Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1

    Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1 Chandra Shaker Sriram, Ashok Jangra, Satendra Singh Gurjar, Pritam Mohan, Babul Kumar Bezbaruah PII: DOI: Reference:

S0031-9384(15)30158-X doi: 10.1016/j.physbeh.2015.10.029 PHB 11079

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

25 May 2015 17 October 2015 27 October 2015

Please cite this article as: Sriram Chandra Shaker, Jangra Ashok, Gurjar Satendra Singh, Mohan Pritam, Bezbaruah Babul Kumar, Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1, Physiology & Behavior (2015), doi: 10.1016/j.physbeh.2015.10.029

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ACCEPTED MANUSCRIPT Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1 Chandra Shaker Srirama, Ashok Jangraa, Satendra Singh Gurjarb, Pritam Mohand, Babul Kumar

Research (NIPER), Guwahati, Assam, India-781032

Department of Biotechnology, National Institute of Pharmaceutical Education and Research

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(NIPER), Guwahati, Assam, India-781032,

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Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education &

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Bezbaruaha, c, *

Department of Pharmacology, Gauhati Medical College, Guwahati, AssamIndia-781032, Department of Pharmacology & Toxicology, College of Veterinary Science, Assam Agricultural

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University, Khanapara, Guwahati, AssamIndia-781022,

* Corresponding author: Dr. BK Bezbaruah Professor & Project Director Department of Pharmacology & Toxicology National Institute of Pharmaceutical Education & Research (NIPER), Narkachal Hilltop Bhangagarh, Guwahati, Assam-781032, India Mobile no: +91-9864066772. Email address: [email protected], [email protected]

Abstract Poly (ADP-ribose) polymerase-1 (PARP-1) is a DNA nick-sensor enzyme, functions at the center of cellular stress response and affects immune system at several key points, thus modulates inflammatory diseases. Our previous study demonstrated that lipopolysaccharide (LPS)-

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Lipopolysaccharide

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Keywords Poly (ADP-ribose) polymerase-1 Depressive-like behavior Anxiety Edaravone

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induced depressive-like behavior in mice can be ameliorated by 3aminobenzamide, which is a PARP-1 inhibitor. In the present study we've examined the effect of a free radical scavenger, edaravone pretreatment aganist LPS-induced anxiety and depressive-like behavior as well as various hippocampal biochemical parameters including PARP-1. Male Swiss albino mice were treated with edaravone (3 & 10 mg/kg i.p.) once daily for 14 days. On the 14th day 30 min after edaravone treatment mice were challenged with LPS (1 mg/kg i.p.). After 3h and 24h of LPS administration we've tested mice for anxiety and depressive-like behaviors respectively. Western blotting analysis of PARP-1 in hippocampus was carried out after 12h of LPS administration. Moreover, after 24h of LPS administration serum corticosterone, hippocampal BDNF, oxido-nitrosative stress and pro-inflammatory cytokines were estimated by ELISA. Results showed that pretreatment of edaravone (10 mg/kg) ameliorates LPS-induced anxiety and depressivelike behavior. Western blotting analysis showed that LPS-induced anomalous expression of PARP-1 significantly reverses by the pretreatment of edaravone (10 mg/kg). Biochemical analyses revealed that LPS significantly diminishes BDNF, increases pro-inflammatory cytokines and oxido-nitrosative stress in the hippocampus. However, pretreatment with edaravone (10 mg/kg) prominently reversed all these biochemical alterations. Our study emphasized that edaravone pretreatment prevents LPS-induced anxiety and depressive-like behavior, mainly by impeding the inflammation, oxidonitrosative stress and PARP-1 over expression.

1. Introduction

Major depressive disorder (MDD) is a regular and severe psychiatric disorder with a life time occurrence of 10–20% (Licinio and Wong, 2011). MDD ranked second among the diseases with the largest number of years lived with disability in 2010 in the United States (US Burden of Disease Collaborators, 2013). So far numerous factors that contribute for the development of mood disorders identified, but still, the present knowledge of aetiology and pathophysiology of mood disorders, including anxiety and depression is still very limited. Thus, numerous studies have been conducted with the aim of better understanding of the neurobiology of mood disorders, as well as finding

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ACCEPTED MANUSCRIPT pharmacological targets with faster actions and more efficiency (Duman et al., 2012; Goswami et al., 2013; Xu et al., 2014).

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Many of recent research findings indicate that an increase in oxidative and nitrosative stress

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(O&NS) is implicated in pathophysiology of mood disorders (Maes et al., 2011). Increased levels

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of reactive oxygen species (ROS) and reactive nitrogen species (NS) including peroxide (Maes et al., 2010) and nitric oxide (NO) (Dhir and Kulkarni, 2011; Suzuki et al., 2001) have been

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reported to be associated with mood disorders. Accordingly, O&NS mechanisms have been

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proposed as targets for novel drugs intended for mood disorders (Li et al., 2012; Lee et al., 2013).

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Further, several research reports suggest that anxiety and depression are accompanied the

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elevation of inflammatory cytokine levels, for example, tumor necrosis factor alpha (TNF α),

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interleukin-6 (IL-6), and IL-1β (IL-1β) (Sluzewska et al., 1996; Dowlati et al., 2010; Lu et al., 2013; Sluzewska et al., 1996; Sulakhiya et al., 2015). Reason for the association of immuneinflammatory pathways association with mood disorders is presently ambiguous. One of such reasons responsible for inflammation could be hyperactivity of the hypothalamic–pituitary– adrenal (HPA) axis system (Holsboer, 2001). Upon the experience of severe stress, overactivation of the HPA axis can take place, and the feedback regulation of HPA axis gets perturbed that could precipitate mood disorders. HPA axis overactivation is an indicator of glucocorticoid resistance. The glucocorticoid resistance implies ineffective action of glucocorticoid hormones on target tissues, which can cause immune system activation. Immune system activation can lead to neuroinflammation via direct action on proinflammatory cytokines (IL-6, TNF-1 etc.) (Zunszain et al., 2011).

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ACCEPTED MANUSCRIPT Moreover, peripheral inflammatory mediators signal the brain to affect behavioral, affective and cognitive changes that are consistent with symptoms of major depressive disorder (Messay et al.,

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2012).

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A well established model to study the physiological and behavioral responses under

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inflammation is the peripheral administration of a single dose of the cytokine-inducer lipopolysaccharide (LPS) (Song and Wang, 2011). LPS is a bacterial endotoxin that causes

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activation of innate immune response and production of pro-inflammatory cytokines (Miller et

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al., 2005). In animals, intraperitoneal (i.p.) administration LPS has been exhibited to cause several depressive-like behaviors including, reduced social interaction and exploratory behavior,

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as well as augmented anhedonia (de Paiva et al., 2010; Jangra et al., 2014b; Salazar et al., 2012).

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LPS-induced depressive-like behavior follows a particular temporal fashion with an earlier “anxiety behavior”, which maximizes in the first 2–6 h and that manifests into “depressive -like

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behavior” after 24h of LPS administration (Dantzer et al., 2008; Jangra et al., 2014b). Moreover, antidepressants including serotonin and norepinephrine reuptake inhibitors (SNRI) and selective serotonin reuptake inhibitors (SSRI) showing anti-inflammatory effects in vitro and in vivo, fortify that aiming inflammation could be an exciting approach to deal with mood disorders (Ohgi et al., 2013). Poly (ADP-ribose) polymerase-1 (PARP-1) is an enzyme catalyzes poly (ADP-ribosyl)ation (PARylation), a post-translational modification implied in DNA repair mechanism. PARP-1 plays a major role in the development of inflammatory responses (Czapski et al., 2013; Jangra et al., 2013). In fact, PARP-1 is the co-activator of Ap -1 (activator protein-1) and nuclear factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells, other transcription factors, which control the inflammatory response genes (Hassa and Hottiger, 2002). PARP-1 inhibitors have been exhibited their protective activity in several neuroinflammatory 4

ACCEPTED MANUSCRIPT disorders (Sriram et al., 2015a). In our previous study we have demonstrated that LPS-induced depressive-like behavior in mice can be ameliorated by the employment of a PARP-1 inhibitor,

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3-aminobenzamide (Sriram et al., 2015b). In the present study we examine the effect of

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edaravone (EDV) on LPS-induced anxiety and depressive like behavior and as well as neuroinflammation and PARP-1 overactivation. EDV is a free radical scavenger; possess anti-

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inflammatory activity against ischemic injury (Yuan et al., 2014). Moreover, EDV has been

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found to produce its protective effect against various LPS-induced implications, such as liver, lung and kidney injury (Kono et al., 2003; Zong et al., 2014; Tajima et al., 2008; Liu et al.,

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2015). In addition, EDV possesses a potential neuroprotective activity (Qi et al., 2004; Qin et al., 2014; Srinivasan and Sharma, 2011). Indeed, EDV has been approved for the treatment of

2.1. Animals

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2. Materials and methods

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cerebral ischemic stroke in Japan (Kikuchi et al., 2011).

We’ve performed the study in Swiss albino mice (weighing: 25-30 g), which were procured from the Central animal facility of the institute (Gauhati Medical College, Guwahati). The study was approved by the Institutional Animal Ethics Committee (IAEC) (Approval No: MCI/05/2014/2). The animals were housed 3 per cage in standard polycarbonate cages (42 × 20.5 × 20 cm) and provided with standard environmental conditions (24 ± 1 °C; humidity 65 ± 5%; reversed 12-h light/dark cycle). Moreover, animals provided with abundant access to food and water ad libitum. All the tests carried out between 9:00 and 14:00 h. We’ve followed the guide-lines of the Committee for the purpose of control and supervision of experiments on animals (CPSCEA), New Delhi, India for performing the experiments of the present study.

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ACCEPTED MANUSCRIPT 2.2 Chemicals LPS (Escherichia coli; strain 055: B5) and EDV purchased from Sigma Aldrich Corp., St Louis,

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USA. PARP-1 (Cat# 9542; Cell signaling technology) antibody and horseradish peroxidase-

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conjugated secondary antibody (Santa Cruz) were used for western blotting analysis. All the

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other chemicals and reagents were of analytical grade from Sigma-Aldrich unless otherwise

2.3 Drug treatment and experimental design

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indicated.

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Mice were randomly divided into four experimental groups.

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1. Group I was treated with normal saline for 14 days. This group served as a normal

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control group.

2. Group II was treated with normal saline for 14 days and, LPS (1 mg/kg, i.p.) was

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administered on the 14th day.

3. Group III was treated with EDV (3 mg/kg, i.p.) for 14 days and LPS (1 mg/kg, i.p.) was administered on the 14th day. 4. Group IV was treated with EDV (10 mg/kg, i.p.) for 14 days and LPS (1 mg/kg, i.p.) was administered on the 14th day. The study design is diagrammatically illustrated in the Fig. 1. EDV prepared freshly in saline every day just before the treatment. The doses of EDV (3 & 10 mg/kg) were chosen based on the previous studies (Qi et al., 2004; Srinivasan and Sharma, 2011). Each experimental group was continued with their respective treatment for 14 days. On the 14th day, LPS (1 mg/kg, i.p.) or saline was injected into mice 30 min after drug administration. After 3 h of LPS challenge anxiogenic behavior was assessed by performing elevated plus maze (EPM) test, light–dark box 6

ACCEPTED MANUSCRIPT test and open field test (OFT). The time point 3 h was considered for the anxiety-like behaviour assessment is based on the previous studies (Jangra et al., 2014b; Sulakhiya et al., 2015).

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Western blotting analysis of hippocampal PARP-1 was carried out after 12 h of saline or LPS

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challenge. The time-point 12 h was regarded for the western blotting analysis of PARP-1 is based on our previous study (Sriram et al., 2015b). After 24 h of LPS or saline administration

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depressive-like behavior was tested by performing tail suspension test (TST) and forced

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swimming test (FST). The time-point 24 h was regarded for testing depressive like behavior, since the previous studies suggest that depressive like behavior peaks after 24 h of the LPS

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challenge (Custódio et al., 2013; Dantzer et al., 2008). In addition, the anhedonic response was examined by performing sucrose preference test after 24 h of the LPS or saline administration.

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Finally, the animals intended for biochemical parameter estimation were killed after 24 h of LPS or saline administration. Hipocampus was isolated quickly from each mouse brain and stored at -

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80˚C and used for various biochemical parameter estimations. All the experimental protocols used in the present study were approved by Institutional Review Committee for animal subjects. 2.4. Assessment of anxiety-like behavior Anxiety-like behavior was assessed by performing OFT, light-dark box test and EPM test. 2.4.1. Open field test (OFT) OFT is an important experiment to evaluate behavioral and motor changes in mice. In this test an acrylic transparent box (72 x 72 x 36 cm3), floor seperated into 16 equal sized squares (18 x 18 cm2) was employed. Four center squares were measured as the centre, and the 12 squares along the walls were measured as the periphery. While testing, mouse was placed in the centre of the

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ACCEPTED MANUSCRIPT box, and number of peripheral and central crossings, the immobility time and rearing movements were observed for 10 min. (Bassi et al., 2012).

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2.4.2. Light-dark box test

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The light-dark box test apparatus consisted of two different compartments: a light side (42 x 30 x

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20 cm3; white walls and brightly illuminated with 40 W bulb) and a dark side (42 x 30 x 20 cm3;

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opaque black walls and dark), with a gap (6 x 6 cm2) between the two compartments. While testing, mouse was kept on a dark side with head facing towards the light side and allowed to

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travel around for 10 min. The number of light-dark transitions, % time spent in the light compartment, and % time of risk assessments were recorded, evaluated and presented (Lacosta et

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al., 1999).

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2.4.3. Elevated plus maze test (EPM)

EPM consists of two closed arms (35 x 5 cm2) perpendicular to two open arms of the same size with a small central square (5 x 5 cm2) between arms. The maze was elevated 50 cm from the floor in a dim room. While testing, mouse was positioned at the centre of EPM with head facing in the direction of the open arm and 5 min free exploration of mice was recorded. The % time spent in open arm, end-arm explorations and total number of entries into the open arm throughout

the test was examined and presented (Espejo, 1997). 2.5. Assessment of depressive-like behavior Depressive-like behavior was assessed by performing tail suspention (TST) test and forced swimming test (FST). 2.5.1. Tail suspension test (TST) 8

ACCEPTED MANUSCRIPT The total period of immobility in tail suspension test was tested based on the earlier protocol (Steru et al., 1985). Acoustically and visually isolated mice were suspended 40 cm above the

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floor by an adhesive tape placed approximately 1 cm from the tip of the tail. For testing, mouse

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was suspended for 6 min, and the immobility time in seconds was calculated during the last-5

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min period analyzed and presented.2.5.2. Forced swimming test (FST)

FST was performed following the standard procedure with minor modification (Porsolt et al.,

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1977). In brief, mice were separately forced to swim in an open cylindrical container (10 cm

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diameter, 25 cm height), containing water (25 ± 1 °C) to a depth of 20 cm. The immobility time defined as the absence of escape-oriented behaviors. Each animal was forced to swim for 6 min,

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and the total time of immobility in seconds was calculated during the last 5 min. The water in the

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containers was changed after each trial and the observer was blind to the group treatment of the

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mice. 2.5.3. Sucrose preference test

Sucrose preference test was performed to examine the anhedonic response after LPS injection. We employed two-bottle paradigm in which mice could select between two bottles, one with 2% sucrose solution and other one having water. A whole week before the initiation of the experiment, all the mice incorporated in this test were given daily 2% sucrose solution and drinking water for 24 h period to determine the baseline consumption and to minimize the response to novelty. Water and food were given to the animals ad libitum before and during the test. Sucrose preference test was performed after 24 h of saline or LPS administration by keeping the bottles containing drinking water and sucrose solution for the next 24 h. To avoid a side preference, the bottles were switched after every 6 h. Sucrose consumption was measured by

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ACCEPTED MANUSCRIPT weighing the bottles and calculated by using an equation: % Sucrose consumption = sucrose intake/ total fluid intake (water + sucrose intake) ˟ 100 (Jangra et al., 2014b).

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2.6. Assessment of oxido-nitrosative stress parameters

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Oxido-nitrosative stress was assessed by performing superoxide dismutase (SOD), catalase

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(CAT), reduced glutathione (GSH), lipid peroxidation and nitrite assays.

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2.6.1. Superoxide dismutase (SOD) assay

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The SOD content was estimated quantitatively by employing SOD assay kit (Sigma-Aldrich, St. Louis, MO, USA). Hippocampus was separated from the mice brain and homogenized in

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phosphate buffer saline (pH 7.4, 8% w/v). Homogenates were centrifuged at 15,000 g for 20 min

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at 4 ˚C. The Supernatants were collected and SOD content was determined according to the

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manufacturer’s instructions. Absorbance was read at 440 nm on microplate reader and results were demonstrated as U/mg of protein. Protein level was estimated by the method of Bradford method (Bradford, 1976).

2.6.2. Catalase (CAT) assay

CAT was determined by means of commercially available catalase Assay Kit, following the instructions provided by the manufacturer (Sigma Aldrich Corp., St Louis, USA). Hippocampus was separated from the mice brain and homogenized in phosphate buffer saline (pH 7.4, 8% w/v). Supernatants were taken for the CAT assay after centrifugation of homogenates at 15,000 g for 20 min at 4 ˚C. Absorbance was read at 520 nm and the results were exhibited as mMol/min/mg of protein.2.6.3. Reduced glutathione (GSH) assay

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ACCEPTED MANUSCRIPT Reduced glutathione level was estimated by employing the method of Beutler (Beutler et al., 1963). In Brief, trichloroacetic acid (10% w/v) was mixed with supernatant of hippocampal

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homogenate and centrifuged at 1000 g for 10 min at 4 ˚C. The resultant supernatant obtained after centrifugation mixed with 0.3 M disodium hydrogen phosphate and then added 0.001 M

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freshly prepared DTNB [5, 5’-dithiobis (2-nitro benzoic acid) dissolved in 1 % w/v sodium

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citrate] and absorbance was read at 412 nm. The results were shown as µg/g of tissue.2.6.4. Lipid

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peroxidation assay

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Lipid peroxidation assay was performed based on the method of Ohkawa (Ohkawa et al., 1979). In Brief, 0.1 ml of hippocampal homogenate was added to a mixture of 0.2 ml of 8.1% SDS

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(sodium dodecyl sulphate), 1.5 ml of 20% acetic acid solution adjusted to pH 3.4 with NaOH

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(Sodium hydroxide) and 1.5 ml of 0.8% thiobarbituric acid. The final mixture was then heated on a water bath at 95˚C for 60 min, and centrifuged at 10,000 rpm for 5 min. The supernatant was

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separated and its absorbance was read at 532 nm. Protein estimation was carried out according to Bradford method (Bradford, 1976). Results were expressed as mM/mg of protein concentration. 2.6.5. Nitrite assay

The hippocampal nitrite level was tested by employing Griess reagent commercially obtained from Sigma Aldrich Corp., St Louis, USA. Equal volume of sample and Griess reagent was mixed and set aside for 15 min at room temperature. Absorbance was read at 540 nm. The results were demonstrated as µMoles/mg of tissue. 2.6.6. Nicotinamide adenine dinucleotide (NAD) assay The hippocampal NAD content was estimated by employing an enzyme cycling assay (Nisselbaum and Green, 1969). In brief, the isolated hippocampus homogenized in nine volume 11

ACCEPTED MANUSCRIPT potassium phosphate buffer and placed in boiling water bath for 5 min. Then the homogenate was centrifuged at 1000 g on 4˚C. The NAD content was estimated by using an enzyme cycling

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2.7. Cytokines, BDNF and corticosterone assessments

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shown as ng/mg of protein concentration (Jangra et al., 2014a).

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mixture containing alcohol dehydrogenase and the absorbance was read at 556 nm. Results were

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Immunoassays of Interleukin1-β (Thermo Fischer Scientific, India), tumor necrosis factor (TNF)-α (Invitrogen, Carlsbad, CA, USA) and BDNF (Promega, Madison, WI, USA) performed

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according to manufacturer’s protocol available with the respective ELISA kits and the absorbance was read by a microplate reader at 450 nm. Concentration of IL-1β and TNF-α were

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shown as picogram per millilitre (Pg/ml). Concentration of BDNF was demonstrated as

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nanogram per milligram of protein (ng/mg of protein). Circulating level of corticosterone

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(CORT) in the serum was estimated by corticosterone assay ELISA kit (Abnova Corporation, Taiwan). Serum corticosterone concentration was estimated according to the manufacturer's protocol and expressed as nanogram per millilitre (ng/ml). 2.8 Protein estimation and western blotting analysis of PARP-1 After 12 h of LPS administration mice were decapitated and their brains were rapidly removed and hippocampus was dissected out from the each brain. Hippocampus tissue (approx 50 mg) was homogenized with ice-cold 500 µL mixture of radioimmunoprecipitation assay (RIPA) buffer supplemented with a cocktail of protease inhibitor and incubated on ice for 20 min. Tissue lysates were centrifuged at 10000 g for 10 min, at 4˚C, and supernatants were stored at -80˚C. Protein concentrations of each sample were measured by the Bradford method (Bradford, 1976). For western blot analysis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis 12

ACCEPTED MANUSCRIPT (PAGE) was carried out using a vertical mini gel system . Briefly, tissue protein samples were suspended in a sample buffer containing 2% SDS, 50 mM Tris–HCl buffer (pH 6.8), 10%

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glycerol (v/v) and 2-mercaptoethanol (5%) and heated at 100 °C for 5 m on a water bath.

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Equivalent (40 µg) amount of protein of protein was loaded onto 10% SDS-PAGE, and run at 100 V for around 120 min. Protein was transferred onto a polyvinylidene difluoride (PVDF)

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membrane using Thermo Scientific™ Pierce™ Power Blotter (Cat# 22834). The membrane was

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blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 3% bovine serum albumin (BSA) for 2 h at room temperature. After blocking the membrane the blots were probed

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over night at 4 ˚C, with PARP-1 Antibody (Cat# 9542; Cell signaling technology). After three washes in TBST for 5 min each, the membranes were incubated for 2 h, with horseradish

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peroxidase-conjugated secondary antibody (Santa Cruz). Following the post-secondary washes (3 × 5 min), the resulting antigen–antibody–peroxidase complexes visualized by incubating the

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membrane with 3,3′,5,5′-Tetramethylbenzidine (TMB) blotting solution (Cat# 37574, Thermo scientific) until the color was developed. Blot images were scanned and quantitative analysis of image was done using Image J software. Results were normalized with respect to β-actin. 2.9. Statistical analysis

All the experimental results were expressed as the mean ± standard error of mean (S.E.M.). All data were processed in Jandel Sigma Stat Version 3.5 software for the statistical analysis and P ˂ 0.05 was considered as significant. Comparisons between experimental and control groups were performed by one-way analysis of variance (ANOVA) followed by Tukey's test when appropriate. 3. Results

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ACCEPTED MANUSCRIPT 3.1. Effect of EDV pretreatment on LPS-induced anxiety-like behavior After 3 h of LPS administration anxiety-like behavior was assessed by performing EPM test,

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light-dark box test and OFT. In EPM test, LPS administration produced a marked decrease in the

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no. of closed arm entries (P < 0.01), no. of open arm entries (P < 0.05), % time spent in open arm

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(P < 0.001) and end-arm explorations (P < 0.01). However, EDV (10 mg/kg) pretreatment significantly reversed the LPS-induced alterations. When compared to the LPS-control group,

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EDV (10 mg/kg) showed a marked increase in the no. of closed arm entries (P < 0.01), no. of

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open arm entries (P < 0.05), % time spent in open arm (P < 0.01) and end-arm explorations (P < 0.05) (Table 1).In light-dark box test also, LPS administration exhibited a considerable effect on

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% time spent in the light compartment (P < 0.001), light-dark transitions (P < 0.01) and % time

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of risk assessment (P < 0.001) (Table 2). Whereas the pretreatment with EDV (10 mg/kg) exerted a notable ameliorating effect on % time spent in the light compartment (P < 0.01), light-

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dark transitions (P < 0.05) and on risk assessment time (P < 0.01) (Table 2). Similarly in the OFT, LPS injection produced a noteworthy difference between the number of central (P < 0.001) and peripheral crossings (P < 0.001), rearing (P < 0.001), and immobility time (P < 0.001). On the other hand pretreatment with EDV (10 mg/kg) notably reversed the number of central (P < 0.01) and peripheral crossings (P < 0.001) and rearings (P < 0.001). However, EDV (3 mg/kg) pretreatment didn’t produce any significant effect in all these tests, except on no. of central crossings in the OFT (P < 0.01) (Table 3). 3.2. Effect of EDV pretreatment on LPS-induced depressive-like behavior After 24 h of LPS administration depressive-like behavior was assessed by performing TST and FST. We found that LPS injection significantly (P < 0.01) increased the immobility time in TST

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ACCEPTED MANUSCRIPT as well as FST (Fig. 2A & 2B). Conversely, pretreatment with EDV (10 mg/kg) showed a marked (P < 0.01) decrease in the immobility time in FST and TST as well (Fig. 2A & 2B). In

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addition, LPS challenge produced marked (P < 0.001) anhedonia, which is evident from the

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reduction in the sucrose preference of LPS-control group as compared to normal control group (Fig. 2C). However, the pretreatment of EDV (10 mg/kg) significantly (P < 0.001) prevented the

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anhedoina (Fig. 2C).

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3.3. Effect of EDV pretreatment on LPS-induced oxido-nitrosative stress

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Oxido-nitrosative stress was assessed by performing, SOD, catalase (CAT), reduced glutathione (GSH), lipid peroxidation and nitrite assays. Single dose of LPS (1 mg/kg) administration

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significantly diminished the reduced glutathione level (P < 0.05), and markedly (P < 0.001)

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decreased the activities of SOD as well as CAT (Table 4). Conversely, treatment with EDV (10

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mg/kg) notably reversed the altered levels of reduced glutathione (P < 0.01), and markedly restored the activities of SOD (P < 0.01) and CAT (P < 0.001) (Table 4). Further, MDA and nitrite levels were found to be significantly (P < 0.001) increased by the treatment of LPS (Table 4). On the other hand, pretreatment with EDV (10 mg/kg) significantly diminished the MDA (P < 0.001) and nitrite (P < 0.01) levels as compared to LPS-control group. Moreover, pretreatment of EDV (3 mg/kg) didn’t produce any significant effect (Table 4). 3.4. Effect of EDV pretreatment on LPS-induced pro-inflammatory cytokines in the hippocampus To study the anti-inflammatory effect of EDV, we have estimated the pro-inflammatory cytokine levels in the hippocampus. LPS treatment significantly (P < 0.001) increased the IL-1β and TNFα level in the hippocampus (Table 5). However, pretreatment with EDV (10 mg/kg) significantly 15

ACCEPTED MANUSCRIPT (P < 0.01) decreased the LPS-induced hippocampal IL-1β and TNF-α level (Table 5). But, the pretreatment of EDV (3 mg/kg) didn’t produce any marked effect on LPS-induced pro-

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inflammatory cytokines.

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3.5 Effect of EDV pretreatment on LPS-induced alterations of corticosterone and BDNF level

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Single dose of LPS (1 mg/kg) administration exhibited a marked decline in BDNF (P < 0.001)

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and a prominent increase in corticosterone (P < 0.001) level (Fig. 3A & 3B). On the contrary, pretreatment with EDV (10 mg/kg) significantly (P < 0.01) reversed LPS-induced alterations of

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BDNF and corticosterone level (Fig. 3A & 3B).

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3.6. Effect of EDV pretreatment on LPS-induced alterations of hippocampal NAD level

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LPS administration produced a marked depletion in hippocampal NAD level. However,

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pretreatment with EDV (3 & 10 mg/kg) prominently restored the LPS-induced alteration in NAD level in the hippocampus (Fig. 4).

3.7. Effect of EDV pretreatment on the LPS-induced alteration of PARRP-1 Protein expression was detected by western blots analysis in the hippocampus tissue after 12 h of LPS administration. LPS injection (1 mg/kg) induced a marked (P < 0.001) increase in the activity of PARP-1. Conversely, pretreatment with EDV (3 & 10 mg/kg) significantly prevented the overactivation of PARP-1 (Fig. 5). 4. Discussion and conclusion In our previous study, we have reported that anti-inflammatory property of a PARP-1 inhibitor, 3-aminobenzamide

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effectively ameliorate

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LPS-induced

neurobehavioral

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neurochemical anomalies (Sriram et al., 2015b). The ameliorating effects of 3-aminobenzamide 16

ACCEPTED MANUSCRIPT were likely through attenuating the oxido-nitrosative stress and reducing neuroinflammation, as well as inhibiting PARP-1 over expression in the hippocampus (Sriram et al., 2015b). In the

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present study, we have tested the effect of a free radical scavenger, EDV pretreatment on LPS-

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induced anxiety and depressive-like behavior. Moreover, we have examined the effect EDV pretreatment on LPS-induced alterations of serum corticosterone level, hippocampal PARP-1

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expression, pro-inflammatory cytokines, oxido-nitrosative stress and BDNF. Results of the

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present study indicated that pretreatment of EDV (10 mg/kg) could efficiently block LPSinduced anxiety and depressive-like behavior. In addition, pretreatment of EDV showed the

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reversal of LPS-induced alterations of hippocampal PARP-1 over expression, pro-inflammatory

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cytokines, oxido-nitrosative stress, BDNF and serum corticosterone.

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Several studies demonstrated that oxido-nitrosative stress and neuroinflammation can play key roles in the development of LPS-induced anxiety and depressive-like behavior (Biesmans et al.,

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2013; Henry et al., 2008; Jangra et al., 2014b). It is evident that LPS causes oxido-nitrosative stress by the augmentation of pro-inflammatory cytokines production and via inducing the generation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) by numerous mechanisms (Cadenas and Cadenas, 2002). During the balanced physiological conditions the harmful effects of ROS and RNS are neutralized by the endogenous antioxidant defense systems which include nonenzymatic and enzymatic antioxidants, such as glutathione, vitamin C, bilirubin, superoxide dismutase, catalase etc (Khassaf et al., 2003). Upon the persistent exposure of stress, the antioxidant levels can diminish in turn that may result in several central nervous system (CNS) implications such as neuroinflammation, anxiety and depressive like behavior as well as depletion of neurotrophic factors in the brain (Biesmans et al., 2013; Dantzer et al., 2008; Henry et al., 2008; Salim et al., 2012). This condition could be ameliorated by employing 17

ACCEPTED MANUSCRIPT potential antioxidants (Jangra et al., 2014b; Tomaz et al., 2014; Wang et al., 2014). We have hypothesized that being a strong antioxidant, EDV can produce ameliorating effect on LPS-

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induced alterations of endogenous antioxidant mechanisms. As evident from the results, in the

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present study LPS injection produced a significant decrease in the hippocampal SOD, CAT, and GSH levels, which is a manifestation of oxido-nitrosative stress caused by LPS. But in contrary,

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pretreatment of EDV (10 mg/kg) prevented the alterations of SOD, CAT and GSH. Similarly,

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LPS injection caused an increase in the MDA and nitrite levels of hippocampus, when compared normal control group. Conversely, pretreatment of EDV (10 mg/kg) effectively attenuated the

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MDA and nitrite levels as compared to LPS-control group. This protective effect of EDV can be

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attributed to its strong antioxidant activity (Qi et al., 2004; Srinivasan and Sharma, 2011).

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Previous preclinical and clinical evidences showed that the development of mood disorders such as depression and anxiety was directly associated with incessant activation of innate immune

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system and inflammatory processes (Lotrich, 2014; Patki et al., 2013; Salim et al., 2012; Vogelzangs et al., 2013; Zeugmann et al., 2013). In the present study too, LPS-induced anxiety and depressive-like behavior was found to be associated with the raised levels of inflammatory mediators (IL-1β & IL-6). It has been corroborated that there is a CNS cytokine network which not only produces cytokines but also amplify cytokine signals which in turn have profound effects on neurotransmitters (Raison et al., 2006). In our study, pretreatment of EDV significantly prevented the LPS-induced elevation of IL-1β and IL-6 levels. Thus, it is sensible to infer that EDV is having strong anti-inflammatory effect (Qi et al., 2004). Further, LPS-induced pro-inflammatory cytokines may contribute to the development of mood disorders through numerous fundamental pathways; one of such pathways is to influence the HPA-axis (Miller et al., 2009). Results from clinical and preclinical studies show that impaired 18

ACCEPTED MANUSCRIPT glucocorticoid receptor expression and function is related to the HPA axis hyperactivity in depressed subjects (Schüle et al., 2009), which are further confirmed by previous postmortem

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studies (Cubała and Landowski, 2005; Webster et al., 2002). In our study, after 24 h of LPS

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administration, serum corticosterone level was found to be significantly increased when compared to the normal control group. But, the pretreatment of EDV (10 mg/kg) was found to

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reverse the LPS-induced alteration of serum corticosterone. We can infer that the potential

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antioxidant activity of EDV can be the reason behind its ameliorating effect on LPS-induced

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alteration of corticosterone.

Several research reports indicate that neurotrophic factors, particularly BDNF, might mediate the

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clinical effects of the drugs which are intended to treat anxiety and depressive disorders (Autry

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and Monteggia, 2012). It has been widely reported that BDNF could promote neuronal survival, growth and plasticity in the brain. As evident from various research reports, the decrease in the

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BDNF content of hippocampus could result in the reduction of hippocampal neurogenesis and in the development of mood disorders (Karege et al., 2005; Kimpton, 2012). In accordance, with earlier research reports, our study witnessed a significant decrease in the hippocampal BDNF content by LPS challenge. However, pretreatment of EDV (3 & 10 mg/kg) remarkably reversed the LPS-induced alterations of hippocampal BDNF content. By inhibiting the LPS-induced loss of hippocampal BDNF, EDV produced a significant neuroprotection. PARylation, a post-translational protein modification catalyzed by PARP-1, also found to be connected with inflammation. Previous studies of PARP-1 inhibition found to produce protective effect in several neurological implications (Sriram et al., 2014). In accordance with the existing literature, our previous study found that a PARP-1 inhibitor, 3-aminobenzamide could effectively reverse all the LPS-induced neurobehavioral and neurochemical anomalies (Sriram et 19

ACCEPTED MANUSCRIPT al., 2015b). In the present study, to check effect of LPS and EDV on PARP-1 activity, we performed western blotting analysis of hippocampal PARP-1 expression. Besides to the

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hippocampal PARP-1 expression, we also examined hippocampal NAD content. NAD is a

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substrate for PARP-1, thus by checking the NAD levels we can indirectly measure the activity of PARP-1. In our study, we observed that LPS produces a significant increase in hippocampal

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PARP-1 expression and decreases the NAD level as compared to normal control group.

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Depletion of hippocampal NAD level indicates the overactivation of PARP-1 enzyme. However, pretreatment of EDV (3 & 10 mg/kg) found to prevent the LPS-induced over expression of

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hippocampal PARP-1 and also prevents the depletion of hippocampal NAD pool. Therefore, we can presume that EDV could efficiently block the LPS-induced over activity of PARP-1, by

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virtue of its free radical scavenging activity. Several research reports indicate that there is co-morbidity between depression and sickness

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behaviors since inflammatory pathways are alike in the pathogenesis of these behavioral disorders (Berk et al., 2013; Sulakhiya et al., 2015). As evident from the results of previous studies and our present investigation, peripheral LPS challenge can cause a systemic inflammation by increasing the production of pro-inflammatory mediators, such as TNF-α, IL-6, IL-1β which produce both sickness behavior and depressive-like behavior (Henry et al., 2009; Sulakhiya et al., 2015). In the present study, peripheral LPS administration found to produce anxiety-like behavior, which is evident from the results of behavioral experiments. In the lightdark test, mice exhibiting higher levels of anxiogenic-like behavior will make fewer transitions between the light area and the dark compartments (Buccafusco JJ, 2009). Stretch attend postures are “risk-assessment” behaviors which indicate that the animal is hesitant to move from its present location to a new position (Blanchard et al., 2001) and thus a high frequency of these 20

ACCEPTED MANUSCRIPT postures indicates a higher level of anxiety. In the present study, we’ve observed a marked reduction in the % time spent in the light compartment, increase in the % time of risk assessment

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in the light-dark test and reduction in light-dark transitions which clearly exhibit the anxiety-like

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behavior of LPS-treated group as compared to the control group. Similarly, in the OFT, an increase in central and peripheral crossings or an increase in time spent in the central part of the

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open field can be interpreted as an anxiolytic-like effect (Prut et al., 2003). In the present study,

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LPS exposure exhibited a significant decrease in the central and peripheral crossings, rearing movements and raised immobility time in the OFT, which certainly corroborate the anxiety-like

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behavior caused by LPS challenge. Similarly, in the literature we can observe that, in EPM test, anxiolytic drugs specifically increase, and anxiogenic drugs specifically decrease, the number of

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entries into the open arms and the time spent there (Komada et al., 2008). In the present study also, during EPM test LPS injection showed anxiety-like behavior, which is evident from the

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significant reduction in the number of entries in open and closed arms, % time duration in open arm, and end-arm explorations as compared to the vehicle-treated group. The anxiety-like behavior of LPS-treated mice observed in behavioral test might be due to eventual increase in pro-inflammatory cytokines, oxidative stress or by increase incorticotrophin-releasing hormone (CRH) that is evoked by the exposure of LPS (Agelaki et al., 2002; Koo and Duman, 2009; Risbrough and Stein, 2006). In all of these anxiety testing paradigms pretreatment with EDV (10 mg/kg) exhibited significant ameliorating effect against LPS-induced anxiety-like behavior possibly by reversing the LPS-induced alterations of pro-inflammatory cytokines, oxidonitrosative stress, corticosterone and BDNF. From the findings of numerous studies, it is clear that LPS exposure can cause depressive-like behavior eventually due to its aggressive pro-inflammatory activity (Song and Wang, 2011). In 21

ACCEPTED MANUSCRIPT the present study, LPS injection found to cause depressive-like behavior which is evident from the increased immobility time both in TST and FST. Pretreatment of EDV (10 mg/kg) for 14

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days was found to prevent the LPS-induced increase in the immobility time, via inhibiting of

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oxido-nitrosative stress as well as decreasing the inflammation. Anhedonia is a basic typical feature of depressive-like behavior (Gorwood, 2008). It is the loss of interest in previously

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rewarding or enjoyable activities. In the present study we’ve assessed anhedonic behavior by

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measuring the intake of sucrose solution. As anticipated, upon LPS injection, we found a significant decrease in the sucrose solution consumption. Conversely, EDV (10 mg/kg)

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pretreatment exhibited marked preference for sucrose solution as compared to LPS-control group. It could be inferred from the findings of sucrose preference test, that the anhedonia caused

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by the LPS challenge was prevented by the pretreatment of EDV (10 mg/kg). In summary, the results of the present study demonstrated the protective effect of EDV in LPS-

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induced anxiety and depressive-like behavior in mice. This protective effect is mainly due to its interference with oxido-nitrosative stress and inflammation cascade by the inhibition of PARP-1 (Sriram et al., 2015b). Moreover, eventual prevention of the alterations in corticosterone and BDNF are also having prominent role in the protective effect of EDV against LPS administration. Thus, our present study unravels the perspective mechanisms of protective effect of EDV against neuropsychiatric disorders associated with inflammation and oxido-nitrosative stress. However, presently one could not suggest the employment of EDV alone for mood disorders, as further studies are still in need to establish its safety and efficacy for treating anxiety and depressive-like behavior. Acknowledgement

22

ACCEPTED MANUSCRIPT We would like to express our sincere gratitude to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for the project grant. The authors are

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enormously thankful to the Institutional Level Biotech hub, NIPER Guwahati and State Biotech

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Hub, College of Veterinary Sciences, Guwahati for providing technical support. Authors are immensely thankful to Mr. Ravi Kishore Neredumelli, MS (Pharmacology & Toxicology),

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NIPER, Guwahati for offering unconditional support throughout the laboratory work.

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Zunszain, PA, Anacker, C, Cattaneo, A, Carvalho, LA, Pariante, CM. Glucocorticoids, cytokines and brain abnormalities in depression. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2011;35:722-729.

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ACCEPTED MANUSCRIPT Figure legends:

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Fig.1. Diagrammatic representation of study plan. Saline and edaravone (3 & 10 mg/kg) treatment was continued for 14 days. On the 14th day, 30 min after the saline and edaravone treatment, LPS (1 mg/kg, i.p.) was administered. After 3 h of LPS challenge, anxiety-like behavior was assessed by performing elevated plus maze test, open field test and light–dark box test. After 12 h of LPS administration, hippocampal PARP-1 expression was detected by western blotting analysis. After 24 h of LPS administration depressive-like behavior was tested by performing tail suspension test (TST) and forced swimming test (FST). All the biochemical analyses were carried out after 24 h of LPS injection. In addition, the anhedonic response was examined for 24 h, by performing sucrose preference test after 24 h of LPS administration.

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Fig.2. Effect of edaravone pretreatment on the LPS-induced depressive-like behavior and anhedonia which was assessed by: (A) tail suspension test (TST), (B) forced swimming test (FST) and (C) Sucrose preference test. All values are expressed as mean ± SEM (n = 6). ##P < 0.01, ###P < 0.001, compared with control group; *P < 0.05, **P < 0.01 and ***P < 0.001 compared with LPS-control group.

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Fig.3. Effect of edaravone pretreatment on the LPS-induced changes in (A) hippocampal BDNF and (B) serum corticosterone. All values are expressed as mean ± SEM (n = 6). ###P < 0.001 compared with control group; *P < 0.05, **P < 0.01 compared with LPS-control group.

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Fig.4. Effect of edaravone pretreatment on the LPS-induced changes in the hippocampal NAD level. All values are expressed as mean ± SEM (n = 6). ##P < 0.01 compared with control group; * P < 0.05, **P < 0.01 compared with LPS-control group. Fig.5. Effect of edaravone pretreatment on the LPS-induced PARP-1 expression. Protein expression was detected by western blots analysis in the hippocampus tissue after 12 h of LPS administration. Statistical analysis was performed using a one-way ANOVA followed by the Tukey’s test. All values are expressed as mean ± SEM (n = 6). ###P < 0.001 compared with control group; **P < 0.01, ***P < 0.005 compared with LPS-control group.

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Fig.2. (A)

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Fig.1

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Fig.2. (C)

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Fig.2. (B)

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Fig.3. (A)

Fig.3. (B)

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Fig.4

Fig. 5. PARP-1(116KDa) PARP-1 (89KDa) Β-actin (42KDa)

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Table 1

Control

LPS-Control

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Parameter

EDV 10 mg/kg + LPS 9.8 ± 0.89**

3.6 ± 0.54#

4.8 ± 0.64

7.2 ± 0.95

19.45 ± 0.69###

20.66 ± 1.32

29.97 ± 1.72

1 ± 0.12##

2.23 ± 0.92

4.39 ± 0.59*

5.13 ± 0.9

No. of entries in open arm

7.5 ± 1.23

% Time spent in open arm

35.12 ± 1.51

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* **

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10.9 ± 1.07

5 ± 0.97

EDV 3 mg/kg + LPS 5.87 ± 0.93

No. of entries in closed arm

End-arm explorations

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Effect of edaravone pretreatment on LPS induced changes on the exploratory behavior of mice in the elevated plus-maze test. Values are expressed as the mean ± SEM (n = 8 mice/group). #P < 0.05, ##P < 0.01 and ###P< 0.001 compared with control group; *P < 0.05 and **P < 0.01 compared with LPS-control group

Table 2

Parameter % Time spent in light compartment Light-dark transitions % Time of risk assessment

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Effect of edaravone pretreatment on LPS induced changes on the exploratory behavior of mice in the light-dark transition test. Values are expressed as the mean ± SEM (n = 8 mice/group). ##P < 0.01 and ###P < 0.001 compared with the control group; *P < 0.05 and ** P < 0.01 compared with LPS-control group.

Control

LPS-Control

EDV 3 mg/kg + LPS

EDV 10 mg/kg + LPS

29.20 ± 1.51

10.96 ± 1.10###

14.03 ± 0.67

19.37 ± 1.06**

58.89 ± 6.23

25.64 ± 4.89##

34.12 ± 6.87

53 ± 8.21*

3.285 ± 0.21

8.16 ± 0.40###

7.55 ± 0.50

5.38 ± 0.26**

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Table 3

46 ± 2.87

19.25 ± 3.39###

20 ± 1.29 32.75 ± 1.49

8.5 ± 0.64### 19.5 ± 1.04###

EDV 3 mg/kg + LPS

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Control

EDV 10 mg/kg + LPS

23.25 ± 1.88

33.70 ± 2.04**

16.75 ± 2.21** 20.25± 0.85

20.5 ± 1.91*** 29.19 ± 2.13***

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Parameter Peripheral crossings Central crossings Rearings

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Effect of edaravone pretreatment on LPS induced changes on the exploratory behavior of mice in the open-field test. Values are expressed as the mean ± SEM (n = 8 mice/group). ###P < 0.001 compared with the control group; **P < 0.01 and ***P < 0.001 compared with LPS-control group

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Effect of edaravone pretreatment on the LPS-induced oxido–nitrosative stress in the hippocampus which was assessed by: superoxide dismutase (SOD) activity, catalase activity, reduced glutathione estimation, malondialdehyde (MDA) estimation, and nitrite estimation. All values are expressed as mean ± SEM (n = 6). #P < 0.05 and ###P < 0.001 control group; **P < 0.01 and ***P < 0.001 compared with LPS-control group

Parameter Superoxide dismutase (SOD) activity (U/mg of protein) Catalase (mMol/min/mg of protein) Reduced Glutathione (µg/g of tissue) Malondialdehyde (MDA) (µM/mg of protein) Nitrite content (µM/mg of tissue)

Control

LPS-Control

EDV 3 mg/kg + LPS EDV 10 mg/kg + LPS

8.46 ± 0.67

4.75 ± 0.41###

6.31 ± 0.80

8.18 ± 0.21**

49 ± 6.06

23.33 ± 2.66 ###

37 ± 2.74

51 ± 2.1***

1958.33 ± 228.80

1239.33 ± 118.36#

1845.83 ± 118.87

2056.83 ± 149.42**

121.83 ± 11.71

230.16 ± 14.72###

196 ± 15.95

126.33 ± 13.60 ***

3.71 ± 0.33

6.91 ± 0.53 ###

5.83 ± 0.53

4.11 ± 0.37**

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Table 5

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Effect of edaravone pretreatment on the LPS-induced alterations of hippocampal IL-1β and TNFα level. All values are expressed as mean ± SEM (n = 6). ###P < 0.001compared with control group; **P < 0.01 compared with LPS-control group

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LPS 13.70 ± 0.92### 10.43 ± 0.68###

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Control 7.23 ± 0.79 5.95 ± 0.75

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Parameter IL-1β in Hippocampus (pg/ml) TNF-α in hippocampus (pg/ml)

EDV 3 mg/kg + LPS EDV 10 mg/kg + LPS 10.68 ± 0.93 8.66 ± 0.45** 9.33 ± 0.49 7.15 ± 0.49**

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Highlights

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Edaravone (EDV) pretreatment reversed the LPS-induced neurobehavioral anomalies EDV pretreatment prevented LPS-induced oxido-nitrosative stress EDV pretreatment ameliorated anomalous expression of PARP-1 and neuroinflammation EDV pretreatment reversed LPS-induced serum high corticosterone and low BDNF levels

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