KET + DOX (50 mg/kg) + RIS

D

EP

0

*

M

** 40

20

*

A

*

TE

% Exploratory preference

80

Treatments

(B)

CC

Fig. 5. Effect of DOX on ketamine-induced impairment in spatial recognition memory in preventive (A) and reversal (B) treatment protocols in mice. Bars represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to vehicle group; * p < 0.05 as

A

compared to KET group; (two-way ANOVA followed by Bonferroni test).

Tables

Table 1. Effects of DOX on ketamine-induced increases in superoxide dismutase (SOD) and

SC RI PT

catalase (CAT) activities in the preventive protocol in mice. Treatments & Dose

SOD (Unit/mg Protein) CAT (Unit/mg Protein)

VEH (10 mL/kg)

7.17 ± 0.36

KET (20 mg/kg)

2.60 ±0.24 **

DOX (25 mg/kg) + KET

3.42 ± 0.23

DOX (50 mg/kg) + KET

7.28 ± 0.36 *

DOX (100 mg/kg) + KET

7.13 ± 0.23 *

RIS (0.5 mg/kg) + KET

7.18 ± 0.28 *

4.33 ± 0.20

1.90±0.18 ** 2.77 ± 0.12 *

U

N

A

M

DOX (50 mg/kg) + RIS + KET 7.67 ± 0.33 *

4.03 ± 0.26 * 4.33 ± 0.28 * 3.90 ± 0.27 * 4.82 ± 0.36 *

D

Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

A

CC

EP

Bonferroni test).

TE

vehicle group; *p < 0.05 as compared to KET group (two-way ANOVA followed by

Table 2. Effects of DOX on ketamine-induced alterations in superoxide dismutase (SOD) and catalase (CAT) activities in the reversal protocol in mice. SOD (Unit/mg Protein) CAT (Unit/mg Protein)

VEH (10 mL/kg)

6.97 ± 0.26

KET (20 mg/kg)

2.21±0.24 **

KET + DOX (25 mg/kg)

2.86 ± 0.07

KET + DOX (50 mg/kg)

3.64 ± 0.15 *

KET + DOX (100 mg/kg)

5.51 ± 0.28 *

KET + RIS (0.5 mg/kg)

4.97 ± 0.18 *

SC RI PT

Treatments & Doses

4.28 ± 0.24

1.24±0.24 ** 1.44 ± 0.20

3.44 ± 0.25 *

U

N

A

3.14 ± 0.21 * 4.56 ± 0.19 *

M

KET + DOX (50 mg/kg) + RIS 6.37 ± 0.50 *,#

4.12 ± 0.29 *

Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

D

vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET +

A

CC

EP

TE

RIS group according to two-way ANOVA followed by Bonferroni test.

Table 3. Preventive effects of DOX on brain glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) concentrations and acetylcholinesterase (AChE) activity in ketaminetreated mice. MDA (nmole/g

Nitrite (nmole/g

Protein)

Tissue)

Tissue)

VEH (10 mL/kg)

147.5 ± 4.43

15.02 ± 0.74

105.5 ± 4.94

KET (20 mg/kg)

100.8 ± 3.38 **

26.00 ± 0.68 **

152.3 ± 2.70 **

DOX (25 mg/kg) + KET

109.5 ± 5.48

22.45 ± 1.39

130.7 ± 6.95

DOX (50 mg/kg) + KET

179.2 ± 10.64 *

DOX (100 mg/kg) + KET

165.3 ± 6.40 *

RIS (0.5 mg/kg) + KET

U

83.83 ± 4.72 *

10.30 ± 1.67 *

78.00 ± 2.91 *

151.8 ± 6.81 *

11.13 ± 0.50 *

90.17 ± 6.08 *

M

SC RI PT

GSH (nmole/mg

8.37 ± 0.55 *,#

53.60 ± 4.33 *,#

N

12.52 ± 0.93 *

A

Treatments & Doses

D

DOX (50 mg/kg) + RIS + KET 200.0 ± 5.56 *,#

Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

TE

vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET +

A

CC

EP

RIS group according to two-way ANOVA followed by Bonferroni test.

Table 4. Reversal effects of DOX on brain glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) concentrations and acetylcholinesterase (AChE) activity in ketaminetreated mice. MDA (nmole/g

Nitrite (nmole/g

Protein)

Tissue)

Tissue)

VEH (10 mL/kg)

147.5 ± 4.43

15.68 ± 0.68

99.83 ± 5.31

KET (20 mg/kg)

93.48±3.75 **

25.33±0.80 **

147.8±4.62 **

KET + DOX (25 mg/kg)

99.50 ± 6.64

23.00 ± 0.97

140.5 ± 7.54

KET + DOX (50 mg/kg)

133.8 ± 5.55 *

10.52 ± 1.24 *

KET + DOX (100 mg/kg)

138.8 ± 6.20 *

KET + RIS (0.5 mg/kg)

165.3 ± 6.41 *

KET + DOX (50 mg/kg) + RIS

175.3 ± 5.49 *

106.3 ± 5.37 *

10.52 ± 0.71 *

111.8 ± 5.78 *

9.03± 0.38 *

104.5 ± 6.47 *

8.03 ± 1.34 *,#

66.30±8.58 *,#

D

M

A

N

U

SC RI PT

GSH (nmole/mg

Treatments & Doses

Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

TE

vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET +

A

CC

EP

RIS group according to two-way ANOVA followed by Bonferroni test.

Table 5. Effects of DOX on Ketamine-induced increase in brain concentrations acetylcholinesterase (AChE) activity in the preventive treatment protocol in mice. AchE (μmole/min/g Tissue)

VEH (10 mL/kg)

3.412 ± 0.23

KET (20 mg/kg)

7.75 ± 0.25 **

DOX (25 mg/kg) + KET

7.27 ± 0.29

DOX (50 mg/kg) + KET

5.72 ± 0.15 *

DOX (100 mg/kg) + KET

5.45 ± 0.21 *

U

N

A

4.73 ± 0.31 *

M

RIS (0.5 mg/kg) + KET

SC RI PT

Treatments & Doses

DOX (50 mg/kg) + RIS + KET 3.75 ± 0.36 *

D

Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

A

CC

EP

Bonferroni test).

TE

vehicle group; * p < 0.05 as compared to KET group (two-way ANOVA followed by

Table 6. Effects of DOX on Ketamine-induced increase in brain concentrations acetylcholinesterase (AChE) activity in the reversal treatment protocol in mice. AchE (µmole/min/g Tissue)

VEH (10 mL/kg)

3.50 ±0.22

KET (20 mg/kg)

7.80 ± 0.23 **

KET + DOX (25 mg/kg)

7.57 ± 0.50

KET + DOX (50 mg/kg)

4.38 ± 0.34 *

KET + DOX (100 mg/kg)

5.08 ± 0.26 *

KET + RIS (0.5 mg/kg)

4.95 ± 0.20 *

A

N

U

SC RI PT

Treatments & Doses

M

KET + DOX (50 mg/kg) + RIS 3.28 ± 0.36 *,# Values represents the mean ± S.E.M (n = 7 animals/group). ** p < 0.05 as compared to

D

vehicle group; * p < 0.05 as compared to KET group; # p < 0.05 as compared to KET +

A

CC

EP

TE

RIS group according to two-way ANOVA followed by Bonferroni test.