Edaravone alleviates cisplatin-induced neurobehavioral deficits via modulation of oxidative stress and inflammatory mediators in the rat hippocampus

Author’s Accepted Manuscript Edaravone alleviates cisplatin-induced neurobehavioral deficits via modulation of oxidative stress and inflammatory mediators in the rat hippocampus Ashok Jangra, Mohit Kwatra, Tavleen singh, Rajat Pant, Pawan Kushwah, Sahabuddin Ahmed, Durgesh Dwivedi, Babita Saroha, Mangala Lahkar

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To appear in: European Journal of Pharmacology Received date: 23 December 2015 Revised date: 29 July 2016 Accepted date: 1 August 2016 Cite this article as: Ashok Jangra, Mohit Kwatra, Tavleen singh, Rajat Pant, Pawan Kushwah, Sahabuddin Ahmed, Durgesh Dwivedi, Babita Saroha and Mangala Lahkar, Edaravone alleviates cisplatin-induced neurobehavioral deficits via modulation of oxidative stress and inflammatory mediators in the rat h i p p o c a m p u s , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.08.003 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 galley proof before it is published in its final citable 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.

Edaravone alleviates cisplatin-induced neurobehavioral deficits via modulation of oxidative stress and inflammatory mediators in the rat hippocampus Ashok Jangra1, Mohit Kwatra1, Tavleen singh1, Rajat pant1, Pawan Kushwah1, Sahabuddin Ahmed1, Durgesh Dwivedi1, Babita Saroha2, Mangala Lahkar3* 1

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education

and Research Guwahati, Guwahati, Assam, India 2

Department of Biotechnology, University Institute of Engineering & Technology (UIET),

Maharshi Dayanand University, Rohtak, Haryana, India 3

Department of Pharmacology, Gauhati Medical College, Guwahati, India

[email protected] [email protected] *

Corresponding author at: Department of Pharmacology, Gauhati Medical College, Guwahati,

Assam- 781032, India Tel: +91 9706806533; Fax: +91 3612337700.

Abstract Cisplatin is a chemotherapeutic agent used in the treatment of malignant tumors. A major clinical limitation of cisplatin is its potential toxic effects, including neurotoxicity. Edaravone, a potent free radical scavenger, has been reported to have the neuroprotective effect against neurological deficits. The aim of the present study was to determine the neuroprotective effect of edaravone against cisplatin-induced behavioral and biochemical anomalies in male Wistar rats. Our results showed that cisplatin (5 mg/kg/week, i.p.) administration for seven weeks caused marked 1

cognitive deficits and motor incoordination in rats. This was accompanied by oxido-nitrosative stress, neuroinflammation, NF-κB activation and down-regulation of Nrf2/HO-1 gene expression level in the hippocampus. Edaravone (10 mg/kg/week, i.p.) treatment for seven weeks inhibited the aforementioned neurobehavioral and neurochemical deficits. Furthermore, edaravone was found to up-regulate the gene expression level of Nrf2/HO-1 and prevented the cisplatin-induced NF-κB activation. These findings demonstrated that oxido-nitrosative stress and inflammatory signaling mediators play a key role in the development of cisplatin-induced neurobehavioral deficits which were prevented by edaravone treatment. Keywords: Cisplatin; Edaravone; Oxido-nitrosative stress; Neurobehavioral deficits; Neuroinflammation

1. Introduction Cisplatin (cis-diamminedichloridoplatinum(II)-CDDP), is a well-known platinum-based chemotherapeutic agent that is extensively used for the treatment of various malignant tumors in the breast, bladder, head, neck, ovary, and testicles (McWhinney et al., 2009; Stathopoulos, 2010). The mechanism of action of cisplatin has been associated with its ability to interfere with DNA replication and/or transcription process and DNA repair mechanism that eventually leads to oxidative stress and mitochondria-dependent apoptosis (Podratz et al., 2011). Moreover, the cisplatin-induced neurotoxicity occurs due to increased oxido-nitrosative stress, proinflammatory cytokines, mitochondrial dysfunction, DNA damage and apoptotic cell death resulting in various morphological changes in the neurons such as axonal shrinkage and demyelination (Sayre et al., 2008; Tuncer et al., 2010). Cisplatin also affects the function of neural progenitor cells in the 2

hippocampus region by repressing cell proliferation and neurogenesis (Piccolini et al., 2012; Hinduja et al., 2015) Increased level of oxidants causes the imbalance between inflammatory and anti-inflammatory mediators. The cisplatin-induced toxicity which is mediated through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) which in turn increases inflammatory mediators that further responsible for altering the long-term potentiation (LTP) in the hippocampus region (So et al., 2007; Kim et al., 2014). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a key role in the protection against free radical scavenger by regulating the expression of several antioxidant/detoxification genes such as heme oxygenase-1 (HO-1) and glutathione S-transferases. Therefore, oxidative stress-mediated neurotoxicity can be alleviated by activating the Nrf2/HO-1 pathway (Ye et al., 2016). Previous experimental studies in animals demonstrated that cisplatin treatment-induced cognitive dysfunction and motor impairment due to dysregulation of hippocampal and cerebellar functions (Shabani et al., 2012a, b). Moreover, cisplatin-induced oxidative stress raises the level of acetylcholinesterase (AChE) in the hippocampus (Chtourou et al., 2015). The exact mechanism underlying the cisplatin-induced behavioral deficits still remains elusive. Several antioxidant molecules such as coenzyme Q10 (Da Silva et al., 2013), cyanidin (Li et al., 2015), walnut (Shabani et al., 2012 b), D-Methionine (Gopal et al., 2012), alpha lipoic acid, melatonin (Tuncer et al., 2010) and curcumin (Mendonca et al., 2013) have been reported to prevent cisplatin and its derivatives-induced neurotoxicity. Edaravone (3-methyl-1-phenyl-2pyrazolin-5-one) is a novel antioxidant with the potent free radical scavenging property (Fig. 1). Previous reports suggested its protection in various animal models such as diabetic stroke (Srinivasan and Sharma, 2012), ischemic reperfusion injuries (Yoshida et al., 2006), restraint 3

stress (Jangra et al., 2016d), lipopolysaccharide-induced behavioral anomalies (Sriram et al., 2016), Alzheimer’s disease (Jiao et al. 2015), Parkinson’s disease (Xiong et al., 2011), and amyotrophic lateral sclerosis (Nagase et al., 2015). Based on significant protective profiles of edaravone, it is worthwhile to evaluate its neuroprotective potential against cisplatin-induced neurotoxicity. Therefore, in present study we have investigated the possible effect of edaravone in cisplatin induced-neurotoxicity model of learning and memory deficits by carrying out certain behavioral studies and measured oxidative stress markers, proinflammatory cytokines, acetylcholinesterase (AChE) activity, brain-derived neurotrophic factor (BDNF) and gene expression levels of NF-κB, Nrf2, HO-1 in the hippocampus. 2. Material and Methods 2.1 Chemicals Edaravone, cisplatin, Griess reagent, thiobarbituric acid, 5,5′-dithiobis-(2-nitrobenzoic acid) SOD assay kit, catalase assay kit were purchased from Sigma-Aldrich, St. Louis, MO, USA. Interleukin-1β, tumor necrosis factor-α (Thermo Fisher Scientific, India) ELISA kits, BDNF Emax® ImmunoAssay kit (Promega, Madison, WI, USA) were used. Total RNA extraction kit (Hi-Media India), RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, India), and primers (Imperial life sciences (P) Limited India) were purchased. All other chemicals used in the experimental study were of analytical grade. 2.2 Animals Male Wistar rats (weight 150-200 g; 5-6 weeks old) were used in the present study. Animals were procured from the Gauhati Medical College, Guwahati, and acclimatized to laboratory conditions for 7 days before commencement of the experiment. Rats were housed in groups of

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four per cage and were given food and water ad libitum. The study was approved (approval no. MC/05/2015/58) by the Institutional Animal Ethics Committee (IAEC), Gauhati Medical College and Hospital (CPCSEA Registration No. 351, 3/1/2001). All the experiments were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The animals were housed at room temperature (24 ± 1 °C), with 65 ± 5% humidity, and 12 h light and dark cycles. 2.3 Experimental Design Cisplatin (5 mg/kg) and edaravone (10 mg/kg) were prepared freshly in saline (0.9% NaCl) and were administered intraperitoneally (i.p.) once in a week for 7 weeks. The animals were randomly divided into four experimental groups: Group1 as Control group: normal saline (0.9%) was administered per week for 7 weeks. Group 2 as Cisplatin group: Cisplatin-5 mg/kg was administered per week for 7 weeks. Group 3 as edaravone and cisplatin treated group: Edaravone and cisplatin at the dose of 10 and 5 mg/kg respectively were administered per week for 7 weeks. Group 4 as only Edaravone treated group: Edaravone at the dose of 10 mg/kg was administered per week for 7 weeks. The doses of cisplatin and edaravone were selected based on previous experimental reports (Sriram et al., 2012; Oz et al., 2015). Behavioral studies were performed from 46th to 50th day of the study (Fig. 2.). The experimental groups consisted of 18 animals each. Due to animal’s mortality in certain groups, 12 animals were randomly taken further to carry out the different behavioral assessment, biochemical evaluation and gene expression studies of NF-κB, Nrf2, and HO-1 by reverse transcriptase PCR. Moreover, a gap of 3-4 h was maintained for different behavioral assessments in different sets of animals. At the end of the experiment, animals were killed by cervical dislocation. The hippocampus was quickly dissected

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out from the isolated brain and homogenized in ice-cold phosphate buffer saline (pH 7.4). Homogenates samples were preserved at -80 °C for further biochemical estimation. 2.4 Behavioral Studies 2.4.1 Morris water maze (MWM) test MWM test was performed to assess the learning and memory function after cisplatin administration (Vorhees and Williams, 2006). In brief, circular tank (145 cm diameter and 50 cm height) consisted of four equal quadrants containing opaque water (26 ± 1 °C) was used. The platform (10 cm diameter) was placed 2 cm below the water level in the acquisition phase. The animal was placed in the pool facing towards the tank wall and allowed to search platform for 120 s during the acquisition phase. If the animal failed to locate the platform in 120 s, then the animal was guided towards the way to platform and remain on the platform for further 30 s. Each animal was trained through four different quadrants to locate the platform for four consecutive days. On the fifth day, the single probe trial was performed for 120 s by removing the platform. The retention memory of trained animals was recorded in terms of time spent in the target quadrant. 2.4.2 Novel object recognition test (NORT) NORT was performed to investigate the recognition memory according to the method described by Bevins and Besheer, 2006. NORT consisted of three phases: habituation, familiarization, and test phase. A black-colored open field box (50 × 50 × 36 cm3) was used in this test. In the habituation phase, each animal was habituated to an empty open field by placing it in the open field arena and allowed to explore for 5 min twice in a day. After habituation phase, familiarization phase was performed by placing the two objects (a rectangular wooden block and 6

a small rubber ball) at the left and right position in the open field apparatus. The rat was placed in an open field with its head position opposite to the objects and allowed to explore freely for 10 min. After 24 h, test session was conducted by replacing the wooden block with a novel object (a plastic box). Each rat was allowed to explore for 3 min in the open field. In all phases of the test, objects and open field arena were repeatedly wiped with alcohol (70% v/v) to avoid the olfactory cues. Time spent to explore the familiar and novel object was recorded during the experiment. Recognition index, a ratio of the time spent in exploring novel object (TN) over the total time spent in exploring familiar object (TF) and novel object (TN) during test session was calculated, and results were represented as a recognition index in percentage (%). 2.4.3 Rotarod test Rotarod test was performed to assess the effect of cisplatin treatment on motor coordination using a rotarod apparatus. Rotating speed and cut-off time were fixed at 15 rpm and 180 seconds respectively (Jangra et al., 2014a). Initially, Rats were acclimatized for two days and on the third day test session were carried out after 3 hrs of the last dose of cisplatin and edaravone. The time taken by the animal to drop off the rod was measured. The incidence of ataxia, i.e. the ability of the rat to fall was recorded in different experimental groups. 2.4.4 Open field test The Open field test was performed to determine the effect of cisplatin on locomotor and exploratory alterations in rodents. The test apparatus consisted of acrylic transparent box (72 × 72 × 36 cm3) with its floor divided into 16 equal sized squares (18 × 18 cm2). The four middle squares were considered as the center region, and the other 12 outer squares were considered as the peripheral region. Each rat was put in the center of the box, and number of central as well as

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peripheral crossing and rearing movements of the rat were observed for 10 min by a video camera (Jangra et al., 2014b). 2.5 Biochemical parameters estimation 2.5.1 Malondialdehyde (MDA) level MDA content in the hippocampus was determined quantitatively by using the method of Ohkawa et al. 1979. In brief, 0.1 ml of hippocampal homogenate sample was added to a mixture of 0.75 ml of 0.8 % thiobarbituric acid, 0.1 ml of 8.1 % Sodium dodecyl sulfate (SDS), and 0.75 ml of 20 % acetic acid solution (pH 3.4). The resultant mixture was kept on a water bath at 95 °C for subsequent one hour, and then cooled and centrifuged at 9600 g for 10 min. The absorbance was measured at 532 nm, and results are expressed as micromoles (µM) of MDA per gram tissue weight. 2.5.2 Nitrite assay Nitrite content in the hippocampus was determined quantitatively by the method of Green et al. (1982). Equal amount of sample and Griess reagent (containing 0.1% N-(1- naphthyl) ethylene diamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) were mixed and then incubated in a dark place. After 15 min, absorbance was measured at 540 nm. The nitrite level was calculated and expressed as micromoles of GSH per milligram of protein. 2.5.3 Reduced glutathione (GSH) level The reduced glutathione content in the hippocampus was estimated by using the method of Beutler et al. 1963. Briefly, the supernatant sample was mixed with 10% trichloroacetic acid (TCA) in 1:1 ratio. Afterwards, the resulted mixture was centrifuged at 1000g for 10 min at 4 °C. The obtained supernatant was mixed with 400 µl of 0.3 M disodium hydrogen phosphate and 50

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µl of 0.001 M 5, 5′-dithiobis (2-nitro benzoic acid) (DTNB). The absorbance was taken at 412 nm and the GSH level was calculated and represented as µM of GSH per milligram of protein. 2.5.4 Superoxide dismutase (SOD) activity The SOD assay kit was used for the quantitative determination of Superoxide dismutase (SOD) in the hippocampus of rat brain. Protein level was measured by the method of Lowry et al., (1951). SOD activity was determined according to the manufacturer's instructions. Absorbance was recorded at 440 nm in the microplate reader, and the results were expressed as U/mg of protein. 2.5.5 Catalase activity The catalase assay kit was used to investigate the enzymatic activity in the peroxidation function of catalase in the hippocampus of rat brain. Catalase activity was estimated using the manufacturer's instructions provided with the kit. The absorbance of red quinoneimine dye formed by oxidative coupling reaction of 4-aminophenazone with 3,5-dichloro-2hydroxybenzenesulfonicacid was recorded at 520 nm. Results were calculated and represented as mMol/min/mg of protein. 2.5.6 Acetylcholinesterase (AChE) assay Acetylcholinesterase (AChE) activity was determined by using the method of Ellman et al. (1961). Briefly, 0.4 ml of homogenate was mixed with 2.6 ml phosphate buffer (0.1 M, pH 8) and 100 μl of DTNB in a cuvette. Thereafter, 20 μl of acetylthiocholine was added, and changes in absorbance were recorded for a period of 5 min at intervals of 1 min on 412 nm. The AChE enzyme activity was calculated using the formula R=A×106/13,600, where R=micromoles of

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substrate hydrolyzed per minute and A=change in absorbance per minute. The AChE activity was calculated and expressed as µM/min/mg of protein. 2.5.7 Estimation of proinflammatory cytokines, BDNF level Proinflammatory cytokines (IL-1β and TNF-α) and BDNF level in the hippocampus was measured by performing enzyme-linked immunosorbent assay (ELISA). The hippocampus was quickly isolated on the chilled Petri dish and was homogenized in ice-cold phosphate buffered saline (pH 7.4) containing protease inhibitor cocktail and phenylmethane sulfonyl fluoride (PMSF). Then, the homogenate was centrifuged at 18,800 g at 4 °C and the supernatant was collected. Interleukin-1β and tumor necrosis factor-α ELISA kit protocols were followed to determine the proinflammatory cytokines level. BDNF level was estimated through the BDNF Emax® ImmunoAssay System. The absorbance was taken at 450 nm. Proinflammatory cytokine levels were calculated and expressed as picogram per ml (pg/ml), while BDNF content was expressed as nanogram (ng) per mg of protein. 2.6 Determination of mRNA expression level of Nrf2, HO-1 and NF-κB by reverse-transcriptase PCR Total RNA was isolated from the freshly isolated hippocampus of rat brain using HiPuraA™ Total RNA Miniprep purification kit. 1 μg of RNA was diluted in 20 μl of retrotranscription reagent and used to synthesize cDNA with the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, India) and the tube was incubated at 25 °C for 10 min and then at 42 °C for 1 h in a thermal cycler. The reaction was terminated by heating at 70 °C for 10 min, and the cDNA was stored at -80 °C for further use. The cDNA samples were used as templates for subsequent PCR amplification using gene-specific primers. Initially, annealing temperature, the number of cycles, and annealing conditions were optimized for each primer pairs. The primer 10

sequences used for Nrf2 forward 5’-GAGACGGCCATGACTGAT-3’ & reverse 5’GTGAGGGGATCGATGAGTAA-3’, HO-1 forward 5′-AAGAGGCTAAGACCGCCTTC-3′ & reverse 5′-GCATAAATTCCCACTGCCAC-3′, NF-κB forward: 5′-GCTTTGCA AACCTGGGAATA-3′ & reverse 5′-CAAGGTCAGAATGCACCAGA-3’ and β-actin, forward 5’-GGGAAATCGTGCGTGACATT-3’ and reverse 5’-GCGGCAGTGGCCATCTC-3’ (Ueyama et al., 2009; Palsamy et al., 2011; Wang et al., 2012; Pakzad et al., 2013). The PCR products were resolved on 2% agarose gel amplified using optimal PCR conditions. Gel image was taken by Bio-Rad's Gel Doc XR+ system and image analysis was carried out with the help of Bio-Rad's Image Lab software. 2.7 Statistical analysis All the results are expressed as mean ± standard error of mean (S.E.M.). The intergroup variation was analyzed statistically using one-way analysis of variance (ANOVA) followed by post hoc analysis with Tukey’s test. Two-way ANOVA followed by Bonferroni analysis was applied to body weight changes and escape latency (MWM test). The value of P < 0.05 was considered statistically significant. GraphPad Prism 5.0 Version Software (San Diego, CA, USA) was used for statistical analysis. 3. Results 3.1 Body weight changes and mortality during the study The health condition of the animals was observed on the daily basis and body weight of the animals in all groups was observed once in a week. In cisplatin group, one animal in a week died starting from fourth to seventh week of the study. Furthermore, cisplatin + edaravone group also exhibited some effects of toxicity where one animal in a week died from fifth to seventh week of

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the study (Fig. 3A and B). These animals were found sick and had intense weight loss throughout the study. Rest of the groups was found healthy with no mortality. Two-way ANOVA revealed that the animal group treated with cisplatin had significant (P < 0.001) lower body weight from the fourth week until the end of the experiment when compared with control group animals (Fig. 4). Edaravone treatment significantly (P < 0.01) ameliorated the cisplatin-induced weight loss on the sixth and seventh week when compared with cisplatin treated group. Furthermore, the significant difference in the body weight was found in the fifth (P < 0.01), sixth (P < 0.001) and seventh week (P < 0.001), respectively between control group and cisplatin + edaravone group. This relevant comparison provides the evidence of edaravone efficacy against cisplatin-induced weight reduction. 3.2 Behavioral assessment 3.2.1 Effect of edaravone treatment on the cognitive function in cisplatin-treated animals In order to determine the cisplatin-induced memory impairment, the MWM test was performed. Two-way ANOVA analysis revealed that mean escape latency of cisplatin-treated group was significantly (P < 0.001) higher from the second day to the fourth day trial as compared to the control group animals (Fig. 5A). The edaravone treatment in cisplatin rats caused marked reduction in mean escape latencies on the second day (P < 0.01), third (P < 0.001) and the fourth day (P < 0.001) of the training period. On the fifth day of single probe trial (50th day of experiment), the time spent by the animal in the target quadrant was calculated. Cisplatin-treated animals spent significantly (P < 0.001) less time in the target quadrant as compared to the control group (Fig. 5B). However, edaravone treated rats spent significantly (P < 0.01) more time in the target quadrant as compared to cisplatin treated animals. The swimming speed across all groups did not alter in the probe trial (Fig. 5C). 12

Additionally, novel object recognition test (NORT) was performed to determine the effect of cisplatin treatment on recognition memory. The cisplatin-treated group showed significantly (P < 0.001) lower preference towards novel object as compared to control group which indicates recognition memory impairment (Fig. 6A). However, edaravone treatment significantly (P < 0.05) increased the recognition index (%) when compared with cisplatin-treated animals. Edaravone alone did not alter the recognition memory and thus, the recognition index (%) was found to be unaltered and similar to control group. On the other hand, cisplatin + edaravone group exhibited lower (P < 0.001) recognition index compared with control group. We also observed the total exploration time for both objects (familiar object and novel object) in both training as well as in the test phase and found to be unaltered among all experimental groups (Fig. 6B). 3.2.2 Effect of edaravone treatment on the motor coordination in cisplatin-treated animals Fig. 7 shows that cisplatin treatment significantly (P < 0.001) reduced the time of fall that depicts the motor incoordination in cisplatin-treated group. While edaravone treatment significantly (P < 0.01) improved the muscle coordination as evident by the increase in the latency to fall from rotarod apparatus as compared to the cisplatin-treated group. Edaravone alone did not alter the motor co-ordination in the rotarod test compared to normal control group. In addition, significant difference (P < 0.001) was observed between control and cisplatin + edaravone groups. 3.2.3. Effect of edaravone treatment on the open field test parameters in cisplatin-treated animals The open-field test was carried out to evaluate locomotor activity and anxiety-related behavior after cisplatin administration. We found that cisplatin significantly affected the number of central crossings (P < 0.001), peripheral crossings (P < 0.001) and rearings (P < 0.01) as compared to normal control group (Table 1) Edaravone treatment significantly increased the number of 13

central crossings (P < 0.01), peripheral crossings (P < 0.05), rearings (P < 0.05) as compared to cisplatin treated group. Edaravone alone treatment did not affect any of these parameters compared to normal control group. However, the significant difference was observed in central (P < 0.001) and peripheral crossings (P < 0.01) between the control group and cisplatin + edaravone groups. 3.3 Biochemical estimation 3.3.1 Effect of edaravone treatment on hippocampal oxido-nitrosative stress in cisplatin treated animals Cisplatin administration significantly raised the level of oxidative stress markers in the hippocampus region as indicated by increased MDA (P < 0.001) and nitrite (P < 0.001) level as compared to normal control group (Table 2). In addition, reduced glutathione level, SOD activity, and catalase activity were found significantly (P < 0.001) lower in the hippocampus of the cisplatin-treated group when compared with control group (Table 2). Concomitant treatment with edaravone (10 mg/kg) significantly reduced the cisplatin-induced elevated MDA (P < 0.001) and nitrite (P < 0.001) level. Moreover, edaravone treatment significantly raised the reduced glutathione (P < 0.001) level, SOD activity (P < 0.05) and catalase activity (P < 0.001) when compared with cisplatin treated group. Edaravone treatment alone did not alter any of the above mentioned oxidative stress parameters. Furthermore, significant difference were found in MDA (P < 0.01), nitrite (P < 0.001), reduced glutathione level (P < 0.01) and catalase activity (P < 0.05) between control and cisplatin + edaravone animals. 3.3.2 Effect of edaravone treatment on hippocampal acetylcholinesterase (AChE) activity in cisplatin treated animals

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Table 2 shows that cisplatin administration caused marked (P < 0.001) elevation of the AChE activity in the hippocampus. However, concomitantly treatment with edaravone significantly (P < 0.01) lowered the cisplatin-induced AChE activity as compared to the cisplatin group. Edaravone treatment alone did not alter the AChE activity compared to the control group. 3.4 Effect of edaravone treatment on hippocampal proinflammatory cytokines (IL-1β and TNFα) and BDNF level in cisplatin treated animals Fig. 8A shows the effect of edaravone treatment on the proinflammatory mediators i.e. IL-1β, and TNF-α in the cisplatin-treated rats. We found significant elevation of IL-1β (P < 0.01) and TNF-α (P < 0.001) level in the animals exposed to cisplatin when compared with the control group. However, treatment with edaravone significantly (P < 0.05 and P < 0.01) prevented the elevation of TNF-α and IL-1β respectively in the hippocampus region of cisplatin-treated rats. Furthermore, significant difference (P < 0.001) was found in the TNF- α level when compared control and cisplatin + edaravone groups. The cisplatin treatment caused marked (P < 0.001) reduction in the hippocampal BDNF level. However, concomitant treatment of edaravone with cisplatin for seven weeks significantly (P < 0.001) prevented the depletion of BDNF level as compared to cisplatin treated group (Fig. 8B). 3.5 Effect of edaravone treatment on hippocampal NF-κB, Nrf2 and HO-1 gene expression level in cisplatin treated animals The reverse-transcriptase PCR results showed that cisplatin administration significantly (P < 0.001) increased the expression level of NF-κB and caused marked (P < 0.001 and P < 0.001, respectively) reduction of the expression level of Nrf2 and HO-1 when compared with the control group (Fig. 9B-D). The edaravone treatment significantly prevented the cisplatin-induced 15

NF-κB activation (P < 0.01) and thereby reducing inflammatory mediators. Moreover, edaravone treatment potentiated the antioxidant defense system by upregulating Nrf2 (P < 0.001) and HO-1 (P < 0.001) expression level as compared to the cisplatin-treated group. 4. Discussion Chemotherapy is one of the most effective strategies for the treatment of cancer. Apart from treating cancer, chemotherapeutic drugs often cause severe adverse effects such as cardiotoxicity, ototoxicity, nephrotoxicity, neurotoxicity, thereby limiting its clinical application (Li et al., 2006; Kwatra et al., 2016a, b). Cisplatin exhibits hydrophilic property and therefore unable to transit through the blood-brain barrier (BBB) under normal physiological conditions. However, under altered physiological milieu such as hypoxic condition, cisplatin easily pass through the BBB (Oz et al., 2015). Some experimental reports evidently suggested that the neurotoxic effects of cisplatin mediated through alteration in the structural architecture of BBB which permit drug across it (Abou-Elghait et al., 2010). Cisplatin eventually causes damage in certain brain regions such as cerebral cortex, cerebellar cortex, and hippocampus that ultimately elicits neurotoxic consequences such as seizures, cognition deficits, peripheral and autonomic neuropathy. The detrimental changes induced by cisplatin in the hippocampus region leads to cognitive dysfunction (Chtourou et al., 2015; Golchin et al., 2015; Oz et al., 2015). There is no experimental report which suggests the role of NF-κB and Nrf2/HO-1 signaling pathway in cisplatin-induced neurobehavioral and neurochemical deficits in the hippocampus. The present study evaluated the gene expression levels of Nrf2, HO-1 and NF-κB along with oxidative stress markers and proinflammatory cytokines level in the hippocampus of cisplatin-treated rats. Moreover, our aim of the present study was to assess the neuroprotective action of edaravone, a potent antioxidant against cisplatin-induced behavioural anomalies. Some studies have been 16

carried out that suggests the contrasting overview about the use of antioxidant which causes tumor protection and decreased survival rate of patients (Lawenda et al., 2008). However, edaravone was found to boost the anti-tumor potential of the anticancer agent (irinotecan) against the colon cancer and also thwarted the multiple cyst formations in ascites cancer-bearing rats through apoptotic pathway without altering the anticancer activity of cisplatin chemotherapy (Kokura et al., 2005; Iguchi et al., 2005). In the present study, progressive body weight loss was observed in cisplatin treated animals which is in concordance with the previous studies (Oz et al., 2015). However, edaravone treatment significantly prevented the cisplatin-induced body weight loss. Gastrointestinal toxicity, renal toxicity and altered lipid metabolism in liver may be the probable factors responsible for the weight loss after cisplatin treatment (Sugihara and Gemba, 1986; Garcia et al., 2013). Nrf2 is a basic leucine zipper transcription factor that activates the antioxidant response element (ARE) in the promoter regions of many antioxidant and detoxification genes. Therefore, Nrf2 is a key regulator of anti-oxidative defense responses which plays an important role in protection against oxidative stress (Vargas et al., 2008). HO-1 also provides a strong defense system against oxidative stress and its transcription is controlled by Nrf2 gene level. HO-1 exerts a neuroprotective effect by accelerating the degradation of heme molecules that results in the formation of biliverdin and bilirubin. We found upregulation of NF-κB expression level and downregulation of Nrf2 and HO-1 expression levels in the hippocampus of cisplatin-treated animals. The administration of edaravone exerts a neuroprotective effect against neurobehavioral anomalies provoked by cisplatin treatment via upregulation of Nrf2 and HO-1 (Fig. 9C and 9D) and down-regulation of NF-κB (Fig. 9B).

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The brain is a very complex and delicate organ which is greatly affected by several chemotherapeutic agents treating various cancers. Several studies reported that neurotoxicants negatively affects the hippocampus by activating the neuroinflammatory cascade, which in turn causes behavioural deficits (Wang et al., 2009; Jangra et al., 2015; 2016a). In the present study, we found that administration of cisplatin caused significant impairment in spatial and recognition memory evident by MWM and NORT results. Edaravone treatment concomitantly reversed the whole paradigm i.e reduced the escape latency and increased the time spent in the target quadrant in the animals. Furthermore, NORT result revealed that cisplatin treated animals showed lower percentage of recognition index. Whereas, edaravone treatment in cisplatin group significantly prevented the reduction of recognition index which indicates the protective effect of edaravone against recognition memory impairment. The resulted protective action of edaravone against spatial and recognition memory impairment may be mediated through restoration of BDNF and AChE activity in the hippocampus region. The augmentation as well as the reduction of AChE activity has been reported to be involved in the pathogenesis of cognitive dysfunction (Jangra et al., 2014a; Kasbe et al., 2015). A recent study demonstrated that cisplatin increases the AChE expression level in the hippocampus of aged rats (Chtourou et al., 2015). Similarly, in our study, we found that increased AChE activity in the hippocampus region of cisplatin-treated rats. The raised ROS level and disturbed calcium homeostasis might result into increase in AChE activity (Melo et al., 2003; Huh et al., 2014; Mao et al., 2014). A recent study has reported that the ameliorative effect of edaravone against cognitive dysfunction mediated thorugh reduction of hippocampal AChE level (Yang et al., 2015). In our study, concomitant treatment of edaravone prevented the augmentation of AChE activity in the hippocampus that contributes to its ameliorative effect against cognitive dysfunction. Cisplatin also affects the motor function by

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disrupting the normal functioning process of neurons in the cerebellar cortex region. In the present study, we found marked motor in-coordination in the cisplatin-treated rats evident by results of rotarod and open field test. Studies have shown that cisplatin administration causes motor incoordination by affecting the growth of Purkinje cells and morphology of granule cells (Bernocchi et al., 1990; Pisu et al., 2005). In our study, edaravone treatment significantly alleviated cisplatin-induced motor incoordination. Cisplatin raised the reactive oxygen species (ROS) level, which in turn affects the brain mitochondria that leads to disruption of the electron transport chain function. The generated free radicals further damage mitochondrial and nuclear DNA which eventually causes cell death (Marullo et al. 2013). These raised free radical species further cause lipid peroxidation and raise the level of MDA and reduce the reduced glutathione level. In our study, similar finding was observed in the cisplatin-treated group. Edaravone treatment significantly prevented the cisplatin-induced depletion of glutathione level and augmentation of MDA level in the hippocampus. A recent report suggested that cisplatin evoked the inducible nitrite oxide synthase (iNOS) level in the hippocampus that further causes nitrosative stress. iNOS elicits the formation of nitrite oxide (NO) which is an unstable product. The cellular metabolism produces the stable products of nitrogen as of nitrates (NO3-) and nitrites (NO2−). These nitrogen species further causes apoptosis and neuronal cell death (Sawicka et al., 2013). In addition, elevated nitrite level disturbed the normal functioning of mitochondria. We found the significant elevation of nitrite level in the hippocampus of the cisplatin-treated group which was prevented by concomitant treatment with edaravone. A previous study has shown that edaravone exerted neuroprotection in transient ischemia by reducing the iNOS expression (Otani et al., 2005). Furthermore, we found that antioxidant enzymes (SOD and CAT) activities were reduced in the cisplatin-treated group. 19

The depletion of these antioxidant enzymes were prevented by concomitant treatment with edaravone. The prevention of antioxidant enzymes depletion by edaravone may be due to the upregulation of Nrf2/HO-1 axis. The increased expression of defensive genes such as Nrf2 and HO-1 leads to prevention of cellular oxidative insult by free radicals. Pro-inflammatory cytokines are one of the major culprits involved in behavioural deficits. Several pharmacological interventions mitigated behavioural deficits by attenuation of proinflammatory cytokines in the hippocampus (Jangra et al., 2016b-d; Sriram et al., 2016; Sulakhiya et al., 2016). NF-κB plays a critical role in the activation of neuroinflammatory pathway which causes induction of iNOS gene expression level and proinflammatory cytokines level (Li et al., 2006; Lawrence et al., 2009). The raised level of proinflammatory level of cytokines such as TNF-α and IL-1β were observed after weekly treatment of cisplatin for seven weeks which was prevented by concomitant treatment of edaravone. These results indicate the anti-inflammatory property of edaravone against cisplatin-induced neuroinflammation. The antiinflammatory property of edaravone in different animal models has been demonstrated earlier in our laboratory (Jangra et al., 2016d; Sriram et al., 2016). BDNF is an important neurotrophic factor which involves in neuronal survival, maintaining synaptic plasticity, learning and memory (Vaynman et al., 2004; Sriram et al., 2016). Furthermore, BDNF involves in hippocampal longterm potentiation and effectiveness of synaptic ability to trigger cognition (Lu et al., 2014). Inflammatory mediators have been shown to reduce hippocampal BDNF level after administration of cytokine-inducer lipopolysaccharide (Jangra et al., 2014b, Sulakhiya 2015). In our study, cisplatin treatment caused BDNF depletion along with neuroinflammation in the hippocampus region. The reduction of BDNF level in the hippocampus may be responsible for the cisplatin-induced learning and memory deficits. A number of previous studies showed 20

reported similar findings with cisplatin treatment (Sun et al., 2013; Andres et al., 2014). Edaravone treatment significantly prevented the BDNF depletion which is corroborated with previous experimental reports (Okuyama et al., 2015; Jangra et al., 2016d; Sriram et al., 2016). The protective effect of edaravone against cisplatin-induced BDNF depletion may be attributed to its ability to reduce proinflammatory cytokines via inhibition of NF-κB. 5. Conclusion In summary, our results showed the neuroprotective potential of edaravone against cisplatininduced neurotoxicity in experimental rats. The systemic administration of cisplatin caused motor-incoordination, learning and memory deficits that involved upregulation of NF-κB, AChE and downregulation of Nrf2, HO-1, and BDNF level in the hippocampus. Edaravone treatment upregulated the Nrf2/ARE pathway and thereby reducing oxido-nitrosative stress. Moreover, Edaravone inhibited the NF-κB activation and thereby suppresses proinflammatory cytokines level. The ameliorative effect of edaravone may be attributed to its ability to reduce the AChE activity and augment the BDNF content in the hippocampus region. Hence, our study states the utilization of edaravone during cisplatin chemotherapy which may effectively prevent the chemotherapy-induced cognitive deficits. Edaravone may be an intriguing therapeutic approach that is capable to provide the better quality life to cancer patients under cisplatin chemotherapy with neurobehavioral deficits. Conflict of Interest The authors declare no financial or commercial conflict of interest Acknowledgments

21

We would like to thank the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, for the financial support. The authors are immensely thankful to the Institutional Level Biotech hub, NIPER Guwahati and State Biotech Hub, College of Veterinary Sciences, Guwahati for providing technical support for providing technical support. References Abou-Elghait, A. T., El-Gamal, D. A., Abdel-Sameea, A. R., Mohamed, A.A., 2010. Effect of cisplatin on the cerebellar cortex and spinal cord of adult male albino rat and the possible role of vitamin E: light and electron microscopic study. Egypt. J. Histol. 33, 202-212. Andres, A.L., Gong, X., Di, K., Bota, D.A., 2014. Low-doses of cisplatin injure hippocampal synapses: a mechanism for 'chemo' brain? Exp. Neurol. 255:137-144. Bernocchi, G., Scherini, E., Nano, R., 1990. Developmental patterns in the rat cerebellum after cis-dicholordiammineplatinum treatment. Neuroscience. 39, 179-188. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882-8. Bevins, R. A., Besheer, J., 2006. Object recognition in rats and mice: a one-trial non-matchingto-sample learning task to study “recognition memory”. Nat. Protoc. 1, 1306-1311. Chtourou, Y., Gargouri, B., Kebieche, M., Fetoui, H., 2015. Naringin abrogates cisplatin-induced cognitive deficits and cholinergic dysfunction through the down-regulation of AChE expression and iNos signaling pathways in hippocampus of aged rats. J Mol Neurosci. 56, 349-362.

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Fig. 1. Structure of Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) Fig. 2. Illustration of experimental timeline and study plan. BW, Body weight; MWM, Morris water maze test; OFT, Open field test; NORT, Novel Object Recognition Test Fig. 3. Mortality in animals (A) percentage of mortality during the study and (B) Percentage of mortality in different groups. Fig. 4. Effects of cisplatin and edaravone on body weight of animals. The data are expressed as the mean ± S.E.M. (n = 6). ###P < 0.001 compared with control group, **P < 0.01 compared with cisplatin-treated animals. $$$P < 0.001, $$P < 0.01 compared with control group. Fig. 5. Effect of edaravone on cisplatin-induced cognitive impairment in Morris Water Maze Test. (A) Escape Latency in training trials (B) time spent in the target quadrant (C) swimming speed. The data are expressed as the mean ± S.E.M. (n = 6). ###P < 0.001 compared with control group, ***P < 0.001, **P < 0.01 compared with cisplatin-treated animals. Fig. 6. Effect of edaravone on novel object recognition task (NORT) in cisplatin-treated animals (A) recognition index (%) (B) total exploration time (sec). Values are expressed as mean ± S.E.M. (n = 6). ###P < 0.001 compared with control group, *P < 0.01 compared with cisplatintreated animals. $$$P < 0.001 compared with control group. Fig. 7. Effect of edaravone on rotarod test in cisplatin-treated animals. Values are expressed as mean ± S.E.M. (n = 6). ###P < 0.001 compared with control group, **P < 0.01 compared with cisplatin-treated animals. $$$P < 0.001 compared with control group. Fig. 8. Effect of edaravone on (A) interlekin-1β and tumor necrosis factor-α (B) BDNF level in the hippocampus of cisplatin-treated animals. Values are expressed as mean ± S.E.M. (n = 6). 33

###

P < 0.001, ##P < 0.01 compared with normal control group; ***P < 0.001, **P < 0.01, *P <

0.05 compared with cisplatin-treated group. $$$P < 0.001 compared with control group. Fig. 9. Effect of cisplatin and edaravone on hippocampal gene expression levels (A) Representative pictures of agarose gels showing the RT-PCR expression (B) NF-κB (C) Nrf2 (D) HO-1 gene expression level was expressed in terms of relative intensity normalized by β-actin expression level. Results are expressed as mean ± S.E.M. (n = 6). ###P < 0.001 compared with normal control group; ***P < 0.001, **P < 0.01 compared with cisplatin-treated group.

34

Fig. 1.

Fig. 2.

35

Fig. 3. (A)

(B)

36

Fig. 4.

Fig. 5.

(A)

37

(B)

(C)

38

Fig. 6.

(A)

(B)

39

Fig. 7.

Fig. 8. (A)

40

(B)

Fig. 9. (A)

Control

Cisplatin

Cisplatin + Edaravone Edaravone Edaravone

NF-κB Nrf2 HO-1 β-actin

41

(B)

(C)

42

(D)

43

Tables Table. 1. Effect of edaravone on cisplatin induced changes in the exploratory behavior of rats in the openfield test. Values are expressed as the mean ± S.E.M. (n = 6). Cisplatin + Control

Cisplatin

Edaravone Edaravone

Central Crossings

a

14.83 ± 0.79

4.50 ± 0.43

41.33 ± 1.14

29.00 ± 1.32

37.33 ± 1.20

30.00 ± 1.26

7.67 ± 0.56

a,c

14.67 ± 0.42

(Frequency) Peripheral crossings

a

b,d

34.50 ± 1.12

40.00 ± 1.07

(Frequency) Rearings

b

d

35.50 ± 1.18

36.17 ± 0.95

(Frequency) a

P < 0.001 compared with control group.

b

c

P < 0.01 compared with control group.

P < 0.01 compared with cisplatin treated group.

d

P < 0.05 compared with cisplatin treated group.

44

Table. 2. Effect of edaravone on cisplatin induced changes in biochemical parameters n the hippocampus. Values are expressed as the mean ± S.E.M. (n = 6).

Control

MDA level

47.06±2.78

Cisplatin

#

Cisplatin + Edaravone

101.9±3.28

a

64.42±2.86

b,d

Edaravone

46.43±2.70

###

(µM/g of tissue wt) Nitrite level

a

20.43±1.59

62.8±2.08

4.31±0.23

1.79±0.19

7.10±0.29

4.85±0.28

43.65±1.37

26.50±0.87

7.83±0.44

13.18±1.01

42.69±3.06

a,d

21.65±1.87

(µM/mg of protein) Reduced Glutathione

a

3.14±0.16

b,d,

4.29±0.12

(µM/mg of protein) Superoxide dismutase (SOD) activity

a

6.21±0.32

f

6.90±0.29

(U/mg of Protein)

Catalase activity (mMol/min/mg of

a

37.98±1.40

c,d

42.37±1.17

protein) Acetylcholinesterase activity (µM/min/mg

a

9.33±0.66

e

8.11±0.65

of protein) a

P < 0.001 compared with control group.

b

c

P < 0.01 compared with control group.

P < 0.05 compared with control group.

d

P < 0.001 compared with cisplatin treated group. 45

e

P < 0.01 compared with cisplatin treated group.

f

P < 0.05 compared with cisplatin treated group.

46